Note: Descriptions are shown in the official language in which they were submitted.
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PURIFICATION AND CHARACTERIZATION OF HLA
PROTEINS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims Convention priority and benefit under 35 U.S.C.
~ 119(e) to U.S. Patent Application No. 60/347,906, filed 2 January 2002,
entitled "sHLA ASSAY METHODOLOGIES," the contents of which are hereby
expressly incorporated herein in their entirety by this reference.
This application is also a U.S. continuation-in-part of U.S. Patent
Application No. 10/022,066, filed 18 December 2001, entitled "METHOD AND
APPARATUS FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES
THEREOF," the contents of which are hereby expressly incorporated herein in
their entirety by this reference.
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BACKGROUND
1. Field
The present invention relates generally to the production and use of
functionally active soluble HLA molecules that are isolated and purified
substantially away from other proteins, and methods of purifying same.
2. Description of the Background Art
Class I major histocompatibility complex (MHC) molecules, designated
HLA class I in humans, bind and display peptide antigen ligands upon the cell
surface. The peptide antigen ligands presented by the class I MHC molecule
are derived from either normal endogenous proteins ("self") or foreign
proteins ("nonself") introduced into the cell. Nonself proteins may be
products of malignant transformation or intracellular pathogens such as
viruses. In this manner, class I MHC molecules convey information
regarding the internal fitness of a cell to immune effector cells including
but
not limited to, CD8+ cytotoxic T lymphocytes (CTLs), which are activated
upon interaction with "nonself" peptides, thereby lysing or killing the cell
presenting such "nonself" peptides.
Class II MHC molecules, designated H~A class II in humans, also bind
and display peptide antigen ligands upon the cell surface. Unlike class I MHC
molecules which are expressed on virtually all nucleated cells, class II MHC
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molecules are normally confined to specialized -cells, such as B lymphocytes, -
macrophages, dendritic cells, and other antigen presenting cells which take
up. foreign antigens from the extracellular filuid. via an endocytic pathway. -
- The peptides they.,.bind and present arederived from extracellular foreign
antigens, such as products of bacteria .that multiply outside of cells,
wherein
such products include -protein toxins secreted by the bacteria that often .
times have deleterious and even lethal effects on the host (e.g. human). in
this manner, class.-iI molecules convey information regarding the fitness of
the extracellular space in the vicinity of the cell displaying the class II
molecule to immune effector cells, including but not limited to, CD4+ helper
T .cells, :thereby, helping to eliminate such pathogens. The examination of
such pathogens is accomplished by both helping B cells make antibodies
against microbes, as well as toxins produced by such microbes, and -by
activating macrophages to destroy ingested microbes.
Class I and class II HLA molecules exhibit extensive polymorphism
generated by systematic recombinatorial and point mutation events; as
such, hundreds of different HtA types exist throughout the world's
population, resulting in a large immunological diversity. Such extensive HLA
diversity throughout the population results in tissue or organ transplant
rejection between .individuals. as well as differing susceptibilities and/or
resistances to infectious diseases. H1~1 , molecules also contribute
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si9~ificantly to autoimmunity and cancer. Because HLA molecules mediate
most, if not all, .adaptive immune responses, large quantities of pure
isolated
HL~4~ proteins are required in order to effectively study transplantation; .
autoimmunity disorders, and for vaccine development.
. ~ These are several applications in which purified, individual class ~I and
class II MHG proteins are highly useful.. ~ Such . applications include using
MHC-peptide multimers as iiiimunodiagnostic reagents for disease
resistance%autoimmunity; assessing the binding of potentially therapeutic
peptides; elution of peptides from MHC molecules to identify vaccine
candidates; screening transplant patients for preformed MHC specific
antibodies; and removal of anti-HLA antibodies from a patient. Since every
,.
individual has differing MHC molecules, the testing of numerous individual
MHC molecules is a prerequisite for understanding the differences in disease
susceptibility between individuals. Therefore, isolated and purified MHC
molecules that are representative of the hundreds of different HLA types
existing throughout the world's population are highly desirable for unraveling
disease susceptibilities and resistances, as well as for designing
therapeutics
such as vaccines.
Class I HLA molecules alert the immune response to disorders within
host cells. Peptides, which are derived from viral- and tumor-specific
proteins within the cell, are loaded into the class I molecule's antigen
binding
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groove in the .endoplasmic reticulum of the cell and subsequently carried to
the cell surface.. Once the class I HLA molecule and its loaded peptide ligand
are on the cell surface, the class I molecule -and its peptide ligand are
accessible to cytotoxic T lymphocytes (CTL). CTL survey the peptides
presented by the class I molecule and destroy those cells harboring liga.nds
derived from. infectious or neoplastic agents within that cell.
While specific C'fL targets have been idebtihed, little is known about
the breadth and nature of ligands presented, on the surtace of a diseased
cell. From a basic science perspective, many outstanding questions have
permeated through the art regarding peptide exhibition. For instance, it has
been demonstrated that a virus can preferentially block expression of HLA
class I molecules from a given locus while leaving expression at other loci
intact. Similarly, there are numerous reports of cancerous cells that fail to
express class I HLA at particular loci. However, there is no data describing
how (oc if) thethree classical HLA class I loci differ in the immunoregulatory
ligands they bind. It is therefore unclear how class I molecules from the
different loci vary in their interaction with viral- and tumor-derived ligands
and the number of peptides each will present.
Discerning virus- and tumor-specific ligands for CTi_ recognition is an
important component of vaccine design. Ligands unique to tumorigenic or
infected cells can be tested and incorporated into vaccines designed to evoke
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a protective CTL response. Several methodologies are currently employed to
identify potentially protective peptide ligands. One approach uses T cell
lines
or clones to screen for biologically active ligands among chromatographic
fractions of eluted peptides (Cox et al.; Science, vol 764, 1994, pages
716-719, which is expressly incorporated herein by reference in its entirety);
This approach has been employed to identify peptides ligands specific to
cancerous cells. A second technique utili2es predictive algorithms to identify
peptides capable of binding to a particular class I molecule based upon
previously determined motif and/or individual ligand sequences (De Groot et
al.,_. Emerging Infectious Diseases, (7) 4, 2001, which is expressly
incorporated herein by reference in its entirety). Peptides having high
predicted probability of binding from a pathogen of interest can then be
synthesized and tested for T cell reactivity in precursor, tetramer or ELISpot
assays.
However, prior to the presently claimed and disclosed inventions)
there has been no readily available source of individual isolated and purified
HLA molecules, The quantities of HLA protein previously available have been
small and typically consist of a mixture of different HtA molecules.
Production of HLA molecules, traditionally involves growth and lysis of cells
expressing multiple HLA molecules. Ninety percent of the population is
heterozygous at .each of the FiW loci; codominant expression results in
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multiple HLA proteins, expressed at each HlA locus. .To purify native class I
orclass II molecules from mammalian cells requires time-consuming and
cumbersome purification methods, and since each cell typically, expresses
multiple surface-bound HLA class .I or class II molecules; Hl~ purification
results in a mixture of many different HLA class I or class II molecules.
When performing. experiments using such a mixture of HLA molecules or
performing experiri~ents using _a cell having' multiple surface-bound HLA
molecules, interpretation of results cannot directly distinguish between the
different HLA molecules, and one cannot be certain that any particular HLA
molecule is responsible for a given result. Therefore, prior to the presently
claimed and disclosed invention(s), a need existed in the art for a method of
producing substantial Quantities of individual HLA class I or class II
molecules so that they can be readily purified and isolated independent of
other HLA class I or class Il~molecules. Such individual isolated and purified
HLA molecules, when provided in suffcient quantity and purity as described
herein, provide a powerful too( for studying and measuring immune
responses.
Therefore, there exists a need in the art for improved methods of
isolating and purifying individual HLA molecules substantially away from
other proteins. In one exemplary embodiment, the present invention solves
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this need by coupling the production of soluble HLA molecules with a
purification methodology involving affinity chromatography.
SUMMARY
The present invention is directed to a functionally active, individual
soluble HLA molecule purified substantially away from other proteins such
that the individual soluble HLA molecule maintains the physical, functional
and antigenic integrity of the native HLA molecule. The term "physical,
functional and antigenic integrity of the native HLA molecule", as used
herein, will be understood to mean that the soluble HLA molecules exhibit
the same structure (including primary, secondary, tertiary and quaternary)
as the extracellular portion of the native HLA molecules, that they are
identical in functional properties to an HLA molecule expressed from the HLA
allele mRNA or gDNA and thereby bind peptide ligands in an identical
manner as full-length, cell-surface-expressed HLA molecules, and that they
are recognized by the cellular machinery responsible for responses to
specific HLA-peptide complexes, that is NK and T cells.
The functionally active, individual soluble HLA molecule is a Class I HLA
molecule or a Class II HLA molecule, and may have an endogenous peptide
loaded therein.
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The peptide may be produced by several methods, including but not
limited to the following. In one embodiment, HLA allele mRNA from a source
is isolated and reverse transcribed to obt8in allelic cDNA. Iri a separate
ern6adiment, gDNA encoding a HLA allele is obtained. The allelic cDNA or
gDNA is amplified by PCR -utilizing at least one locus-specific primer that
truncates the allelic cDNA or gDNA, thereby resulting in a truncated PCR
product having the coding regions encoding cytoplasmic and transmembrane
domains..of the allelic cDNA removed such that the truncated PCR product
has a coding region encoding a soluble Ht,.A molecule. The at least one
locus-specific primer may include a stop codon incorporated into a 3' primer,
or,the at least one locus-specific primer may include a sequence encoding .a
tail such that _ the soluble IiLA molecule encoded by the truncated PCR
product contains a tail attached thereto that facilitates in purification of
the
soluble HLA molecules produced therefrom.
The truncated PCR product is then inserted into a mammalian
expression vector to form a plasmid containing the truncated PCR product
having the coding region encoding a soluble HLA molecule, and the plasmid
is electroporated into at least one, suitable host cell. The mammalian
expression vector contains a promoter that facilitates increased expression
of the truncated PCR product. The host cell may lack expression of Class I
HLA molecules.
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A cell pharrn is inoculated with the at least one suitable host cell
containing the plasmid containing the truncated PCR product such that the
cell pharm produces soluble HLA molecules, wherein the soluble NI.A
molecules are folded naturally and are trafficked through the cell in . such a
way that-.tliey are identical 'in functional properties .to an HLA molecule
expressed from .the HlA allele mRNA and thereby bind peptide ligands in an
identical manner as full-length, cell-surface-expressed Hl~4~molecules. The
individual, soluble HLA molecules are then harvested from the cell pharm
and purified substantially away from other proteins: The purification process
involves affinity column purification and filtration. The purified individual
soluble HLA, molecules maintain the physical, functional and antigenic
integrity of the native HLA molecule.
When HLA allele mRNA is used, the source is selected from the group
consisting of mammalian DNA 'and an immortalized cell line. When gDNA
which encodes an HI.A allele is used, the gDNA is obtained from blood,
saliva, hair, semen, or sweat.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. i is a graphical representation of a Class I location and sHl~4 class
I construction strategy. (A) Simple map of the human MHC region with the
class I HLA-B, -C, and -A loci noted. Genetic distances are in kilobases. (B)
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The basic exon structure of HLA class I gene transcripts. Seven exons
encode the class I heavy chain. (C) PCR strategy for truncating the class I
molecule so that it is secreted rather than surface bound.
FIG. 2 is a pictorial representation of native and recombined truncated
form of sHLA which differ in the presence of a transmembrane and cytosolic
region in the native molecule. Both forms show no differences in their
ambiguity and peptide presenting properties.
FIG. 3 is a three dimensional pictorial representation of a truncated
molecule. The by view is visualizing the al and a2 domains harboring the
peptide. The side view shows the full molecule with a detailed view of a3 and
~32m domains.
FIG.4 is a pictorial representation showing the peptide binding
platform in more detail where two a helices form the rim and seven ~3 sheets
form the bottom of the binding groove.
FIG S is a graphical representation of an ELISA procedure
demonstrating that W6/32-coupled affinity column can be saturated with
crude harvest containing sHLA-B*0702His.
FIG.6 is a graphical representation of an ELISA procedure
demonstrating the wash step for the W6/32-coupled affinity column of
FIG. 5.
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FIG:7 is a. graphical representation of an ELISA procedure . .
demonstrating the elution of sHLA-B*0702His from the W6/32~coupled
affinity column of FIG. 5.
.FIG.8 is a chart showing the buffer exchange and concentration
procedure using MACROSEP'~'" filters. ELISA performed during the filtration
steps confirm minimal loss of protein.
FIG, 9 is a chart showing the final sterile , filtration step optimized to
remove remaining particles within the filtrate.
FIG. 10 is a tabular representation showing a summary of values
measured during the purification procedure directly related to the efficiency.
FIG. 11 is a pictorial representation illustrating the protein Sequence
Data for MHC Class I-HLA-A*0201T.
FIG._ 12 is a pictorial , representation showing the protein Sequence
Data for MHC Class I-HLA-B*0702T.
FIG. 13 is a pictorial representation illustrating the Protein Sequence
Data for MHC Class I-Hll1-B*1512T.
FIG. 14 is a tabular representation illustrating the amino acid analysis
of B*1512 following proteolysis of whole molecule.
FIG.15 is a graphical representation showing SuperdexT'"
chromatography to demonstrate sample purity of stiLA-8*i512T.
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FIG.- 16 is a wgraphical representation illustrating a Triple analysis of
B*1512T: It shows.a separation of sHLA under denaturing and under native
conditions.
FIG. 17 is a graphical representation showing a SuperdexT"' profile of
A*0201T.
FIG. l8 is.a.=pictorial ~epresentatiowof an. SDS-PAGE gel analysis. of
several purii~ied sHLA samples .confirming the purity with this procedure.
FIG. 19 is a .pictorial representation of a Western blot analysis to
follow the HC and ~2m subunits of sHLA.
FIG. 20 is a chart depicting an activity confirmation of sHLA using
s.~andard sandwich ELISA procedure.
FIG. 21 is a pictorial scheme of antibody binding scenarios for the
direct ELISA procedure. Several antibodies were tested on intact as well as
denatured sHIA, Direct .finding of sHl.A molecules causes partial
denaturization of the molecules and thus no specific denaturation step is
necessary.
FIGS. 22-27 are, charts showing reaction panels for conformation-
specific Ab binding assays using the direct ELISA procedure.
FIG. 28 is a pictorial scheme of the two antibody binding scenarios
using W6/32 or anti-b2m . as capturing antibodies in a sandwich El.,ISA
procure. Several detection antibodies were used.
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FTGS. 29-32 are charts showing reaction panels for conforrnation-
specific Ab binding assays using several Pan=Class I monoclonal antibodies in
the sandwich ELISA procedure..
FIGS. 33-34 are charts illustrating various antibody combinations to
test for artificial structural forms such as aggregation or dimeric structures
showing A, B, and C alleles.
FIGS. 35-36 are charts illustrating neutralization experiments to verify
antigenic integrity using sHLA-A*0201T and A2 alloantiserum M102 as well
as Ab MA2:1.
FIG. 37 is a pictorial representation illustrating anti-calreticulin blot of
full-length HLA-B27 (+), Ht~1 negative cell line 721.221 (-) and various
constructs of soluble HLA-B15 molecules immunoprecipitated with the HLA-
specific antibody HC-10.
FIGS. 38-51 are charts showing ELISA reactions testing a panel of
selected sHI.A alleles using different commercially available single
specificity
monoclonal antibodies.
. FIGS. 52-53 are charts illustrating E~ISA Reaction panels testing
antibodies Bw6 and Bw4.
FIG. 54 is a pictorial representation depicting a motif comparison
between sHLA-B*1501 and membrane bound B*1501 from another
laboratory..
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FIG.55 is a pictorial representation showing a fluorescence
polarization sche~ie allowing the detection- of bound and free peptides to the
sNLA complex in solution without separation using radiometric
measurement. of parallel and perpendicular -fluorescent. intensities. Free
pepti~s create a low FP signal where bound peptides show.high FP values.
FIGS. 56-57 are g~aphicai representations showing a one phase
exponential association curve using the sHLA allele A*0201T combined With
the FITC-labeied peptide P5 (A*0201).
FIGS. 58-59 are graphical representations showing saturation
experiments generating saturation curve wherein sHLA (binder) is held
constant to determine the dissociation constant (Kp).
FIGS. 60-61 are graphical representations showing competition
experiments of, fixed concentration of fluorescent-labeled synthetic peptide
in
the presence of various concentrations of unlabeled test competitor-peptides
r f:.: _ .-
to determine the ICSO value. -
FIG. 62 is a graphical representation showing an ELISA procedure
demonstrating the binding of a HBV peptide to sHLA molecules and
successful-replacement of the endogenous peptide with the HBV peptide.
FIGS: 63-66 are charts showing ELISA procedures demonstrating
stability of sHLA-B*1512T in different buffers and solutions during different
days with a summary given in FIG. 66.
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FIG. 67 ,is a- graphical representation showing an (LISA procedure ~.
demonstrating the influence of temperature on stability of sHLA complex.
FIG.. 68 is a graphical representation ,showing the - influence of freeze-
thaw cycle on stability. . .
FIG.69 is a pictorial representation showing the experimental
procedure for determining .loss of complex reactivity due to nonspecific
adhesion to.surfaces of tubes.-
FIG. 70 is a chart showing the effects of different microcentrifuge
tubes or cryo vials on reactivity of sHLA.
FIG. 71 is,a chart showing the effects of larger tubes on reactivity of
sHLA.
FIG. 72-73 are charts depicting the effects of blocking agents on
reactivity of sHLA, including PVP and PEG.
FIG. 74~ is a chart showing the effects of non-ionic detergents on
reactivity~of sHLA.
FIG: 75 is a chart showing the effect of different BSA concentrations
on reactivity of sHlA.
FIG. ~ 76 is a ~ chart showing the effect . of different StabilguardTM
concentrations on reactivity of sHLA.
FIG. 77 is a chart showing the effect of PEG concentrations on
reactivity of sHL~I.
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FIG.78 is a chart showing, the effect of PVP concentrations on
reactivity of sHfll.
FIGS. 79-85 are charts illustrating a sera screen assay that utilizes .
HlA to identify antigen-specific antibodies in human sera.
FIG: 86 is a chart showing SHLA A*0201T reactivity on beads sampled .
through the EDC method:
FIG:.87 is a graphical representation depicting .the screening of test
competitors for ability to inhibit FITC-labeled standard peptide from binding
to sHLA.
FIG. 88 is a graphical representation showing constructed ICso binding
curves ,using a single inhibition value obtained at 100 uM competitor
concentration.
FIG. 89 is a graphical- representation showing ICSO values obtained
during the single value procedure as well as the more accurate 9 point
procedure. sorted according to their measured affinities.
FIGS. 90-9i are graphical representations illustrating the
;.. ,
improvement of binding of modified peptides to sHI.A-A2 as compared to the
native test-peptides Vac 104 and Vac 105.
FIG. 92 is a graphical representation summarizing the purification and
characterization procedures for soluble human HI.A proteins of the present
invention.
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DETAILED DESCRIPTION
Before explaining at least one embodiment of the invention in detail by
way of exemplary drawings, experimentation, results, and laboratory
procedures, it is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of the
components set forth in the following description or illustrated in the
drawings, experimentation and/or results. The invention is capable of other
embodiments or of being practiced or carried out in various ways. As such,
the language used herein is intended to be given the broadest possible scope
and meaning; and the embodiments are meant to be exemplary - not
exhaustive. Also, it is to be understood that the phraseology and
terminology employed herein is for the purpose of description and should not
be regarded as limiting.
The present invention combines methodologies for the production of
individual, soluble MHC molecules with novel and nonobvious methodologies
for the isolation and purification of individual, soluble MHC molecules
substantially away from other proteins. The method of production of
individual, soluble MHC molecules has previously been described in detail in
parent application U.S. Serial No. 10/022,066, filed December 18, 2001,
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entitled "METHOD AND APPARATUS FOR THE PRODUCTION OF SOLUBLE
MHC ANTIGENS AND USES THEREOF," the contents of which are hereby
expressly incorporated in their entirety by reference herein. A brief
description of this methodology is included herein below for the purpose of
exemplification and should not be considered as limiting. One of ordinary
skill in the art, given the disclosure in the 10/022,066 application would be
truly capable of producing individual soluble MHC molecules to be used with
the presently disclosed and claimed isolation and purification methodologies.
It should be preliminary noted, however, that the presently claimed and
disclosed isolation and purification methodologies can be used with HLA
molecules (soluble or non-soluble) obtained by any means and should not be
regarded as being limited to soluble HLA molecules produced according to
the methodologies claimed and disclosed in the 10/022,066 application. In
the event HLA molecules produced according to methodologies other than
those produced according to methodologies disclosed and claimed in the
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10/022,066 application are used in the isolation and purification
methodologies disclosed and claimed herein, one of ordinary skill in the art
(given in the present specification, drawings and claims) would be capable of
making any necessary modifications or derivations to such HLA molecules
such that they may be used in the isolation and purification methodologies
presently claimed and disclosed herein in an efficient and accurate manner.
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Exemplarv Production of Individual, Soluble MHC Molecules
The methods of the present invention may, in one embodiment, utilize
a method of producing MHC molecules (from genomic DNA or cDNA) that are
secreted from mammalian cells in a bioreactor unit. Substantial quantities of
individual MHC molecules may be obtained in the manner by more
particularly modifying class I or class II MHC molecules so that they .are
capable of being secreted, isolated, and purified. Secretion of soluble MHC
molecules overcomes the disadvantages and defects of the prior art in
relation to the quantity and purity of MHC molecules produced. Problems of
quantity are overcome because the cells producing the MHC do not need to
be detergent lysed or killed in order to obtain the MHC molecule. In this
manner, the cells producing secreted MHC remain alive and therefore
continue to produce MHC. Problems of purity are overcome because the
only MHC molecule secreted from the cell is the one that has specifically
been .constructed to be secreted, Thus, transfection of vectors encoding
such secreted MHC molecules into cells which may express endogenous,
surface bound MHC provides a method of obtaining a highly concentrated
form of the transfected MHC molecule as it is secreted from the cells.
Greater purity is assured by transfecting the secreted MHC molecule into
MHC deficient cell lines.
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Production of the MHC molecules in a hollow fiber. bioreactor unit
allows . cells to be cultured at a density substantially .greater than
conventional liquid phase. tissue culture permits; Dense culturing of cells
secreting MHC ri~olecules further amplifies. the ability to continuously
harvest
the transfected MHC molecules. Dense bioreactor cultures of MHC secreting
cell lines. allow for high concentrations of individual MHC proteins to be .
obtained.r Highly c9ncentrated individual MHC proteins provide an advantage
in that most downstream protein purification strategies perform better as the
concentration of the protein to be purified increases. Thus, the culturing of
MHC secreting cells in bioreactors allows fior a continuous production of
individual MHG proteins. in a ,concentrated form.
While hollow fiber bioreactor units or cell pharms have been described
~., :,.~~
herein for utilization in the culturing methods of the present invention, it
is
to- ba understood that any large scale mammalian tissue culture system
evident to a person having ordinary skill in the art may be utilized in the
methods of the present -invention, and therefore the present invention is not
specifically limited to the use of a hollow fiber bioreactor unit or a cell
pharm.
The method of producing MHC molecules utilized in the present
invention. and described in ~~ detail in parent application U.S. Serial
No. 10/022;066 begins by obtaining genomic or complementary DNA which
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encodes the desired MHC class I or class II molecule. Alleles at the locus
which encode the desired MHC -molecule are PCR amplified in a locus specific
manner. These locus specific PCR products may include the entire coding
region of. the MHC molecule or a portion thereof. In one embodiment a
nested or hemi-nested PCR is applied to produce a truncated form of the
class I or class Ih gene so that it will be secreted rather than anchored to
the
cell surface. FIG., 1 illustrates the PCR products resulting from such nested
PCR reactions.: In another embodiment the PCR will directly truncate the
MHCrmolecule.
Locus specific PCR products are cloned into a mammalian expression
vector and screened with a variety of methods to identify a clone encoding
the desired MHC molecule. The cloned MHC molecules are DNA sequenced
to ensure fidelity of the PCR, Faithful truncated clones of the desired MHC
molecule are then transfected into a mammalian cell line. When such cell
line is transfected with a vector encoding a recombinant class I molecule,
such cell line may either lack endogenous class I MHC molecule expression
or express endogenous class I MHC molecules.
Qne of ordinary skill in the art would note the importance, given the
present invention, that cells expressing endogenous class I MHC molecules
may spontaneously release MHC into solution upon natural cell death. In
cases where this small amount of spontaneously released MHC is a concern,
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the tra,nsfected class I MHC molecule can be "tagged" such that it can be
specifically purifred away from spontaneously released endogenous class I
molecules in cells that express class h molecules. For example, a DNA
fragment encoding a HIS tail ~ may be attached to the protein by the PCR
reaction or may be .encoded by the vector into which the. PCR fragment is
cloned, and such HIS tail, therefore, further aids in the purification of the
cl~ss~ I MFiC molecules away from endogerious class I molecules. Tags
beside a histidine tail have also been demonstrated to work, and one of
ordinary skill in the art of tagging proteins for downstream purification
would
appreciate and know how to tag a MNC molecule in such a manner so as to
increase the ease by which the MHC molecule may be purified.
Cloned genomic DNA fragments contain both axons and introns as well
as other non-translated regions at the 5' and 3' termini of the gene.
Following transfection into a cell line which transcribes the genomic DNA
(gDNA) into RNA, cloned genomic DNA results in a protein product thereby
removing introns and splicing the RNA to form messenger RNA (mRNA),
which is then translated into an MHC protein. Transfection of MHC molecules
encoded by gDNA therefore facilitates reisolation of the gDNA, mRNA/cDNA,
and protein. Production of MHC molecules in non-mammalian cell lines such
as insect and bacterial cells requires cDNA clones, as these lower. cell types
do not have the ability to splice introns out of RNA transcribed from a gDNA
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clone, . In these instances the mammalian gDNA transfectants of the present..
invention provide a valuable source of RNA which can be reverse transcribed .
to form MHC cDNA. The cDIVA can then be cloned, transferred into cells, and
then translated , into protein. In addition to producing secreted MHC, such
gDNA transfectants therefore provide a ready souree of mRNA, and therefore
cDNA clones, which can then be transfected~ into non-mammalian cells for
produrCion of MHC: Thus; the present invention which starts with MHC
genomic DNA clones allows for the production of MHC in cells from various
species.
A. key advantage of starting from gDNA is that viable cells containing
the . MHC molecule of interest are not needed. Since all individuals in the
population have a different MHC repertoire, one would need to search more
than 500,000 individuals to find someone with the same MHC complement
as a desired individual - such a practical example of this principle is
observed- when trying to find a donor to match a recipient for bone marrow
transplantation. Thus, if it is desired to produce a particular MHC molecule
for use in an experiment or diagnostic, a person or cell expressing the MHC
allele of interest would first need to be identified. Alternatively, in the
method of the present invention, only a saliva sample, a hair root, an old
freezer sample, or less than a milliliter (0.2 ml) of blood would be required
to isolate the , gDNA. Then, starting from gDNA, the MHC molecule of
~,
2s
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interest could be obtained via a gDNA clone as described herein, and
following transfection of such' clone into mammalian cells, the desired
protein
coufd be, produced directly in mammalian cells or from cDNA iri several
species of~cells using the methods-described herein.
Current experiments to obtain an MHC, allele for protein expression
typically start from mRNA, which requires a fresh sample of mammalian cells
that express the MHC molecule of interest. Working . from gDNA does not
require gene expression or a fresh biological sample. It is also important to
note~that RNA is inherently unstable and is not as easily obtained as is
gDNA. Therefore, if production of a particular MHC molecule starting from a
cDNA clone .is desired, a person or cell line that is expressing the allele of
interest must traditionally first be identified in order to obtain RNA. Then a
fresh sample of blood or cells must be obtained; experiments using the
methodology of the present invention show that > S milliliters of blood that
is
less -than 3 days old is required to obtain sufficient RNA for MHC cDNA
synthesis. Thus, by starting with gDNA, the breadth of MHC molecules that
can be readily produced is expanded. This is a key factor in a system as
polymorphic as the MHC system; hundreds of MHC molecules exist, and not
all MHC molecules are readily available. This is especially true of MHC
molecules unique to isolated populations or of MHC molecules unique to
ethnic minorities. Starting class I or class II MHC molecule expression from
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the point of genomic DNA simplifies,the isolation of the gene of interest and
insures a more equitable means of producing MHC molecules for study;
otherwise,. one would be left to determine whose MHC molecules are chosen
and not chosen .for study, as wel( as to determine which ethnic population
from which fresh .samples cannot be obtained 'and therefore should not have
thei~~,MHC.molecules- included in a diagnostic :assay.
While cDNA may be substituted .for genomic DNA as the starting
material, production of cDNA for each of the desired HLA class I types will
require hundreds of different, HLA typed, viable cell lines, each expressing a
different HLA class I type. , Alternatively, fresh samples are required from
individuals with the various desired MHC types. The use of genomic DNA as
the starting material allows for the production of clones for many HLA
molecules from a single genomic DNA sequence, as the amplification process
can _be manipulated to mimic recombinatorial and gene conversion events.
Several mutagenesis strategies exist whereby a given class I gDNA clone
...:,.:~: , . . .
could be modified at either the level of gDNA or at the cDNA resulting from
this. gDNA clone. The process of producing MHC molecules utilized in the
present invention does not require viable cells, and therefore the
degradation which plagues RNA is not a problem.
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Purification of Individual. Soluble MHC Molecules
'The ability to produce large quantities of single specificity sHLA
molecules allows for assay procedures to be quantitative' and resistant to
interferences encountered in biological matrices as well as also being
reliable, highly reproducible, sensitive, and therefore applicable for; high-
throughput systems. Alternative economical ~ methodologies . for obtaining
large quantities of_sHLA molecules do not currently exist since: (1) there is
no readily available source of individual HLA molecules; (2) purification of
native class I molecules from mammalian cells requires time-consuming and
cumbersome purification methods and does not deliver sufficient quantities;
and (3) native molecules from. mammalian cells typically consist of a mixture
of different HIJa molecules. Such a mixture of specificities is not useful
and/or applicable for single specificity studies.
Ht.A class I.molecules are antigen-presenting glycoproteins expressed
universally in nucleated cells. In humans, heavy chains are encoded at 3 loci
(6, C, and A) within the' MHC on the short arm of chromosome 6 (FIG. lA).
FIG. 1B illustrates each a-chain comprised of al, a 2, and a 3 domains, as
well
as a transmembrane domain, which tethers the molecule to the cell surface
and a short C-terminal cytoplasmic domain. In contrast, the light chain is
encoded outside of the MHC (on chromosome 15 in humans) and bears no
such anchoring 'dorriain; it .instead associates noncovalently with the as
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domain of the heavy chain.' FIG, iC illustrates the approach for creating
sHl~4 class I transcripts. The PCR primers truncate the class .I heavy chain
following exon 4, just before the transmembrane doniain and cytoplasmic
domains. Using this PCR truncation strategy, we have successfully. created
sHt.A class I gene products for a series of fifty divergent HLA-molecules.
Class I sHLA genie constructs created as in l=IG. 1C are cloned arid DNA
sequenced to insure fidelity of each clone. The individual class i constructs
are then subcloned into a suitable protein expression vector.
Produced in transfected B cells, sHLA molecules have close to identical
primary structures as papain solubilized Hl~s. Truncated molecules have
been shown by the present inventors to maintain' their structural integrity.
In
addition, HI.A-Aw68, ~ from which the complete alpha 3 domain has been
proteolytically removed, shows no gross morphological changes compared to
the intact protein. A decameric peptide complexed with the intact HLA-Aw68
is seen to bind to the proteolized molecule . in the conventional manner,
demonstrating that the alpha. 3 domain is not required for the structural
integrity of the molecule or for peptide binding. Pictures of sHLA graphics
(FIG. 2) and 3D structures (FIG. 3) more clearly visualize how the molecules
look like.
HLA/MHC genes are the most polymorphic system in mammals,
generated ~ by systematic recombinatorial and' point mutation events; as
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such, hundreds of different HLA types exist throughout the world's
population, resulting in a large immunological diversity. Individuals inherit
a
set of three class I genes from' each parent, and since their expression is
codominant, a single person may therefore display up to . six different HIJ~
class I' molecules .upon his or her nucleated cells. Such extensive HLA
diver~Sity results in differing susceptibilities and/or resistances between
individuals in infectious diseases. Depending .upon allelic composition, two
individuals' molecules may not necessarily bind the same peptides with equal
affinity or even at all. Therefore, despite the overall structural
conservation
illustrated among class I heavy chains, their peptide binding grooves can
vary drastically from one allelic form to another; as a result various
isoforms
are capable of associating with distinct arrays of peptides. A binding
platform
is shown in FIG. 4. The first two domains (alpha i, alpha 2) of the heavy
chain create the, peptide binding cleft and the surface that contacts the T-
cell
receptor. X-ray crystallographic analysis indicates that a processed antigen
is
presented as a peptide bound in a cleft between the two o-helices of the
heavy chain of the HLA complex (Bjorkman P.J., 1987; Nature 329: 506-512
& 512-518 / Garett T.7. 1989: Nature 342; 692-696 / Saper M.A.; 1991; J.
Mol. Biol. 219; 277-319 / Madden D.R. (1991) Nature 353; 321-325; the
contents of each are hereiri expressly incorporated by reference in their
entirety.). The third domain (alpha 3) associates with the T-cell co-receptor,
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CDB, during T=cell recognition. Availability of: a wide spectrum of
recombinant sHIA - molecules overcomes the current art limitations on
population coverage imposed by the rules of MHC restriction. In most cases, v
.
a single-peptide epitope will be useful only for treating a small subset . of
patients, who express the MHC allele product that is capable of binding that
specific peptide. Since every individual has differing MHC molecules, . the
testing of numerous individual MHC molecules is a prerequisite . for
understanding the difference in disease susceptibility between individuals.
PURIFICATION METHODOLOGY
There are many purification methods available for the separation of
macromolecules. To effectively resolve a crude mixture of substances, it may
be necessary to use a combination of techniques. In most cases, a
purification procedure will involve some chromatographic techniques.
Affinity chromatography occupies a unique place in separation
technology since it is the only technique which enables purification of almost
any biomolecule on the basis of its biological function or individual chemical
structure. Affinity chromatography makes use of specific binding interactions
that occur between molecules. It is a type of adsorption chromatography in
which the molecule to be purified is speciFcally and reversibly adsorbed by a
cor~nplementary binding substance (Ilgand) immobilized on an insoluble
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support (matrix). A single pass through an afFnity column can achieve a
1,000-10,000 fold.purification of ligand from ~a crude mixture. It is possible
to. isolate a compound in a form pure enough to obtain a single band upon
SDS-poiyacryfamide gel electrophoresis: Any component that .has an
interacting counterpart can be attached to a support andwused for affinity
purification.
Successful : separation _ by affinity chromatography requires .that a
biospecific ligand is available and that it can be covalently attached to a
chromatographic bed material called a matrix: It is important that the
biospecific ligand (antibody, enzyme, or receptor protein) retains its
specific
binding _ affinity for the substance of interest (antigen, substrate, or
hormone). Methods must also include removing the bound material in active
form with low pH, high pH, or high salt. The selection of the ligand for
affinity chromatography is influenced by two factors. Firstly, the tigand
should~exhibit specific and reversible binding affinity for the substance to
be
purified. Secondly, it should have chemically modifiable groups, which allow
it to be' attached to the matrix without destroying its binding activity. The
ligand should ideally have an affinity for the binding substance in the range
10'4 to 10'$ M in free solution.
The protocol herein discussed provides a method to couple protein to a
commercially available CNBr-activated Sepharose 4B (APB #17-0430-01).
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An alternative option would be running the procedure' with Sepharose 4 Fast
flow '(APB #17-0981-01). Sepharose Fast Flow is more highly crosslinked
than Sepharose 4B. As a result, Fast Flov~r beads are more' stable and can
withstand higher flow rates than the 4B beads. CNBr-activated 5epharose 4B
is~ better suited for batch cliromatography and small columns with gravity
flow. Another difference is in coupling capacities. The coupling .reaction
proceeds most efficiently in the pH range 8-10 where the amino groups on
the ligand are predominantly in the unprotonated form. A buffer at pH 8.3 is
most frequently used for coupling proteins. IgGs are often coupled at a
slightly higher pH, for example in a NaHC03 buffer (0.2-0.25 M) containing
0.5 M NaCI, at pH 8.5-9Ø Carbonate/bicarbonate and borate buffer
systems with the addition of NaCI may be used. The coupling buffer solution
should have a high salt content (about 0.5 M NaCI) to minimize protein-
protein adsorption caused by, the polyelectrolyte nature of proteins. Coupling
at - low pH is less efficient but may be advantageous if 'the ligand loses
biological activity when it is fixed too firmly, e.g. by multi-point
attachment,
or because of steric hindrance between binding sites which occurs when a
large amount ,of high molecular weight ligand is immobilized. A buffer of
approximately pH..6.is used. Tris and other buffers containing amino groups
must not be used at this stage since these buffers will couple to the gel.
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Protein coupled to CNBr-activated ~epharoseT" 4i3 is ~ssual4y more
stable to_ .denaturation than the protein in free solution; but reasonable
care
in the choice of. storage conditions should be exercised. Suspensions should
be stored in a refrigerator below 4~C in the presence of a suitable
bacteriostatic agent. The choice of buffer solution depends on the properties
of the particular coupled .protein.
In . affinity chromatography, nonspecific proteins flow through the
column while the specific protein is retained by the column. The protein is
then eluted , and individual fractions are tested for specific-binding
activity
and purity. Several different approaches can be taken to allow efficient
binding of antigens to immunoaffinity columns. Because the antibody is not
in solution, the time required for the antibody-matrixJantigen interaction
will
have different kinetics than soluble interactions. It will take considerably
longer for equilibrium to be reached than for solution assays. Therefore, the
binding protocol should maximize the degree of interaction. The
recommended method is binding by passing the antigen solution down an
antibody-matrix column, keeping the antigen in contact with the antibody for
as long as possible. In this case, high-affinity antibodies will be
signiFcantly
more efficienk at removing the antigen from solution than low-affinity
antibodies. Several small-scale columns can be used to determine the best
conditions for binding and collecting the antigen.
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Although the exact affinity of an antibody for an antigen can be ;
calculated, for most work the crucial criterion is whether the antibodies will
remove.the antigen from solution quantitatively. The easiest, method to test .
this is to set up small-scale reactions and examine the first wash buffer for
the presence of the antigen. The. amount of bound -antigen may be increased .
by using higher amounts of antibodies ~on the beads,. by increasing the
number of 'beads, or by i~icreasing the amount of time for binding. ~,
Unfortunately, all of these conditions will raise the nonspecific background,
so a compromise normally will result in the highest yields with the lowest
acceptable background. Use of high-affinity antibodies solves the problem of
efficiently collecting the antigen. Consequently, they can be used in dilute
sotutions,.,, at relatively Power antigen. Consequently, they can be used in
dilute solutions, at relatively lower concentrations, and for shorter times.
A titration can be performed as a first step in estimating the ratio of
column matrix needed to bind a given amount of antigen. This can be
handled where an .equal volume of the antibody/Sepharose 4B matrix is
added to samples containing increasing concentrations of the antigen. The
-;; .
slurry is mixed at 4°C for 1 hr and then processed: This will yield a
rough
idea of the volume of column matrix needed to collect the desired amount of
antigen. If the supernatants from the binding reaction are assayed for the
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presence of the antigen; the extent of antigen depletion also can be .
determined.
Developing the-best elution conditions is a,n empirical task determined
by=.,.testing a series -of buffers: Three types of elution are possible. The
antigen-antibody interactions can be broken b~y (1) treatirig with harsh
conditions, (2) adding a saturating amount. of a small compound that mimics
the 'binding site,.and/or (3) treating with an~agent that induces an
allosteric ~.
change 'that releases ~ the antigen. The most commonly used elution
procedure relies on breaking the bonds between the antibody and antigen.
The elutions may be harsh, denaturing the antibody and the antigen, or mild,
leaving both the antigen and antibody in active states.
The mildest elution conditions are required if the protein of interest is
labile. Avoid dithiothreitol and other reducing . agents, as they will break
disulfide linkages , Any buffers that fail to elute the antigen should be
considered as good , candidates for wash buffers, Some noneluting buffers
may, in fact, drive the antibody-antigen equilibrium toward complex
formation. The usual procedure when elution conditions have not been
defined is to try the mildest elution conditions first and proceed to harsher
treatments. If trying for the gentlest elution conditions, start with acid
conditions first, then check basic elution buffers. If these conditions do not
36
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Brute the antigen, try others. A general order to check the various conditions
would be:
Low pH acid, pH 3=1.5
0,1 M glycine-HCI (pH 2.5)
0.1 M glycine sulfate (pH 2.3)
0.1 M propionic acid (pN 2.3)
3.0 M KSCN (pH 2.3)
High pH base, pH 10-12.5 .
. 0.1 M glycine-NaOH. (pH 12.0)
0.15 M NH40H (pH 10.5)
Chaotropic Agents MgCf2, 3-5 M 4 M MgCi2 iri 10 mM PBS (pH 7.0)
LiCI 5-10 M
Water
Polarity-reducing Agents Ethylene glycol Z5-50%
Dioxane 5-20%
Denaturing Agents ~ Thiocyanate i-5 M
Guanidine 2-5 M
Urea 2-8 M
SDS 0.5-Z%
Microconcentrators are used primarily for removal of excess salts in
protein purification 'or analysis. A variety of materials have been used to
fabricate v these semipermeable membranes, ranging from cellulose and
cellulose esters to polyethersulfone (PES) or polyvinylidene difluoride
(PVDF). All membranes are ,characterized by their molecular-weight cutoff
(MWCO) value. This is usually defined as the molecular weight of a solute
that is 90% prevented from penetrating the membrane under a chosen set
of conditions. How readily a particular protein is rejected by the membrane is
a function of the shape, hydration state, and charge of the protein molecule.
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Moreover, MWCO values are not sharp; rather, there is a gradual increase in
retention as .the size of solute molecules approaches and exceeds the
average membrane pore Size. Only at the point where all pores are.smaller .
than a particular solute molecule is that molecule completely excluded.
The advantage-.~of desalting processes based on ultrafiiltration over
those based on simile dialysis is that the. rate of low.-molecular-weight
solute
removal is. not determined by a concentration'differential, but rather by the
flow. rate of solvent and th,e rejection of the solute by the ultrafiltration
membrane employed. Membranes for ultrafiltration are generally selected on
the basis of the MWCO needed to retain the protein of interest but allow the
maximum amount of other materials to pass through. It is usually best to
choose an MWCO value that is roughly one-half the molecular weight of the
species to be retained. This provides a reasonable margin of retention
whereby almost none of the protein of interest should be lost, but at the
same time provides the largest difference between the MWCO value and the
molecular weight of the salts to be removed, thereby maximizing filtration
rate.
In regard to the degree of nonspecific adsorption of protein to
membranes, losses of 1% to 5% are not uncommon when dealing with total
quantities of protein in the range of 1 to 10 mg using a filter with a 43-mm
diameter. The nature of the buffer can also affect adsorption of protein;
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some membranes exhibit altered-flow properties.when high levels of ions are
present. In this regard, phosphate .buffers seem to present more of a
problem than Tris buffers. The degree of concentration to be achieved by
ultrafiltration should be t~iat required for subsequent work. Recovery of
sample following concentration is .generally 95%; failure to achieve .this
value usually indicates leakage into the fltrate or nonspecific binding to the
membrane and/or concentration apparatus itself.
At a constant temperature and pressure, the flow rate is a function of
the filter area and the degree to which concentration polarization can be
avoided. Buildup of protein on the surface will result in slow filtration,
even
when the protein concentration of the sample is relatively low. Filtration
rates at 4°C are often only one-half those seen at 25 °C.
because of the
influence of viscosity. For .biochemical analysis, monomorphic monoclonal
antibodies are particularly useful for identification of HLA locus products
and
their subtypes.
W6/32 is one of the most common mohoclonal antibodies (mAb) used
to characterize human class I major histocompatibility complex (MHC)
molecules. This antibody recognizes only mature complexed class I
molecules. It is directed against a conformational epitope on the intact MHC
molecule that includes both residue 3 of beta2m and residue 121 of the
heavy chain (Ladasky JJ, Shum BP, Canavez F, Seuanez HN, Parham P.
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Residue 3 of beta2-microglobulin. affects binding of class I MHC molecules by
the W6/32 antibody. Immunogenetics 1999 Apr;49(4):312-20, the contents
of. which is expressly incorporated herein by reference in its entirety.). The
constant portion of the molecule W6/32 binds to is recognized by CTLs and
thus can inhibit cytotoxicity. The reactivity of W6/32 is sensitive to the
amino
terminus of human beta2-microgiob~iin (Shields M), Ribaudo RK. .Mapping of
the .monoclonal antibody bv6/32: sensitivity to the amino terminus of beta2-
microglobulin. Tissue Antigens 1998 May;51(5):567-70, the contents of
which is expressly incorporated herein by reference in its entirety.). HLA-C
could not be clearly identified in immunoprecipitations with W6/32
suggesting that HLA-C locus products may be associated only weakly with
~2m, explaining some of the difficulties encountered in biochemical studies
of HLA-C antigens. The polypeptides correlating with the C-locus products
are recognized far better by HC-10 than by W6/32 which seems to confirm
that at least some of the C products may be associated with ~2m more
weakly than HLA-A and -B.
HC-10 is reactive with almost all N1~1-B locus free heavy chains. The
A2 heavy chains are only very weakly recognized by HC-10. Moreover, HC-
_ reacts only with a few Ht~-A locus heavy chains. In addition, HC-10
seems to react well with free~heavy chains of HLA-C~types. No evidence for
reactivity of HC-10 with heavy-chain/~i2m complex was obtained. None of
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the immunoprecipitates obtained with HC-10 contained (i2m. This suggests
that HC-10 is directed against a site of the HL.A class I heavy chain that
might include the portion. involved in interaction with the ~2m. The pattern
of HC=1Q ~ precipitated material is qualitatively different from that isolated
.
with U116/32. ~'- ~ ~ ~.
TP25.99. detects a 'determinant in Ehe alpha3 domain of HLA-ABC. It is
found on denatured HLA-Bv (in Western) as well as partially or fully folded
HLA-A, B,& C. It doesn't require a peptide or ~i2m, i.e: it works with the
alpha 3 domain . which folds without peptide. This makes it useful for HC
determination.
Anti-human ~32m (HRP) (DAKO P0174) recognizes denatured as well as
complexed ~i2m. Although in principle anti-~i2m reagents could be used for
the purpose of identification of HLA molecules, they are less suitable when
association of., heavy chain and ~32m is weak. The patterns of class I
molecules precipitated with W6/32 and anti-(32m are usually
indistinguishable. .
EXPERIMENTAL EXAMPLES OF THE PRESENT INVENTION
Purification of Individual, Soluble MHC Molecules
The, present invention is directed to a unique method for producing,
isolating, and purifying class ~ I molecules in substantial quantities. As an
example of the method of the present invention, the following graphs show
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that the 'test allele B*070ZHis produced in -static culture can be purified to
homogeneity and eluted as intact molecule. FIG. 5 demonstrates that a
W6/32-coupled affinity column can be saturated with crude . harvest
containing sHLA. Individual values were determined through a standardized
sandwich ELISA procedure using W6/32 as capturing antibody and anti-~2m
as detecting .antibody. This ELISA procedure allows only the detection of
intact sHt~l fnolecutes. After.. successful loading, the column is washed with
PBS. FIG. 6 shows, the washing step. The removal of total protein and active
sHIA measured through ODZ$o and EIISA, respectively, can be followed. It
shows that after S00 ml of wash volume, impurities are successfully
removed from the column. This was also confirmed through SDS-PAGE
analysis of the wash fractions collected. In FIG. 7, we were able to elute
sHI.A molecules with 0.1 M glycine (pH 11.0) and neutrali2e in 1 M potassium
phosphate (pH 7_0) that resulted in fractions of intact molecules as shown
through the standard ELISA procedure. Elution occurred in a single peak
indicating the absence of nonspecifically bound material on the column. SDS-
PAGE analysis confirmed the size of the subunits and their purity. The final
Macrocep procedure was used to remove the neutralization buffer and
replace it with PBS , (0.02% Sodium azide). Experiments presented
hereinafter demonstrate that this buffer is highly suitable to maintain
structural integrity and maintain~the stability of the sHLA complex.
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The same procedure is used to finally concentrate the protein . to
increase the stability of the molecules. Higher concentrations are also more
suitable in most applications. FIG. 8 shows two rounds of buffer-exchange
and confirms minimal loss of protein after the last step. All wash flow-
through's (WFt's) have minimal sNLA content and are usually discarded after
the procedure. The sHLA content was elaborated using the standard ELISA
technique. To remove possible particles or bacterial growth, filtration
through a 0.2 micron filter is standard procedure. FIG. 9 demonstrates that
filter-units tested perform nearly equally good and no decline in total
protein
through absorption to the filters or loss of activity could be detected. The
recovery volume was also highly acceptable and only small amounts of liquid
did remain within the filters. FIG. 10 shows the efficiency of the procedure
measured at each step. A 100% was defrned as the sHLA content directly
bound to~ the column after loading and wash. All Flow-through's and washes
having substantial amounts of sHLA are recovered and can be reused as
loading material for a second round of purification. With this purification
run,
a total efficiency of 75% was achieved.
Chemical and Physical Purity oflndividual, Soluble MHC Molecules
To confirm that the sHLA produced and purified by the method of the
present invention are correctly translated, an Edman degradation was
performed to receive the sequence of the first 10 amino acids. Since an
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intact sHLA molecule is : a corinplex consisting of HC, ø2m_ and a peptide,
sequencing, results gave us several different amino acids at each position.
Since tiC ~and~ ~2m are- present in a ratio of '1:1 each position from 1 to 10
sElould predominantly contain both HC and ø2m amino acids in about equal
amounts. Since both sequences are published and well known, a
comparative analysis can easily be done. Because sHtA molecules. bind a
variety of different 'peptides, .these amino acids are producing noise at each
position rather than delivering 'distinctive recognizable amino acids which
makes it in certain cases impossible to make a proper evaluation. Three
different molecules were sequenced: FIGS. I1-13 illustrate protein sequence
data for MHC Class I HLA-A*0201T, HLA-B*070zT, and HLA-B*1512T,
respectively. The comparison. clearly shows that the sHL~1's are correctly
translated at the amino terminal end. It is also evidence that no other major
...
impurity was present' in those samples.
Proteolysis of the whole molecule complex and analysis of the amino
acid composition was executed on the B*I512T (FIG. 14). The procedure
showed a close relationship between the amino acid content of the calculated
versus the observed residues suggesting a full length molecule. During the
procedure, some amino acids were expectedly degraded and were not taken
into .consideration. The Close match is a further indication of the purity of
our
test-sample.
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The sHLA'S produced a'nd purified by the method of the present
invention were analyzed by Superdex . chromatography to demonstrate
sample purity (FIG. 15). . The Superdex-FPLC analysis under native
conditions for .B*1~5I2T showed a Characteristic ,peak corresponding to the
sHLA complex... No other iiiajor bands can be, detected confirming the pure
nature of our preparation. Under such native conditions, a peak of the size of
39:7 kDa is seen, which is in the area of complexed sHLA. No bands at 31
kD~; representing free HC, or at 12 kDa for ~2m. are visible. However, a
minor band at approximately 94.5 kDa can be seen, which represent
aggregated HCs. Because sHLA samples are filtered through a 10 kDa filter
during the Macrocep procedure, these free. HC molecules remain in the
solution and cannot be removed. Aggregated HC molecules are not
considered an impurity of the sample. Their contribution to the final protein
amount is .less than 1%.. The overall purity of the complex compared to
foreign proteins is more than 99.9%:
A_ triple analysis _ of B*1512T is presented in FIG. 16. It shows a
separation of sHLA under denaturating and under native condition as well as
separation of purified free ~2m (Serotec) alone. A standard curve was run in
parallel to estimate molecular weights.
Using guanidine-HCI as additive to denature the probe, the sample of
B*1512T was run under equal conditions as the other samples. The results
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seen demonstrate that the sHLA complex is unexpectedly stable under such
denaturing. conditions. A clear peak resembling the pure complex can be
identified which is at the same. position as the native peak. As expected, .
sHI.A complexes do fall apart, which resulted. in the increase of aggregated
HC and an increase in free ~2m as their positions are identified through their
overlap rivith the native samples. Surprisingly, the denaturation process did
not deliver a peak at .31 kDa corresponding to free HC. It seems that HC
monomers are not present and immediately aggregate to a higher size
complex. During the denaturing process, several peaks of lower molecular
weight appeared, which correspond not only to aggregated peptides released
from the destroyed complex but also through. fragmentation of ~2m and HC
subunits.
The results of purity are not a unique event and can be demonstrated
with all alleles.. going through our optimized purification procedure. A
Superdex profile of A*0201T is provided as an additional example in FIG, i7.
Several sHLA alleles were loaded on an SDS-PAGE gel and stained with
Coomassie to .assess the purity of the samples (FIG. 18). A band for HC and
a2m, respectively, was detected demonstrating ,the purity of all samples
tested. The antibody W6/32, which is used in the process of affinity
:. _.,.:;.
purification, is, also added. In none of the samples could an equal band be
detected, thus showing that leakage of W6/32 during elution does not occur.
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Western blot analysis to follow. the HC and ~2m subunits of sHI.A were
also performed (FIG, '19): The upper portion shows the results of an. SDS-
electrophoresis performed 'running crude harvest (load), the flow through
(output of the column) and. the wash on the left side, eluate, concentrate
and final sample .on the right.
Using HC10 antibody., visualized with .a secondary mouse antibody
coupled .'to HRP, several .bands could be.. stained resembling different
aggregates of HC. It-appears that the dimeric form is dominant (40,1 kDa)
ove~-the monomeric form (28.7 kDa) after denaturation and SDS treatment.
The lower value for the dimeric form is evidently an artifact and caused by
an aberrant running behavior on SDS-PAGE gels since a consistent amount
of SDS is not anymore bound per unit weight of protein. The carbohydrate
moiety attached to the HC might also be involved. Higher aggregates are
also visual to a minor extent. The results. show that sHl~4 is present in the
crude and binds to the column since there is a drastic reduction in signal
observed in the flow through. Saturation of the column does result in
material leaving the column not captured. Therefore, wash fractions will also
contain some sHt.A , not captured. The protein, is highly concentrated in the
purified sample and concentrates do not look different than eluted
molecules.
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An -anti-~2m antibody directly labeled with HRP was used to visualize
the;.lighter subunit.~.A single band of I1.7 kDa was seen as expected. ~32m
does not seem to aggregate. However, a faint .band at 46.2 kDa could be
observed.,An extended exposure showed a :clear band at this location which
is. in the size of the intact complex. This would suggest that some complexes
.
survived;-the denaturation step and show SDS resistance.
Separation under denaturing conditions. and staining with the.
antibodies HC10 and anti-~2m revealed that both the heavy chain and (32m
are present. The secondary antibody directed against mouse antibodies also
did not; reveal :any addftinnal bands, indicating that the preparation is free
of
possible W6/32 antibody contamination, which was used in the purification
step.
The Sandwich ELiSA procedure was used to follow the sHLA molecule
through all purification steps and confirm activity of the sHLA molecule (FIG.
20). Final analysis confirms that at no time did the sHLA molecules denature
and that. the sHLA molecules always maintain their structural integrity.
Activity can still be detected in highly diluted samples.
Functional Purity of Individual, Soluble MHC Molecules
1. Conformation-Specific Antibody Binding Assays w
The use of Pan class I antibodies gives conclusive results about the
conformational status of the sHW molecules. Thus, sHLA activity tests using
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Fan-class I antibodies such as W6/32, TP25.99, and Pan class I (One
Lambda) were performed. W6/32 only recognizes conformationally intact
molecules; TP25.99 recognizes the complexed sHLA molecule as well as free
HC and the Pan class I ,(One Lambda) which has equal recognition patterns
as 'seen W th W6/32. The. antibody HC10 is useful in distinguishing free from
bound heavy chain. (HC) since this antibody only recognizes the HC of
denatured sHt~4 molecules. Anti-(32m recognizes the ~2m subunit in both
cases, complexed to the HC as well as free in solution and gives
complementary information in addition to the other antibodies,
Illustrated in FIG. 21 is a scheme of antibody binding scenarios, while
FIGS. 22-27 each illustrate. reaction panels for conformation-specific Ab
binding assays using Sandwich ELISA assays. The Sandwich ELISA assays
include. six steps: (1) choice of appropriate support; (2) coating with pan
HLA specific antibodies; (3) blocking procedure to reduce non-specific
protein binding; (4) capturing of single specificity sHLA molecules at
different
epitopes; (5) positive (or negative) SERA binding to presented sHLA alleles;
and (6) detection ,of reactive SERA antibodies using secondary anti-human
IgG (IgM) antibody.
Sandwich assays can be used to study a number of aspects of protein
complexes. If antibodies, are available to different components of a
heteropolymer, a two-antibody assay can be . designed to test for the
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presence of the complex: ' Using a variation of these assays, monoclonal
antibodies can be used to test whether a given antigen is multimeric. If the
sar~ie monoclonal antibody is used for both the solid phase and the label,
monomeric antigens cannot ,be detected. Such combinations, however, may
detect multimeric forms of the antigen. The W6/32 - anti-~32m antibody
sandwich assay is one of the best techniques for determining the presence'
and quantity of sHLA. Two antibody sandwich assays are quick and accurate,
and if a source of pure antigen is available, the assay can be used to
determine the absolute amounts of antigen in unknown samples. The assay
requires two , antibodies that bind to non-overlapping epitopes on the
antigen. This assay is particularly useful to study a number of aspects of
protein complexes.
::.:.,
To detect the antigen (sHLA), the wells of microtiter plates are coated
with the specific (capture) antibody W6/32 followed by the incubation with
test solutions containing, antigen. Unbound antigen is washed out and a
different -antigen-specific antibody (anti-~2m) conjugated to IiRP is added,
followed by another incubation. Unbound conjugate is washed out and
substrate is added. After another incubation,, the degree of substrate
hydrolysis is measured. The amount of substrate hydrolyzed is proportional
to the amount of antigen in the test solution.
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The~major advantages. of this technique are that the antigen. does' not_
need to bepurified ~ prior to use and that the assays . are very specific. The
sensitivity of the assay depends. on four factors: (1) the number of capture
antibodies; (2) the avidity ,of the capture antibody for the antigen; (3) the
.
avidity of the second antibody for the antigen; and (4) the specific activity
of
the labeled 'second antibody.
'In orde~'to demonstrate proper conformation of our produced sHLA
class. I proteins, several Pan-class I monoclonal antibodies were tested.
Utilizing the sandwich ELISA technique, a selection of sHLA-A and -B alleles
captured with anti-(32m or W6/32 were visualized by a variety of detector
aptibodies specific for sHLA. as seen in the scheme of FIG. 28. All results
were confirmed by both assay procedures indicating that antigenic integrity
of purified sHl.~4 molecules is not compromised. HC10 reactivity was not
detected as ,expected since free HC cannot be captured by anti-~32m or
W6/32 (FIGS. 29-32).
To test for artificial structural forms such as aggregation or dimeric
structures, various antibody combinations were tested (FIGS. 33-34). None
of these experiments revealed any other structures than single complexes.
These complexes have been shown before in equilibrium with very low
amounts of free ~2m, HG and endogenous peptides.
2, ~ Neutralization Experiments
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Antigenic 'integrity was .also verified in neutralization experiments
(FIGS. 35-36). An~~, established reaction -of native beads coupled to HLA
molecules interacting with specific human sera could be inhibited by addition
.
of purified sHLA. in various buffers which competed for the sera. Different
native molecules coupled to beads could be equally neutralized.
The experiments sown in FIGS. 35-36 demonstrate that the sHLA
molecule A0201T highly, competes with the A2 allaantiserum M102 as well as
with ~ the monoclonal Ab MA2.1 confirming the correct behavior of the
molecule in this neutralization experiment. This indicates the presence of a
native conformationally correct molecule within the samples. Particularly, the
MA2.1 (1:600) monoclonal Ab recognizing specific epitopes on~A0201T was
93% blocked. Different buffer supplements do not appear to have any
influence on the capability to block. The recognition by conformation-
sensitive mAbs indicates that the recombinant complex contains native
epitopes, consistent with , the presence of a correctly folded molecular
complex.
3. Chaperone interaction experiments
The class I molecule interacts with several chaperones as it traffics
through the cell on its way to the cell surface. These chaperones include,
but are not limited'to, calnexin, calreticulin, Tapasin, and Erp 94. 35S pulse
chase/Immunoprecipitation experiments were performed to demonstrate that
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the sHLA class I proteins produced and purified by the method of the. present
invention interact with chaperones normally. Interaction with calreticulin,
calnexin, and tapasin has been demonstrated; and interaction with
calreticulin- is shown in FIG. 37.
In addition, . several - experiments have been performed which
demonstrate that truncating -the HLA molecules does not alter the class I
protein.:It will be demonstrated herein that the sHLA class.I proteins
produced: and purified by the methods of the present invention interact
normally with antibodies specific for the native class I molecule and with
peptide ligands.
4. Ab binding assays - Single specificity antibodies
A panel of selected sHLA alleles was tested using commercially
available single specificity. , monoclonal antibodies (FIGS. 38-51). All
experiments performed resulted in the recognition of the allele
corresponding to the chosen antibody. The single specificity monoclonal
antibodies act as detecting antibodies. Soluble Hi.A is presented to the
detecting antibodies through W6/32 as well as anti-~i2m capturing to El.ISA
plates. In single cases, no purified sHIA was readily available to be tested.
Thus, crude material marked with (C) was used: Because crude material
does have excess amounts of free ~2m which neutralize binding to anti-~2m,
no signal was expected.
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- In addition, Bw6 and Bw4 Abs were tested (FIGS. 5Z-53).' Each Ab is
known to recogni2e, a conserved epitope on B alleles_ However, Bw6 positive
B alleles are- Bw4 negative and vice versa. These tests confirmed as
expected that all purified. sHI.A tested harbor the Bw6 or Buv4 epitope,
respectively. . - ,
5. Edman and Mass Spec Amino Acid Sequencing
The peptides bound.. in the antigen binding groove - of the class I
molecule impact the conformation anal the antibody reactivity of the class I
molecule. .The peptides eluted from the sHLA class I molecules produced
and purified by the methods of the present invention have been
characterized, and it was found that the peptide motifs match those of
membrane bound class I molecules reported by other laboratories. FIG. 54
shows a motif comparison between sNLA-B*1501 purified by the methods of
the present invention and a membrane bound B*1501 motif from another
laboratory. The motifs are nearly identical. The same result has been seen
with six sHLA class I molecules analyzed. In addition, individual peptide
ligands isolated from the sHIA purified by the methods of the present
invention have been sequenced, and they match ligands found in membrane
bound class I of other laboratories. Thus, the sHLA proteins of the present
invention appear to traffic and bind peptides as do. membrane bound class I.
6. Peptide Binding Assays
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Fluorescence polarizatiosi allows the direct measurement of the ratio
between free and bound labeled ligand in solution without any .separation
steps (FIG.' S5). Ratiometric measurements are an advantage as these types
of measurements can self-correct for variations caused by lamp intensity
fluctuations or i~terfe~ences caused by quenching of the fluorescence. In the
move toviiards a wider adoption of, fluor~scehce technologies, there is the
added benefit of abandoning radioactive tracers, wh9ch are increasingly
becoming liabilities because of their cost and safety profile. Most important,
FP allows real time measurements of single reactions to determine binding
kinetics as well as equilibriums. Furthermore, since no biological system can
show polarization below 0 mP or greater than 500 mP, FP automatically
checks assay validity. Considered a negative point in using FP is that
detected values often. result in the loss of about 10-90% of fluorescence
intensity. This in itself,may reduce the sensitivity of fluorescence
polarization
assay as opposed to assays with direct intensity measurements.
w ~ ~ The technique .of FP is based on the fact that if excited with plane-
polarized light, the light ,emitted by a fluorophore is polarized as well. The
angle between the planes of exciting and emitted light is highly dependent
on the molecular motion of the fluorophore. FP values are defined by the
equation:
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I ~~ _ Il
Polarization =
Iii + I1
where I~~ is the intensity of the fluorescence measured in the parallel (p) or
horizontal direction (S) and I1 is the intensity of the fluorescence measured
in the perpendicular (1) or vertical direction (P).
If a fluorescent-labeled peptide binds to the sHLA molecule of higher
molecular weight, the average angle (composed of the distribution of all
angles between the optical planes) will decrease due to the slower molecular
rotation of the bound probe (FIG. 55). Therefore, the ratio between the
bound and free probe can be measured by FP directly in solution. This
advantage makes FP an excellent tool for the fast and precise determination
of molecular interactions between sHLA and peptide.
A binding assay was developed to demonstrate that the labeled probe
will bind to the molecule of interest. The following criteria, however, must
be met in order to validate the binding assay: (1) binding should be
saturable, indicating a finite number of binding sites; (2) the binding should
have the requisite specificity, where the binding affinity, defined as the
dissociation constant (Kd), should be consistent with values determined for
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physiological molecules; and ~(3) ligand binding should be reversible,
reflecting the dynamic nature of the ~ chemical transmission process and
reaching equilibrium when the ligand association rate is equal. to the
dissociation rate.
Before reaching equilibrium, the- peptide follows the rules. of
association. In this kinetic experiment the forward (ko") rate constants of
the
binding process can be determined if the amount of sHLA and peptide are
held constant and the time varied. Because the reaction mixture can be.
observed over several independent time points, each experiment's
association curve is determined (FIGS. 56-57). .
In the experimental setup shown in FIGS.. 58-59, a saturation curve is
generated by holding the sHLA (binder) constant. Varying the tracer
concentration (dose range: 0.1 nM - 1 mM) in case of constant binder
(concentration of sHLA determined above) was tested in order to determine
the affinity constant (Ka) of the labeled peptide and to obtain a smooth
saturation curve. The lower the Kd value, the higher the affinity of the
peptide for the sHLA molecule. Only values that have reached equilibrium
(Ymax) can .be used for saturation experiments.
Specific binding of a ~ fixed concentration of fluorescent-labeled
synthetic peptides in the presence of various concentrations of unlabeled test
competitor-peptides (dose range: 0.01 pM -100 NM) was tested (FIGS. 60-
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tyl). The concentration of unlabeled competitor peptide that produces
fluorescent-labeled peptide binding half wray between the upper and lower
plateaus of the obtained curve will be defined as the ICSO. The ICSO is
determiried by three. factors: (1} the affinity of sHLA for the. competitor
peptide - if the afFnity ~Is high, the ICSO will be low; (2) the concentration
of
fluorescent-labeled tracer peptide -choosing a higher concentration of tracer
will take a largei~ concentration of unlabeled peptide to compete for half the
binding sites; and (3) the afFnity of tracer peptide for sHLA (Kd). It takes
more unlabeled competitor peptide to compete for a tightly bound tracer
peptide (low Kd) than for a _ loosely bound tracer peptide (high Kd). To
achieve the highest sensitivity and accuracy of the competition assay, the
parameters identified . will be optimized to the point where the lowest
concentration of a competitor test peptide results in.a clearly
distinguishable,
positive. response. No competition should be detected in the case of using an
irrelevant unlabeled competitor peptide.
As seen in FIG. 62, an HBV peptide known to bind strongly to A*0201T
was used to replace the endogenous peptide iri solution. After incubation for
48 hours at room temperature, the sHLA complexes were immobilized on a
solid support through the HLA specific antibody W6/32. The HBV
peptide/A*0201T complex was then detected using a highly specific antibody
only recognizing this particular conformation. Saturation of the W6/32
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coated ELISA plate could be achieved, demohstrating the binding of the HBV.
peptide to sHLA molecules and a -successful replacement of the endogenous
peptides with the HBV peptide. No saturation was detected using the
irrelevant peptide p53, indicating that peptide p53 as well as endogenous ~. v
peptides do not contribute to the specific signal obtained by the HBV
peptide/A*0201T complex selective antibody:
Storage and Handling of Individual, Soluble MHC Molecules
Each protein may have specific requirements once it is extracted from
its normal biological milieu. If these requirements are not satisfied, the
protein can rapidly lose its ability to carry out specific functions, and an
already limited lifetime may be drastically reduced. Thus, failure to
determine and manage these requirements has often been a major hurdle in
obtaining successful protein characterization. In some cases, the difficulty
has been to stabilize the protein against external proteolysis, while in other
cases the problem has been to maintain ligand-binding or enzymatic activity.
Solutions to these problems are highly specific.
A , buffer is defined as a mixture of an acid and its conjugate base
which can reduce, changes in solution pH when acid or alkali are added. The
selection of an appropriate buffer is important in order to maintain a protein
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at the desired pH ~ and to ensure reproducible results. Buffers are often
present at the highest concentration of al! components in a protein solution
and may have significant effects on a protein or enzyme.
The - experimental approach described herein shows that various
buffers are auitable for use herein. PBS, pH: 7:4, was chosen as standard
buffer since it is~ creates. a stable surrounding and does ,not have
supplements that could, possibly . interfere . with downstream applications.
Phosphate=buffered solutions are highly susceptible to microbial
contamination. To prevent buffer contamination during storage, O.OZ% (3
mM) sodium azide .was used. Sodium azide does not interact significantly
with proteins at this concentration. Refrigeration helps to reduce buffer
contamination:
Very dilute protein solutions are highly prone to inactivation and often
lose activity quickly, possibly via denaturation at surtaces such as glass and
plasticware. This is especially true of oiigomeric proteins where dissociation
of subunits can occur at low concentrations. The individual polypeptide
chains comprising the oligomer may, denature. High protein concentrations
(> .~mg/ml) provide some auto-buffering capacity. Thus, protein solutions of
concentration < 1-2 mg/ml are concentrated as rapidly as possible in the
procedure described herein.
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In the stability assay shown in FIGS. 63-66, sHLA B*1512T was
incubated in different buffiers and solutions at a concentration of 55 Ng/ml
over a time period of 1, 4, and 1.8 days at 4'C. After the incubation time, an
ELISA was performed; using W6/32 as the capture antibody and anti-
~2fi(HRP) as the detector antibody. The ELISA results were standardized
using PBS as 100%.
This experiment clearly demonstrates' a high sta.bility_ of sHLA over a
wide range of buffers and solutions. Only 0.1 N NaOH and 0.2 N acetic acid
were able to completely abolish the reactivity of the molecule.
The stability in. elution buffer is only 85% compared to PBS, justifying
an immediate buffer exchange during the purification procedure. Only four
solutions, 20% Dextrose, Citrate buffer, . 10°~ PVP and SO nM DEA were
found to show declining stability over time, whereas the others seem to be
constant over the tune period tested.
The value of Triton X-100 at four days appears to be the highest value
achieved during the .whole assay. However, it also shows a high standard
deviation value. It appears to be more likely to. be an outsider result due to
a
dilution mistake . rather than increased stability of sHLA after 4 days. This
value was not considered in calculating the average.
Generally, PBS seems to be an optimal storage and reaction buffer.
Only buffers containing BSA seem to perform slightly better than PBS alone.
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Choosing'3°Io BSA in our Et~ISA seemed to be a good choice,
confirmed by
the above results:
Kinetic stability is usually measured at elevated temperatures, but the
inactivating events) at high temperatures may not mirror those at the much
tower' temperatures. used for storage. It is not feasible, however, to monitor
stability in real time at the actual storage temperature. Fortunately, there
is
a methodology that can in many cases overcome these difficulties, namely .
accelerated degradation testing, This involves the periodic assay of samples
incubated at different temperatures and use of the Arrhenius equation to
predict shelf lives at temperatures of interest.
Ink = -Ea/RT
where k is the first-order rate constant of activity decay, Ea is the
activation
energy, R is the gas constant, and T is the temperature in Kelvin. This log
form. of the Arrhenius eguation yields a straight-line plot of Ink against 1/T
with slope -Ea/R. Extrapolation of this plot can give the rate constant (and
hence the. useful life) at a particular temperature. Accelerated storage
testing has been used as a, practical means of quality assurance for
biological
standards (7erne, N. ,K. a.nd Perry, W. L. M. (1956) The stability of
biological
standards.: Bull. Wld. . Hlth. Org. 14, 167-182, the contents of which are
herEby expressly incorporated herein in their entirety.).
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Maintaining the stability of the purified sHLA complex by .identifying
optimal storage. and handling parameters was one of the main interests of
the present invention; It has been determined through the above studies
that PBS and concentrations of sHIA above 2 mg/ml are ~ advantageous to
maintaining stability. In the following experiment, the influence of
temperature to the sHLA complex was tested to determine the half-life.of the
purified product (FIG. 67): ~-As in the above studies, the standard sandwich
ELISA procedure (W6/32 / sHLA / anti-~2m-HRP) was used to measure sHLA
activity in solution. Identical samples of sHLA molecules were incubated at
various temperatures over a time period of 300 minutes, After heat
incubation, the samples were immediately cooled to 4°C and assayed to
determine the percentage of lost . activity relative to non=heated samples
(stored at 4°C) tested at equal time points. The results show a rapid
loss of
activity when heated above 53°C. This can be interpreted as
dissociation of
intact sHLA molecules. The. more energy that was applied, the faster was
their dissociation rate. Below:temperatures of 32°C, sHLA molecules
seem to
be very stable. Using an Arrhenius plot, half lives for T=57°C (3.5
min);
T=53°C (8.6, min); and T=47°C (43 min) were calculated.
Extrapolation of
the graph to room temperature resulted in a calculated half live of more than
20,000 years. These results indicate that sHLA molecules are highly stable
and will maintain 'their structural integrity if stored properly. The quality
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seems to be more than appropriate fo.r commercial and other experimental
purposes.
A single . freeze-thaw cycle at -20 ° C or -80 ° C does
reduce activity
and is therefore not recommended (FIG. 68).; A storage temperature of
4°,C is optimal. It is known that loss of purified protein due to
nonspecific
adhesion onto glass surfaces (1 Ng of protein is absorbed on 5 cm2 of a
glass surface) has to. be:.expected and will significantly.diminish the amount
of protein, in a reaction. .To probe for nonspecific adhesion, a tube test was
developed to examine several different storage vessels. To overcome this
problem,.a variety.of.. potential blocking agents were tested.
FIG. 69 demonstrates the experimental procedure. From a protein
stock, a dilution of 300 ng/mt, was mixed in PBS. To equilibrate the diluted
sample, it was mixed, Z6 hours before starting the experiment and stored at
4°C . After this time,.liquid was removed from .one tube to another
every 30
minutes. If sHLA adheres to the tube, a step-wise reduction in concentration
from tube 1 to tube 6 should be observed. Successful blockers should
prevent loss of protein and the step-wise reduction in concentration should
not be observed or be highly diminished.
Addition of a standard sample (tube 0) to a variety of different
microcentrifuge tubes or cryo vials showed profound effects on the reactivity
of,sHLA'(FIG. 70).~ One of the most used 1.5 ml tubes from Fisher (05-402-
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2'S) showed a step=wise reduction in concentration from tube 1 to tube 6 as
expected for vessels binding pfotein, losing,up to 40 % of reactivity during
the frrst transfer. The same effect was seen with several other
microcentrifuge tubes having adhesive .potential for sHI,A, and some of them
showed '~rhore or . less binding. The best pertormer was the "No stick"
hydrophobic RNase/DNase free microcentrifuge tubes (Gene Mate-ISC
Bioexpress, Kaysville, UT). :However, autoclaving, did .partially destroy
these
properties: These "No stick" hydrophobic tubes are specially treated With a
proprietary non-reactive lubricant to have an extremely hydrophobic surface
(e._g., Teflon). Siliconized tubes performed better in conserving the
molecules reactivity than normal uncoated polystyrene tubes.
Tubes with larger volume capacity performed no better than the Fisher
microcentrifuge tube (FIG. 71). Here, an exception was borosilicated glass
tubes, which did not bind protein and only caused a loss of reactivity of
20°to_
To solve the problem~of loss of reactivity, the tubes need either to be coated
with a blocking agent or the blocker should be added directly to any
molecule dilution. Dilute protein solutions are highly prone to inactivation
and lose activity quickly, possibly via denaturation at surfaces such as glass
and plasticware. High protein concentrations provide some auto-buffering
capacity. Where the usage of high concentrations is not possible, inactivation
may be prevented by addition of an exogenous compound.
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Blocking agents used to coat Fisher (05-402-25) microcentrifuge tubes .
were tested for their ability to prevent inactivation and/or adhesion to the
surface (FIGS. 72-73). The tubes were incubated with the blocker overnight
at 4°C, extensively, washed with PBS and finally air dried to remove
any
traces.. of liquid: 10% BSA, 3% gelatine or 5% Blotto (milk) worked best and
did not result in any~.loss .of protein or activity compared to the tube
preincubated with' PBS. Usage of StabiIGuard Biomolecule Stabilizer
(Surmodics, Eden Prairie, MN; SG01-0125) coated to the tube walls highly
protected the protein against tube surfaces. However, the ELISA resulted in
higher concentrations than actually put into the tube. A problem using this .
blocking solution is its unknown composition (the manufacturer was not
willing to reveal all components, but low molecular weight PVP is one of its
components). A possible cause of seeing higher values with Stabilguard
seem to be the enhancement of antibody-antigen (sHt~4) interaction,
increasing the antibody's affinity to its target during the ELISA procedure.
Stabifguard is a possible candidate to be used in reactions of HIJ~ with
allosera. (The optimal % of Stabilguard needs to be, established first).
Using agents such as PVP (FIG. 72) or PEG (FIG. 73) also showed good
results . ,Known as crowding agents,, they push proteins out of solutions in
the mechanical/physical sense and in the thermodynamic sense. The
crowding action, aided by any~degree of affinity of protein molecules for one
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another promote : protein-to-protein association. . Conformationalty loose
protein molecules are "squeezed" on by these agents; promoting protein ~.
molecule tightening and sometimes promoting an ordered protein
conformation. Thus, these are the most potential candidates to be used in
sbls'tion. In addition, 2% BSA and 10°~ FBS also worked, however with
Lesser intensity. The results obtained from 10% FBS compared to PBS also
explains results earlier observed in the ELiSA procedure In that ELISA values
tend to be higher when crude harvest was tested than after purification
testing the pure protein. It also explains why column efFciencies of only 60-
70% were obtained since the efficiency is evaluated by the ratio of purified
sHI.A (measured in PBS) divided by the amount of sHLA loaded onto the
column (measured in crude harvest containing 10% FBS).
Finally, nonionic detergents did not greatly help preventing the loss of
sliLA compared to 10°l° BSA (FIG. 74). However, these agents
should not be
excluded to be considered as supportive compounds since many proteins
retain their activity iri 1 - 3%. In the study presented here a 10 times lower
concentration was used, and the trend of better performance can be seen
(FIG. 74). , where 0.1 % Tween 20 performed better than 0.05%.
In the above experiments, BSA, Stabilguard (StG), PEG and PVP were
identified as potential blockers and/or stabilizers. However, the usage of the
right concentration is important . in the optimization procedure. Thus,
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different concentrations of blbckers were tested by using a sHt_A standard
curve with declining concentrations.
Concentrations of BSA between 2-10% do not show any .difference .in
performance and' are equally good (FIG. 75). The l% BSA showed slightly
higher values probably caused by an incorrect mixing of the stock solution.
The results obtained with BSA suggest that the present usage of 10% BSA is
not necessary and 'can be reduced to a lower percentage. The best choice is
3%, which wilt highly reduce the usage of chemicals and also buffer out
minor mistakes in making the solution or helping to equalize dilution
differer~es to ay certain degree'. Albumin did not interfere with the ability
of
serum and complement to lyse target cells. In standard lysis assay
procedures, it was found that 30% albumin did not affect the ability of HU4
antigens (Springer TA., JBC 1977; 4682-4693, the contents of which are
hereby expressly incorporated by reference herein in their entirety.).
Stabilguard seems to'v~rork better with lower percentages (FIG. 76). A
steady decrease in signal is observed using higher concentrated samples
indicating an interference in protein-protein interaction rather then
inefficiency in blocking, PEG can. be used at concentrations up to 15% (FIG.
77). After that, PEG seems to highly interfere with the recognition of sHLA.
.;.. . . , ... .
PVP seems to be a great blocker at 5% (FIG. 78). However, it is absolutely
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not usable at higher concentrations, as ~it completely abolishes any
interaction with sHLA.
ANTIGENIC INTEGRITY OF sHLA FOR USE IN VARIOUS APPLICATIONS
SERA SCREEN ELISA PROTOTYPE
In the'SERA SCREEN ELISA approach ('described in detail in-US: Serial
No. 6f~j413,842, filed September 24, 2002, the contents of which are hereby
expressly incorporated here'sn by reference), fhe feasibility of a sera screen
assay that utili2es HLA to identify antigen-specific antibodies in human sera
was tested (FIGS. 79-85). The technique is based on an ELISA procedure
utilizing 1N6/32 and anti-~2m as capturing antibody. These capturing
antibodies present a panel of sHLA molecules at different orientations to
guarantee. the successful recognition by sera antibodies. In the final step, a
secondary anti-human antibody coupled to HRP was used to visualize the
positive sHLA-sera antibody interaction. All sHLA molecules used
demonstrate reactivity with sera tested and thus prove the feasibility of this
prototype.
Coupling of sHI.A molecules to LuminexT"' beads to detect HLA
antibodies in human sera can also be used with the individual, isolated, and
purified sHt,A molecules of the present invention. Disclosed herein is the
information used to bind various sHLA alleles produced to a solid support in
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order to obtain specific recognition of the alleles by human sera. Binding to
a solid bead support was accomplished via the EDC method, coupled sHLA to ~.
7.-ethyl-3-(3-dimethylaminoproplyl) carbodiimide-HCI (EDC) activated beads
(FIG..86). The results shown indicate that the isolated and purified sHt:A of
the present invention is indeed of high value in such assays.
Epit pe Discovery
In.this approach (d'escribed in detail in US Serial No. 60/362,799, filed
March 7, 2002, the contents of which are hereby expressly incorporated
herein by,reference in their-entirety), the feasibility of an assay that
utilizes
HL.A technology in ,a 'high=throughput screening' format to rapidly identify
arifigen-specific epitopes of infectious agents was tested. The proposed
assay is based on competitive binding between a peptide of interest and a
fluorescent-labeled standard peptide to a recombinant, soluble HI,A (sHLA)
.:, ~. .. ., .~
molecule. Synthesized. overlapping peptides covering any protein of interest
can be screened far the ability to bind to. their specific allele and their
potential to stimulate immunoreactions. The state of the art fluorescence
polarization (EP) methodology is utilized for monitoring binding in solution;
the method offers an excellent assay format with respect to robustness, data
quality and reproducibility. Equilibrium results obtained lead to an
efficacious
dose (ICSO), which is used to correlate in vitro potency of binding to the
sHLA
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allele used in.. the assay: A, sorting of ICSO values into categories of high,
mediur», tow;:.and no binding capability was used as the ultimate selection
guide for the identification of potentially immunogenic peptides. Thus, the
combination of sHl~4 technology with FP methodology will create a sensitive,
highly reproducible; quantitative assay to~ measure the binding of defined
synthetic antigenic peptides to various MHC class I alleles,
Test Competitors were pre-screened for .their ability to 'snhibit a . FITC-
labeled standard peptide from binding to the sHLA molecule at a competitor
concentration of 100 uM (FIG. 87). After obtaining equilibrium values for
each test-peptide, .ICSO , values are calculated. A single measurement
obtained at 100 NM.competitor concentration can be used to construct such
an,IC50. value without 'support of- additional data (FIG. 88). This
constructed
graph allows us to sort all competitors and easily categorize them into high,
medium, low and no binders (FIG. 89). Additionally, full scale IC50
determinations are pertormed on all candidates identified showing binding
capacity to the allele tested. Usually, both methods are coming very close as
seen in FIG. 89 in which one point IC50 determinations (bottom) are shown
together with 8 point IC50 determinations (boxed, top).
Appropriate modification of the sequence of a peptide epitope can
increase . the affinity for the - MHC molecules) without interfering with
recognition by . the TCR of T cells specific for the natural ligand sequence.
71
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.,.
i~:::% i~ ,. :",~.: ;~ ~ j~ it:'m: ~';°~ ,:"::~ : le ~~. ~.".,~y,
,:r,,~,, jF ,'. °~;:h .:..~P :F,:, , , i:, ;:.:,t: .~:.:f ".~
.. ,:.:~ .r : nd- ~~::y ~~:e ::':~ r' ,...s d..-.I ii''_ ~.- u:ad :& ;i.-..a
,,~ :f ~~ t~'ai' n~.,. :~_.ra~ ~f t~ .-.::.it
Therefore, b this p p p
y process of a ito a enhancement or o timization, one
should be able to create a more potent vaccine. The first step towards a
successful epitope alteration approach is to increase the binding affinity and
HLA-A2 stabilization capacity of HLA-A2-bound peptides. Since many
immunodominant epitopes are high affinity MHC binders (Sette, 1994), one
strategy is to increase the binding affinity of 'intermediate to low' binding
peptides and therefore increase their potential as immunogens.
The second step is that these substitutions preserve the antigenic
specificity and do not interfere with the peptide/TCR interaction. It is
particularly noteworthy that the CTL responses raised against the modified
peptide do cross-react with the naturally occurring epitope. This will depend
upon the nature and position of the modification. Cross-recognition of native
peptides and their modified variants by specific CTL is the most important
issue in the design of optimized vaccines.
FIGS. 90 and 91 show improvement of modified peptides compared to
the native Pest-peptide. FIG. 90 shows the IC50 of a native peptide Vac105
(ITNSRPPAV (SEQ ID N0:1 )) to A*0201 T whose binding capacity was
improved by changing position 2T to 2L or 2M. The addition of an amino acid
residue at the end did not result in a several fold improvement of binding
(Vac104/105). FIG. 91 shows a much higher binding of the decamer
Vac104/105 (KITNSRPPAV {SEQ ID N0:2)) than the two ninemers Vac104
{KITNSRPPA (SEQ ID N0:3)) or Vac105 (ITNSRPPAV {SEQ ID N0:4)).
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in vsumma .ry, shown in FIG. 92 is a general outline of the purification
and characterization procedures. of soluble human HLA proteins, of the
present invention. The first step involves . purification of soluble HLA;
beginning with cell pharm run-large, scale ,production of sHIA followed by
production analysis. The sH(.A is then purified by. affinity column
purification
(which includes the steps : of loading, washing and elution) and buffer
exchange and concentration of purified allele using Macrocep concentration ,
filters. The pure protein is then sterile filtered, aliquoted and stored, and
the
concentration of the stored pure protein is estimated. Finally, quality
control
demonstrating the extent of chemical purification is performed using
techniques known to those of ordinary skill in the art, including but not
limited to, SDS-PAGE, Western blot analysis, SuperdexT~ chromatography to
demonstrate sample purity, and the like.
The second step in the method of th.e present invention involves
characterization of the purified sHLA-peptide complex. Physical purity of the
a,..~ .._
complex can be. demonstrated by one or more of the following: sequence
analysis to demonstrate the presence of all components of the complex;
protein visualization procedures to demonstrate, not only presence of all
components .but also formation of complex (including, but nat limited to,
SDS-PAGE; Western, Superdex~~ chromatography, and the like); and Mass
Spectrometry,data for use in peptide motif comparisons. Functional purity of
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the complex can be demonstrated by one or more of the following:
demo~stratian, ,of antigenic integrity of sHi.A using ELISA assays and .
neutralization experiments; demonstration , of structural integrity using
Chaperone interaction experiments; and demonstration of specificity, peptide
binding capacity, arid , structural integrity . using fluorescence
polarization
based association and saturation experiments.
The sHl.~4 produced by the method of the present invention is feasible
for use in the following various applications: sera screen assay that utilizes
HLA to identify antigen-specific antibodies in human sera; Lurninex bead
approach to identify antigen-specific antibodies in, human sera; competition
assays, such as screening of test competitors for the ability to inhibit FITC-
labeled standard peptide from binding to sHLA; and procedures to improve
binding of modified peptides to sHLA as compared to native test-peptides.
However, it is to be- understood that many other applications for use of the
sHLA produced by the purification method of the present invention will be
evident to a person having ordinary skill in the art, and therefore the use of
the sHLA produced by the purification method of the present invention is not
limited to those listed above.
The final step in the method of the present invention involves
determining the optimum storage and. handling conditions for soluble HLA.
The following factors in storage and handling have been described herein
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previously: stability testing in different buffers; thermodynamic stability of
sHLA complexes; the influence of freeze-thaw cycles on stability; .
determination of loss of complex reactivity due to nonspecific adhesion to
surtaces of storage vessels;.and.identification of appropriate blocking agents
to maintain reactivity of sHLA.
Thus, in accordance with the present invention, there has been
provided herein ~ methods for the purification-: of soluble NLA, as well as
characterization; storage and handling of the soluble HLA complex. FIG. 92
has provided a general outline that indicates. how each of the individual
experiments described herein previously are interrelated to each other in the
methods of purification, characterization, storage and handling of the
present invention.
MATERIALS AND METHODS
Affinity Column Preparation
1: About 5-10 mg. protein/ml swollen gel is recommended in coupling
reactions in a volume of about 5 ml coupling buffer/g freeze-dried CNBr-
activated Sepharose 4B. A carefully estimated ligand concentration is crucial
in the success of the coupling reaction because of the ligand concentration
dependence. Thus, dissolve the antibody or protein to be coupled in coupling
buffer with a final concentration of 3.3 - 6.7 mg/ml.
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Gel size (ml) 1 2 3.5 5 10 50 i00 (coec.J
~
Coupling Buffer 1.5 3 5.3 7.5 15 75 i50 (mg/m1)
(ml)
Ligand (mg) 2.5 5 8.8 12.525 125 . 1.66
250
5 1.0 17.5 25- 50 250 500 3.33
'
6 12 21 30 60 300 600 9.00
7 14 24.5 35 70 350 700 4.67
8 16 28 40 80 400 800 5.33
9 18 31.5 45 90 450 900 6.00. .
IO 20 35 SO 100 500 1000 6.66
. 12.525 43.8 62.5125 625 1250 8.33
.
. ~ 15 - 52.5 70.5150 750 1500 10
30
2. A very high ligand content can have three adverse effects on
affinity chromatography. Firstly the binding efficiency of the adsorbent may
be reduced due to steric hindrance between . the active sites; this is
particularly important when large molecules such as antibodies, antigens and
enzymes are immobilized. ~5econdly, substances are more strongly bound to
the immobilized ligand which may result in difficult elution. Thirdly, the
extent of non-specific binding. increases at very. high ligand concentrations
which can reduce the selectivity of the adsorbent:
3. Most advantageous is to dialyze the protein into coupling buffer the
night before. Protein samples have to be up-concentrated if the mglml
amount is to low For optimal coupling.
4. Calculate the proper dilution to match chosen protein
concentration:
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Original concentration ci = mg/ml
Chosen concentration c2 = mg/ml
Chosen final volume ~l2 = ml
Starting volume V1 -- ml
V = C2 V2
1
5. ~- Before. tarting- the coupling . procedure, calibrate the
spectrophotometer with coupling buffer and estimate the protein
concentration at the beginning of .the reaction. This value (start-value ts)
should be as accurate as possible to allow an estimation of the coupling
efficiency (ligand binding-efficiency). With the knowledge of total amount of
antibody bound, a maxima! antigen loading capacity can be calculated.
However, this is only possible when the molecular weight of all interactive
compounds is known. The reading is performed at A280. Because stray light
can affect the linearity of absorbance versus concentration, absorbance
values >2..0 should not be used for any sample of proteins measured by the
A280 method.
6. To accurately convert A280 to the actual antibody concentration
use the following formula;
A 280 - :A 280 .blank . . . . . . '
1 ~zng/ml x Dil. factor - mg/mI
1.38 -
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start-value ts: A280= ( ~ ) mg/ml
Time: 0 min
Dilutionfactor:
7. Weigh out the required amount of CNBr-activated Sepharose 4B. One g
freeze-dried CNBr-activated Sephiarose 4B swells to give approximately a. 3.5
ml final gel volume. The active product is freeze-dried in the presence of
dextran and lactose. Free cyanogen bromide is absent. (The freeze-dried
.;
material should be stored below 4°C. Under these conditions the shelf
life is
approximately 18 .. months, although further storage is not usually
accompanied by rapid loss of activity. The opened package should be stored
dry below 4°C).
Get Size (ml) 1 2 3.5 5 10 50 100
Matrix (g) ~.29 ~ 0.57~ ~ 1.43~ 2.86i4.3 ~ 28.6
i.0 ~
. 8. ~, Coupling: a ligand to the activated matrix involves first swelling
and washing the gel in :1 mN ,HCI. The protein binding activity of the gel is
preserved better by washing at low pH than by washing at pH's above 7. The
use of HCI preserves the activity of the reactive groups which hydrolyze at
high pH. Dextran and lactose, which are added to the activated gel to .
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preserve its activity under freeze-drying, 'are washed away during the
swelling stage:
9. . Swelling and washing is performed in a sintered glass filter. A
sintered glassfilter is a -glass funnel with a built-in glass frit. The glass
frit is
used instead of a membrane flter. The filter .unit is placed on top of a side-
arm vacuum flask and filtration occurs using suction/vacuum. The' glass frit
is available i.n different poroslties:. Medium porosity (porosity G3) is
recommended for Sepharose.
10. Before starting to swell, clean the sintered glass filter with 0.5 N
HCI and several rinses of ddH20. The final rinse should be done with 1 mN
HCI.
11. The required amount of freeze-dried powder is suspended in 1
mN N,CI. The .gel swells. immediately and should be washed during a time
period of 15 minutes on the sintered glass filter with the same solution. Let
the mixture equilibrate a few minutes during each washing step.
Approximately 210 ml solution is added in several aliquots for each gram of
dry gel. Suck off the supernatant between successive additions.
Gel size ml 1 2 3.5 5 10 50 100
Matrix ~ ~U.290.57 1.0 1.43 2.86 14.3 26.6
1 mN HCI ml 60 120 2i0 300 600 3000 6000
.
12. In SO or 100 ml gel applications, the amount of 1 mN HCI may
be difFcult to handle. Recent studies have shown, however, that by
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increasing the contact time between gel and HCI, the amount of 1 mN HCl
required to wash out these additives can be reduced to one third of this
recommendation, without affecting the coupling reaction.
i3. The, final aliquot of 1 mN NCI is sucked off until cracks appear in
the gel cake. Be sure swelling. and washing is performed immediately before
ligand coupling, because activated groups hydrolyze in aqueous solutions and
coupling. capacity begins to decrease.:Thus, immediately transfer the swollen
gel.to a solution of the ligand without delay. At pH 3, coupling activity is
lost
slowly, whereas at pN 9 activity is lost fairly rapidly.
14. Optional: It is possiblequickly washthe gel 5
to with gel
volumesof couplingbuffer. However,hydrolysis start at same
will the
moment and decrease the coupling efficiency.
15. Transfer the swollen gel into a SO ml Falcon tube or a 250 ml
bottle by scooping the gel out of the sintered glass filter into the
reaction vessel. Add some 1 mN HCI to the sinter, apply vacuum
and collect small residues of the swollen gel.
16. Immediately add the appropriate volume of protein solution to
the gel. A gel: buffer ratio of 1:2 to 2:3 gives a suitable
suspension for coupling. In this protocol we calculated volumes
for a ratio of 2:3. Rinse the filter with a small volume of the
same solution.
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Gel size ml 1 2 3.5 5 10 50 100
Matrix 0.29 0.57 1.0 1.43 2.86 14.3 28.6.
~
Protein Solution 1.5. 3 5.3 7.5 15 75. 150.
nil
17. , Cap the reaction vessel, and agitate the gel gently on a' rocker.
Do not use magnetic stirrers as they usually cause fragmentation of the get
beads.
18:. ,Coupling occurs very fast under.our chosen conditions; and is
usually complete after ~20-3'0 vminutes at room temperature (20-25°C).
'If
cold temperatures .are necessary, coupling can also be performed overnight
at 4°C. The amount of protein which couples under a given set of
conditions
depends mainly on ttie ratio of .protein to get volume, the pH of the reaction
and the protein itself as well as the duration and temperature of the
reaction. A number of conditions can lead to poor coupling: low ligand
concentration, suboptimal pH, impure ligand, improperly prepared matrix,
inaccessibility of ligand or improperly prepared buffers.
19. The coupling reaction may- be conveniently followed by observing
the decrease in the absorbance.. of the supernatant solution at 280 nm. Thus,
rerriove samples at different,times during coupling and assay the buffer for
the presence of antibodies. Measure A280 at intervals of about 5 minutes
and collect, these values as coupling-values ti-x. Since the reaction-
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mechanism is very fast, the staiting values are more important than the
later ones.
20. Aliquots need to be centrifuged for 30 seconds at full speed
before the measurement. (The actual time-point for t1-x is directly before
starting the centrifuge).
21. To bring the protein samples within the spectrometers accuracy
range, dilute them with an . appropriate amount of coupling buffer if
necessary. (Absorbance values >2.0 should not be used).
A280 - A2so blank
x 1 rng/ml x Dil. factor = mg/ml
1:38 .
coupling-valueA280= ( ) mg/ml Time: Dilutionfactor:
ti: ~
coupling-valueA280= ( ) mg/ml Time: Dilutionfactor:
t2:
coupling-value, A280= ) mg/ml Time: Dilutlonfactor:
t3: (
coupling-valueA280= ( ) mg/m) Time: Dilutionfactor:
t4:
coupling-valueA280= ( ) mg/ml Time: Dilutionfactor:
t5:
coupling-valueAZ80= ( ) -mg/ml Time: Dilutionfactor:
t6:
coupling-valueA2g0= ( ) mg/ml Tlme: Dilutionfactor:
t7:
22. After coupling is complete, spin at low speed (700 rpm) for 5
minutes to separate excess protein from the gel. Remove the supernatant
from the gei slurry and save it to determine protein concentration after the
coupling step (end-value te). (Check if pH is still 9.0).
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A 280 = A 280' blank
X 1 mg/ml x Dia. factor - mg/ml
..- 1.3 8
end-value te: _ A280= ( ) mg/ml Time: . Dilutionfactor:
23. The next step is ao wash away the excess ligand with coupling
buffer: Most efficient way to wash the gel is to use the sintered glass
filter.
Gel size ml i 2 3.5 5 10 50 100
Coupling Buffer (ml) >50 >100 >i80 >200 >350 >800 >1500.
24. Block remaining active groups by transfering the gel to a vessel
with 15 gel volumes of 0.1 M Tris-HCI, pH 8Ø Shake in an Erlenmayer flask
at:180 rpm at room temperature for Z hours. (Alternatively, active groups
can also be blocked using 0.2 mM glycine, pH 8.0 or 1 M ethanolamine, pH
8.0).
Gel size ml 1 2 3.5 5 10 50 100
Blockin Buffer ml 15 30 52.5 75 150 750 1500
25. , After the blocking; pour the solution back onto the filter. Rinse
the tube with blocking buffer to collect most of the coupled gel.
26. The final product is. then washed alternately with 10 gel volumes
of low pH wash buffer (0.1 M sodium acetate containing 0.5 M NaCI, pH 4.0)
arid high pH wash buffer (0.1 M Tris-HCI containing 0.5 M NaCI, pH 8.0) for 4
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tiiriies. Thorough washing of the coupled, product is necessary to remove
traces of non-covalently adsorbed materials. The washing-cycle of low and
high pH is essential for the best results. This procedure ensures that no free
ligand remains ionically bound to the immobilized ligand. Let the mixture
equilibrate a few mihutes during each washing step.
Gel size ml 1 2 . 3.5 5 10 50 100
wash Buffers (ml each wash 10 20 36 50 100 500 1000
27. Finally, pass 15 gel volumes of PBS over the sintered glass filter.
Gel size inl ~ 1 2 3.5 5 IO 50 100
P8S ml 15 30 52.5 75 150 750 1500
28. Transfer the gel into 2.5 gel volumes of PBS containing 0.05%
sodium azide. The protein-sepharose conjugate is now ready for packing into
columns.
Gel size ml 1 2 3.5 ~ 5 ~ 10 ~ 50 ~ ~100
Storage BufFer (ml) _.- _ 2.S , 5 , 9 12.5 2S 125 250 l
29:. '. Store at 4°C. The stability of the coupled gel is dependent on
the
attached ligand and storage might be limited.
30. Collect all A280 measurements in the following data chart. This
data collection- .wilt be used to graph the reaction curve and calculate
efficiency of the coupling reaction (ligand binding efficiency) as well as the
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-antigen loading capacity of the column. These values are particularly useful
to be compared to later performed coupling reactions.
start-value A280= ( ) mg/ml Time: 0 min Dilution Factor:
ts:
Coupling-value A280= ( ) mg/ml Time: Dilution Factor;
ti:
Coupling-value A280= ( ) mg/ml Time: Dilution Factor:
t2:
Coupling-value AzgO= ) mg/ml Time: Dilution Factor;
t3;
Coupling-value AZ80= ( )' mg/ml Time: . Dilution Factor:
t4:
Coupling-value A2g0= ( . mg/ml Time; Dilution Factor:
t5;
Coupling-value A280= ( ..) mg/ml Time: Dilution Factor:
t6:
Coupling-value A280= ( ) mg/ml Time; Dilution Factor:
t7:
end-value te: A280= ( ) mg/ml Time: Dilution Factor:
- 31. To estimate coupling efOciency (ligand binding efficiency),
determine the concentration' of the Iigand in solution before and after the
coupling step. Generally, 70 = 80 % binding is optimal: lower binding leads
to reduced column . capacity white higher binding may result in reduced
binding efficiency due to steric hindrance. Coupling 'efficiencies of 70 - 80%
are- normally a good . compromise between good activity and high
concentrations:
[COI1C.]ts- ~COIIC.~te o ~ o
. . 1 ~U /o = /o
[co>tlc.]t
32. Calculate the total amount of antibody bound per ml of gel.
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(amount protein) is coupling eff ciency .
(mg/nil gel;
ml gel ,
33. The total amount of antibody bound per ml of gel is directly proportional
to the antigen loading capacity which will give an estimate of how much
protein maximally can bind per ml gel. To take into consideration is the Mw
of the Ig~ molecule--of~N150 kDa as well as itsvcapability to bind 2 antigens.
In addition, parameters of the molecule to purify are also necessary (i.e.
class I complex (57 kDa): heavy chain; 45 kDa, ~2-microglobulin; 12 kDa,
peptide):
mg IgG,,~""a/m~ get 57 Ic a = mg AntigenmaXf m~ gel
150 kDa
A variety of columns are available for large scale purification. XK
columns are jacketed and available in different dimensions with diameters of
26 mm (XK26) and 50 mm (XK50). These .columns are only used with
adaptors. The column can be used in aqueous and nearly all organic solvents
(exceptions: acetone, chloroform, phenol). Solutions containing more than
10% NaOH, 10% .HCI or 5% acetic acids should not be used. Kontes Flex-
columns are a more simpler version of columns but as effective.
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1. Sterilize the column before loading using either 100% ethanol or
2 N NaOH. It is possible to autoclave columns for' 15 minutes at 121°C,
wet.
or dry:
2. To start loading, ~resuspend the settled gel by gently mixing.
3. Degas using a vacuum aspirator.
4. Transfer the gel slurry into an appropriate column. Do not use
acetone, berizyl-alcohol, ~ chloroform, phenol', or dimethyl formaldahide
because immediate damage will occur. The columns are resistant to acetic
acid or NaOH.
5. Pack the column by pouring the gel into the vertically held
column. Pour the slurry into the column in one continuous motion. Let the
matrix settle by gravity flow until all slurry is transfered.
6. Insert the flow adapter into the packed column. First, purge the
air from the flow adapter tubing and rinse the flow adapter.
7. Carefully insert the flow adaptor into the column unt(I it touches
the buffer. Avoid trapping air bubbles by slightly tipping the column,
allowing
the air to escape.
8. Slowly lower the flow adapter until it touches the top of the
packed gel bed. The seal should be tight enough to allow the buffer to rise
through the adapter instead of leaking around the seal. This will help clear
trapped air in the adapter tubing.
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9. t=inally;~~completely seal the adapter against the column.
10: . Equilibrate the column by passing 10 bed-volumes of PBS over
the matrix.
11. The column is now packed and ready to use. How welt the
column is packed will have. a major effect on the result of the separation.
i2. Depending on the site of the column, different flow rates can be
applied.
AKTAT'" prime system for standard separation ap~~lications
AKTAT'" prime is a .compact, automated liquid chromatography system.
It is designed. for standard separation applications. Flow rates up to 50
ml/min and pressures up to 1000 kPa can be applied: The system includes
components for measuring UV, conductivity, generating gradients and
collecting fractions. The AKTA"" prime system may be utilized in the large
scale purification procedure of the present invention in accordance with
manufacturer's recommendations.
Lar eq_ scale purification procedure
1. To start the chromatography procedure, prepare the AKTAT"' prime
system. The system can be used immediately but the spectrophotometers
full ability will not, be obtained until after 1 hour of lamp warm-up.
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2. To prepare the system for a run, check that the buffer inlet tubings
are immersed . in the correct buffer vessels and the waste tubings are put
into a waste bottle.
3. 'Only use degassed and filtered liquids to make sure that the liquid
remains free from air bubbles: Degass by applying a vacuum to the solution.
4. Prepare and hook up the buffers necessary for an sHl~4
purifcation:
1. PBS, pH 7.4 (Wash buffer)
2. 20 ~'o Ethanol / 70 % Ethanol(Cleaning solutions)
3. 0.1 N NaOH (MOK elution buffer)
4, 50 mM Diethylamine (DEA), (MOK elution buffer)
pH 11.3
5. Protein sample (The line is stored
in PBS/0.05%
Na Azide, pH 7.4)
6. 0.2 N Acetic acid, pH X2.7(Cleaning & MOK solution)
7. 0.1 M Glyclne, pN 11,0 (sHW elution buffer)
5. . It is important to purge the lines after a new hook-up with about
50 ml of .liquid . to get the air out of the system. Purging can be done
manually through the inlets of the buffer valve (A1-A8), while carefully
immersing the tubing in the respective liquid.
6. To remove any trapped air bubbles in the flow path, purge the
pump in the order PBS / 20 % ethanol / PBS / final buffer solution.
7. Next; prepare the recorder to monitor..the purification. Autozero
the built-in UV spectrophotometer with PBS as reference.
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8. Equilibrate all material. to the temperature at which the
chromatography will be pertormed. For large scale purifications, attach the
column entranee/exit to the system;
9_ Equilibrate the cofumn . by passing 10 bed-volumes of PBS over -
the matrix:. -
10. Before starting .any column purification, the protein concentration
in the sample solution should be determined using a quantitative ELISA
procedure. The sample volume loaded v'rill depend on the size and loading
capacity of the column and the concentration of the sample. calculate the
volume of the sample solution maximally saturating the column according to
the columns capacity to bind the antigen.
(A) Antigen concentration: mg/ml antigen
(B) Antigen bihdi~g capacity: mg antigen/ml gel
(C) Matrix volume: ml gel
(D) Maximal amount of antigen: (B*C) mg
(E) Sample volume: (D/A) ml
li. Since the binding capacity of the column will realistically not be
reached, a much lower volume of sample solution should be chosen. A value
between 40 to 50% of the calculated volume is more accurate which also will
not result in the waste of lots of unbound antibody within the flow-through.
12. Prepare the antibody sample solution for purification. Spin crude
harvest at 5,000 rpm for 25 minutes (JA10 rotor) to remove lipid and cell
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debris. The antigen solution . must be free of particulate matter: Pour. the
supernatant into a suitable container. Prevent air bubble formation.
Name of the crude harvest:
Volume used: ml
Amount of sample: mg
13.~ The. simplest . method to bind the antigen to the
antibody/Sepharose 4B matrix is to apply the sample through the system
pump and pass the protein solution down the column.
14. Set appropriate parameters to record the loading conditions on
the recorder.
Chart Speed Conductivity Optical Density
Load 0.1 mm/min 0.5 V 1.0 V
15. Save a 1 ml'probe from 'the starting material (LOAD) before the
purification procedure for analysis purposes.
Set the : buffer vaEve to position 5 and the injection valve to
position LOAD: Make sure the inlet tubing is purged with sample buffer
without any airbubbles present. To have a purged sample line, disconnect
shortly the column before loading and circulate the sample within the system
with higher flow rate.
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17. Pass the solution slowly througEl the column with a flow rate of
approximately 1.0 ml/min or lower to give the protein time to bind more
efficiently. Higher flow rates will decrease efficiency. A disruption in flow
may
cause a rapid rise in back-pressure. Zf. this occurs, immediately shut off the
pump and check the, gel 'bed for compression.
18. Collect the flow-through in an appropriate container. Keep untie ..
you are sure all material has bound.to the; columrf and ~egiigible amounts
are in the flow through. Take a sample at the end of the run (Ft) which
should be analyzed.
19. Wash the column with PBS at 10 ml/min until UV absorbance at
280 nm is zero. For a large column use 2000-3000 ml wash buffer (PBS).
Save the wash in a container until after the purification.
Chart Speed ConductivityOptical Density
.
Wash o.5 mm/min 0.5 V 1,0 V
20. collection
Prepare tubes by
borosilicate- adding 1.2
ml of 1
M
Tris-HCi, pH 7.0 per 4.8 ml of fraction to be collected ( 1:4): Neutralization
is
a safety measure to preserve the activity of the eluted molecule.
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21. Human vMHC class I {sNIA). molecules are best eluted from a v
W!6/3Z~column by 0.1 M glyclne, pH i1Ø Absorbance is used for generating
a ,protein elution profile.
Chart speed Conductivity Optical Density
Elution, O.S mm/sec . 0:2 V 0.1 V
22. Place the collector arm over the frrst collection tube. Elute 4.8 m) .
per fraction at 10 ml/min. Immediately afterwards, mix each tube gently to
bring the pH back to neutral. As with all protein solutions, avoid bubbling or
frothing as this denatures the proteins. If a very low amount of protein is
eitpected; change the conductivity on the recorder to a lower value.
23. Identify the- antigen-containing fractions by absorbance at 280
nm on the chart and combine them during up-concentration.
z4. up-concentrate immediately and buffer exchange into PBS using
MACROSEPTM centrifugal concentrators (Pall Filtron; Northborough, MA;
MACROSEP 10K; ODOlOC37). Keep the protein on ice at all times and
centrifuge at 4°C.
25. After the. buffer exchange, prepare the sample for storage at
4°C. Filter the,pure samples through a 0.2 p filter and aliquot
directly into
sterile, screw cap tubes. label appropriately.
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26. Determine the absorbance at 280 nm as well as the protein
concentration with the Micro BCA kit. Activity can be determined with a
regular ELISA procedure.
27. The purity of the eluted sHLA can .be assessed by SDS-PAGE;
Western blotting or performing a Superdex column analysis.
28. After the elution, quickly re-equilibrate the column with PBS to
avoid denatu~ation of the W6/32 antibody finked to it.
29. For analytical work in which more than one allele will be purified
on the same column, extreme care must be taken. To be able to reuse the
column, start a maintenance procedure after the reequilibration, Cleaning-in-
place is a procedure, which removes contaminants such as lipids,
precipitates or denatured proteins that may remain in the column after
regeneration. Such contaminations are especially likely when working with
crude materials. The procedure helps to maintain, the capacity, flow
properties and general performance.
30. Mock elute the column using buffers with alternating pH. Start
running over 10 gel volumes of 0.2 N acetic acid followed by 10 gel volumes
of SO mM diethylamine, pN 11.3 at a speed of 10 mUmin. Repeat three
times and always equilibrate with 10 gel volumes PBS between buffer
changes.
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Chart-Speed ConducYrvityOptical Density
Mock-elution~~.0 rrim/min 0.2 V . 0.1 V
31. After Mock-elution, store the column at 4"C in PBS/0.05% Na
Azide.
32.. Sanitization inactivates microbial contaminants in the packed
column and related equipment. One generally recommended procedure is to
wash alternately with high and low pH buffers as performed in the coupling
reaction.
33. For sanitization, disassemble the' column and wash the matrix
alternately with low pH wash buffer (0.1 M sodium acetate containing 0.5 M
NaCI, pH 4.0) and high pH wash buffer (0.1_ M Tris-HCI containing 0.5 M
NaCI, pH 8.0) for 3 times followed by re-equilibration with PBS.
34. Reassemble the cleaned and sterilized column and store it at 4°C
in~ PBS containingØ05% sodium azide.
35. After the column is: removed, the i4KTAT"' prime system has to be
cleaned carefully. Start with the cleaning of line S, where the sample was
hooked up. Rinse the system pump and include the fraction collector line.
36. First clean the inlet tubing, by manually running the system
pump and flushing with 0.2 N acetic acid at 30 ml/min followed by 0.1 N
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NaOH. Always equilibrate with PBS. Don't forget to add a tine between the
injection valve and the UV detector as a bridge, as replacement of the
column.
37. . Finally, rise with 20% ethanol. If the column was sanitized
because of bacterial contamination, rinse with 70°~o ethanol.
Buffer exchange and concentrating samples using Pal-Filtron concentrators
MACROSEPTM centrifugal concentrators (Pall Filtron; Northborough,
MA; MACROSEP lOK; ODOlOC37) provide rapid and convenient
concentration, purification, and desalting of 5 ml to 15 ml biological
samples.
A starting sample of 15 ml can be concentrated to 0.5 ml in 30 to 60
minutes without multiple decanting steps. The MACROSEP's ease of use
saves valuable lab time.
Each centrifugal concentrator is constructed of polypropylene and
contains a low-protein-binding OMEGATM membrane, two factors which
significantly reduce non-specific adsorption and enable the device to yield
the highest recoveries. OMEGA membranes are made from polyethersulfone
(PES) specifically modified to minimize protein binding. These membranes
provide equivalent or higher recoveries than comparable regenerated
cellulose membranes. MACROSEP centrifugal devices are ideal for.
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concentrating small peptides, oligonucleotides, nucleic . acids, enzymes,
antibodies, microbes, and other macromolecules:
Centrifugation up to 5,000 x g provides the driving force for filtration,
moving sample towards -the .encapsulated OMEGA membrane. Biomolecules
larger than the nominal molecular weight cutoff of the membrane are
retained in the sample reservoir. Solvent and low molecular weight
molecules pass through the membrane into the filtrate receiver. 'The .
MACROSEP centrifugal concentrator is available with 9 different molecular
weight cutoffs (MWCO): 1K, 3K, 10K, 30K, SOK, 100K, 300K, 1000K, and 0.3
Nm. For maximum retention, select a MACROSEP device with a molecular
weight cutoff that is 3 to S times smaller than the weight of the molecule to
be retained.
For purification of sHLA molecules of the present invention, a 10K
MACROSEPT" centrifugal concentrator is utilized in accordance with
manufacturer's recommendations.
1. Insert the paddle firmly into the bottom of the sample reservoir
of the. 10K. MACROSEPTM centrifugal concentrator (Pall Filtron;
Northborough, MA; ODOlOC37). The "hooks" on the top part of the paddle
must rest firmly in the notches on top of the sample reservoir. For best
alignment, turn the reservoir.upside down on the_bench top and gently press
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the paddle into place. Attach the filtrate receiver to the bottom of the
sample
reservoir.
Pre=Rinsing (Optional): OMEGATM membranes in the MACROSEP
devices contain trace amounts of glycerine and sodium azlde. If these
chemicals interfere . with an assay, they may be removed. Filter 15 ml of
deionized water or buffer through the membrane.
3. Start to up-concentrate °immediately with the .low peak fractions
first. (With some micro-concentrators, adsorption of protein to the walls of
the unit as well as to the Alter itself can be significant when the sample is
very dilute)..
4. Pipette up to 15 ml of sample (protein-eluate in neutralization
buffer) from the fraction-collector glass-tube into the non-membrane side of
the sample reservoirs) using a 10 ml pipette. (Do not decant the samples as
it will result in a higher loss).
5. Do not overfill. Place the cap on the reservoir.
6. Place the devices) into a swinging bucket rotor. (In a fixed-
an~fe rotor, align the MACROSEP so that one of the "hooks" faces the center
of the centrifuge rotor. This prevents a buildup of macromolecules on the
membrane paddle and allows .the device's deadstop to function properly. A
swinging-bucket rotor is self=aligning).
7. Always counterbalance the rotor.
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8. Keep the protein on ice at all times and centrifuge at 4°C. A non-
refrigerated micro-centrifuge may develop temperatures detrimentalao
protein samples when operaied for extended periods; therefore it.is usually
best to have the non-refrigerated micro-centrifuge in a refrigerator or cold
room for this operation, even though the filtration rate is reduced by the .
cold.
9. Spin at 3,500 rpm (1,000-5,000 g) at 4°C, typically for 30 to 60
minutes, to achieve the desired concentrate volume.
10. For desalting and/or buffer exchange, concentrate the sample at
least tenfold. -
11. After the spin, remove the filtrate from the collector and save it
in an appropriately labeled 500 ml bottle. Keep the bottle on ice at all
times.
12. Refill the same macrosep(s) and repeat the procedure until all
fractions are up-concentrated.
13. In parallel to the up-concentration process, centrifuge the empty
fraction collector tubes to recover remaining traces of protein sample. Add
the recovered material to the macrosep(s).
14. After up-concentration, proceed with the buffer exchange by
adding fresh exchange buffer of the desired composition.
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I5. Add exchange buffer (PBS/0.02ofo Na Azide) to the sample
reservoir in a volume equal or lower to that of the ultrafiltrate. collected,
o
that the concentration of macromolecular species remains unchanged.
16. As filtration proceeds, refill the sample reservoir with fresh
exchange buffer to restore the original volume. Continue doing this until the
volume of ultrafiltrate is four times the volume of the original sample,
indicating that removal of diffu,sible material is 95% to 99% complete.
17. After every fresh buffer exchange, make a mark on the top of
the reservoir cap. This will help keeping track of the status of the
procedure.
18. If there is not enough time to finish the whole procedure, it can
be stopped after 2 buffer exchanges. Refill the macrocep with exchange
buffer to prevent the membrane from going dry, put the cap on and store at
4°C until the next day. Thereafter, the procedure can be interrupted
any
time, but always prevent the membrane from ~ going dry by filling the
reservoir.
19. Recombine the buffer exchange flow through with the original
filtrate. Keep on ice.
20. After the buffer exchange, the same process is used to
concentrate samples, except that the retentate volume is allowed to
decrease until , the desired degree of concentration is reached. Over-
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concentration makes sample recovery difficult and may require re-addition of.~
huffier to wash the membrane, thereby adding to the volume.
21. Check .ODZBO to: estimate an approximate concentration of, the
sample. An ~D2BO of 1.0 is in the area. of 0.5 to 0.7 mg/ml.
22. To recover the final sample, remove the liquid from the sample
reservoir with a 1000 NI pipette tip. Add to a labeled 50 ml Falcon tube and
store at 4°C.
23. In regard to the recovery rate of samples following concentration
being generally 95% and the degree of nonspecific adsorption of protein to
membranes, losses of 5% to 10% are not uncommon when dealing with
total quantities of protein in the range of 1 to 10 mg.
24. To recover with a much higher efficiency, add all the saved
frftrate and flowthroughs again to the same macrocep(s) and proceed in the
same way. ~o not save filtrates a second time. Buffer exchange again four
times and finally combine with the first round concentrate. Make sure to
reach an equal concentration before combining.
25. For maximum concentrate recovery, remove filtrate receiver and
screw on the concentrate cup. The center pin will cause the paddle to lift up
and out of the bottom of the sample reservoir, allowing concentrate to flow
into concentrate cup.
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26. Place the MACROSEP device back into the centrifuge and spin at
3,500 rpm (1,000-S,000 g). for 5 minutes. Remove the device and unscrew
the concentrate cup.
27. Finally,-. prepare thesample for storage at 4°C. Filter the pure
sample. through a 0.2 Nm filter and aliquot directly into sterile, screw cap
tubes. Label appropriately:'
ELISA Procedures
i. The experiment is designed using an ELISA protocol template, and
a ,clear 96-well polystyrene.assay plate is labeled. Polystyrene is normally
used as a microtiter plate. (Because it is not translucent, enzyme assays that
will be ~quantitated by a plate reader should be performed in polystyrene and
not PVC plates).
Company Plate Specificity Cat#
Nunc Maxlsorp standard/untreated441653
StarWell Modules
Framed 8-well
stri s
~~ 2. ' Coating of the 1N6j32 should be performed'in Tris buffered saline
(TBS); pH 8.5. Prepare 'a coating solution of 8.0 ug/ml of specific W6/32
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antibody in TBS (pH 8.5). (Use the blue tube preparation stored at
~20°C
with a co~icentration of 0.2 mg/ml and a volume of 1 ml giving 0.2 mg per
tube).
No. of latexTotal VolumeW6 32 a~tibod TBS H 8.5
Mix: 1 3.0 ml 400 I 9.6 ml
2 20 ml 800 I 19.2 ml .
3 30 ml 1200 2B.8 ml
4 40 m) 1600 I 38.4 ml
50 ml 2000 I 48,0 m)
3. Although this is well above the capacity of a microtiter plate, the
binding will occur more rapidly. Higher concentrations will speed the binding
of antigen to the polystyrene but the capacity of the plastic is only about
100
ng/well (300 ng/crn2), so the extra protein will not bind.
4. If using W6/32 of unknown composition or concentration, first
titrate the amount of standard antibody solution needed to coat the plate
versus a fixed, high concentration of labeled antigen. Plot the values and
select the lowest level that will yield a strong signal.
~5. Do n.ot include sodium azide in any solutions when horseradish
peroxidase is used for detection.
6. Immediately coat the microtiter plate with 100 Irl per well using
a multi-channel .pipette. Standard polystyrene. will. bind antibodies or
antigens when the proteins .are simply incubated with the plastic. The bonds
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that hold the proteins.are non-covalent, but the exact. types of interactions
are not known..
7. Shake the plate to ensure that the antigen solution. is evenly
distributed over the bottom of each well:
8. Seal the plate with plate sealers (sealplate adhesive sealing filrin,
nonsterile, 100 per unit; Phenix (1--800 767-0665); LMT-Seal-EX) or sealing
tape to Nunc-ImrrunoT"' Modules (# 236366).
9. Incubate at 4°C overnight. Avoid detergents and extraneous
proteins.
10. Next. day, remove the contents of the well by flicking the liquid
into the sink or a suitable waste container. Remove the last traces of
solution by inverting the plate and blotting it against clean paper toweling.
Complete removal of liquid at~each step is essential for good pertormance.
11. Wash the plate 10 times with Wash Buffer (PBS containing 0.05
Tween-20) using a~ multi-channel ELISA washer,
12. After the last wash, remove any remaining Wash Buffer by
inverting the plate and blotting it against clean paper toweling.
13. After the W6/32 is bound; the remaining sites on the plate must
be saturated by incubating with blocking buffer made of 3% BSA in P6S. Fill
the wells with 200 NI blocking buffer.
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14. Cover the plates with an adhesive strip and incubate overnight at
4°C. Alternatively, incubate for at least 2 hours of room temperature
which
is, however; not the standard procedure.
25. Blocked plates may be stored for at feast 5 days at 4°C,
16: - .Good , pipetting ~ practice is most important to produce reliable
quantitative,~esults.. The tips are just as important a part of the system as
the pipette itself. If they are of inferior quality or do not fit exactly,
even the
best pipette cannot produce satisfactory results.
17,, The. pipette working position is always vertical: Non-vertical
positions may cause too much:l.iquid to be drawn in.
18. The immersion depth should be only a few millimeters.
19.. ,Allow the pipetting button to retract gradually, observing the
filling operation. There should be no turbulence developed in the tip,
otherwise there is a risk of aerosols being formed and gases coming out of
solution.
20. . When maximum levels of accuracy are stipulated, pre-wetting
should be used at all times, To do this, the required set volume is first
drawn
in one or tv~io..times using the, same tip and then returned. Pre-wetting Is
absolutely necessary on the more difficult liquids such as 3% BSA.
21. Do not pre-wet if your intention is to mix your pipetted sample
thoroughly with an already present solution.
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Z2. Mowever, pre-wet only for volumes greater than 10 Nl. In . the
case of pipettes for voludnes less than 10 NI, the residual liquid film is as
a
rule taken into account when designing and adjusting the instrument. The
tips must be changed between each individual sample.
23. With volumes < 10 ~rf special attention must also be paid to .
drawing in the liquid slowly, otheriniise the sample . will be significantly
warmed Up by. the. frictional. heat generated. Then slowly withdraw the tip
from the liquid, if necessary wiping off any drops clinging to the outside.
24. To dispense the set volume hold the tip at a slight angle, press it
down uniformly as far as the first stop.
25. In order to reduce, the effects of surface tension, the tip should
be in contact with the side of the container when the liquid is dispensed.
Z6. After liquid has been discharged with the metering stroke, a
short pause is made to enable the liquid running down the inside of the tip to
collect at its lower end.
27. Then press it down swiftly to the second stop, in order to blow
out, the tip with the extended stroke with which the residual liquid can be
blown out. In cases that are not problematic (e.g~. aqueous solutions) this
brings about a rapid and virtually complete discharge of the set volume. In
more di~cult cases, a slower discharge and a longer pause before actuating
the extended stroke can help.
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28, To. determine:.the absolute amount- of antigen (sHLA), sample
values .are compared with those obtained using known amounts of pure
unlabeled antigen in a standard curve.
29. For accurate quantitation, all samples have to be run in triplicate,
and the standard antigen-dilution series should be included on each plate.
Pipetting should be.preformed without delay to minimize differences in tirtie
of incubation between samples:
30:: All dilutions should be done in blocking buffer.
31. Thus, prepare a standard antigen-dilution series by successive
dilutions of the homologous antigen stock in 3% BSA in PBS blocking buffer.
In order to measure the amount of antigen in a test sample, the standard
antigen-dilution series needs to span most of the dynamic range of binding.
This range spans from S to 100 ng sHt.A/ml.
3Z. A stock solution of 1 ug/ml should be prepared, aliquoted in
volumes of 300 Irl and stored at 4°C. Prepare a 50 ml batch of standard
at
the time. (New batches need to be compared to the ofd batch before used in
quantitation).
33. Use a tube of the standard stock solution to prepare successive
dilutions according i:o the scheme shown in FIG. 93.
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34. While standard curves are necessary to accurately measure. the
amount of antigen in test samples, they are unnecessary for qualitative
"yes/no" answers.
35. For accurate quantitation, the test solutions containing sHl~4
should be assayed over a number of at least 4 dilutions to assure to be .
within the. ia~ge of the standard curve. Prepare serial dilutions of each
antigen test solution in .blocking.Buffer (3% BSA in PBS).
36. Standard dilutions for purified, crude or flow through samples
are given in FIG. 94.
37. After mixing, prepare all dilutions in disposable U-bottom 96 well
microtiter plates before adding them to the ,W6/32-coated plates with a
multipipette. Add 150 NI in each well.
38. Next remove any remaining blocking buffer and wash the plate as
described above. The plates are now ready for sample addition,
39. Add 100 NI of the sHLA containing test solutions and the
standard antigen dilutions to the antibody-coated wells.
40. Cover the plates with an adhesive strip and incubate for exactly
1 hour at room temperature.
41. After incubation, remove the unbound antigen by washing the
plate lOx with Wash Buffer (PBS containing 0.05 % Tween-20) as described.
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42. Prepare the. appropriate developing reagent to detect sHLA. Use
the second specific antibody, anti-human ~2m=HRP (DAKO P0174 / .0:4
mg/ml) conjugated to Horseradish Peroxidase (HRP). Dilute the anti-human.
~2m-HRP in a ratio of 1:1'000 in 3% BSA in PBS. (Do not include sodium
azide in solutions when horseradish perox'idase is used for detection).
No, of fatesTotal VoleameAnti- 2m-HRP antibod3~r'o BSA
in PBS
Mlx: 1 10 mi 10 I 10 ml
2 24 m! 20 t 20 mt
3 30 ml - 30 l 3o ml
4 40 ml 40 I 40 ml
50 ml 50 ~ul SO ml
43. Add 100 NI of the secondary antibody dilution to each well. All
diiutions should be done in blocking buffer.
44. Cover with a new adhesive strip and incubate for 20 minutes at
room temperature.
45. Prepare the appropriate amount of substrate prior to the wash
step. Bring the substrate to room temperature. .
46. ~ OPD (o-Phenylenediamine) is a peroxidase substrate suitable for
use in ELISA procedures. The substrate produces a soluble end product that
is :yellow in color. The OPD reaction is stopped with 3 N H2504, producing an
orange-brown product and read at 492 nm. Prepare OPD fresh from tablets
(Sigma, P6787; 2 mg/tablet). The solid tablets are convenient .to use when
small quantities of the substrate are required.
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47. After second antibody incubation, remove the unbound ,
secondary reagent by washing the plate lOx with Wash Buffer '(PBS
containing 0.05%,Twee:n-20).
48. After the final wash, add 100 NI of the OPD substrate solution to w
each well and allo~iv it to develop at room temperature for 10 minutes.
Reagents of the developing system are light-sensitive, thus, avoid placing
the plate in direct light. .
49~ ~ Prepare the 3 N H2S04 stop solution.
50. After 10 minutes, add 100 NI of stop ~ solution per 100 NI of
reaction mixture to each well. Gently tap the plate to ensure thorough
mixing.
51. Read the ELTSA .plate at a wavelength of 490 nm within a time
period of 15 minutes after stopping the reaction.
52. The background' should be around 0.1. If the background is
higher, the substrate may have been contaminated with a peroxidase. If the
subtrate background is low and the background in you're the assay is high,
this may be due to insufficient blocking.
53, Finally analyze the readings.
54. Prepare a standard curve constructed from the data produced by
serial dilutions of the standard antigen.
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55. To determine the absolute amount of antigen, compare these
values with those obtained from the standard curve. Use the pre-made Excel
template:
Protein Separation
SDS-PAGE
To localize sHLA with SDS-PAGE, proteins were obtained by
denaturating with a solution containing 4% SDS, 20% glycerol, O.OZ%
bromophenol blue, and 200 mM dithiothreitol in 0.5 M Tris-HCI (pH 6.8). For
separation, Sodium dodecyl , sulfate-polyacrylamide gel elec-trophoresis
(SDS-PAGE) was performed by using the procedures described previously by
[Laemmti, 1970] on a 12.5% gel. Gels were stained in Coomassie-staining.
INesrern Blot Analysis
To localize sHLA in Western blots, proteins were obtained by
denaturating with a solution containing 4% SDS, 20% glycerol, 0.02%
bromophenol blue, and 200 mM dithiothreitol in 0.5 M Tris-HCI (pN 6.8).
Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) was
pertormed by using the procedures described previously by [Laemmli,
1970]. Briefly, the proteins were separated on a' 12.5% gel, electroblotted
onto an Immobilon-P mem-branes (Millipore, Bedford, MA), and blocked
m
CA 02514872 2005-07-04
WO 03/057852 PCT/US03/00243
overnight in 3% BSA in Tris-buffered saline/ Tween 20 buffer. All primary
and secondary antibodies were applied in this buffer. The working dilution of
primary antibodies was 1:1,000 for (32m(HRP), and 1:5000 for HC10, and
that of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG
antibody was 1:2,000. To visualize antibody binding, the membranes were
developed using the ECLplus reaction according to the manufacturers
recommendation.
Thus, in accordance with the present invention, there has been
provided a method for purifying Class I and Class II MHC molecules
substantially away from other proteins that includes methodology for
producing and manipulating Class I and Class II MHC molecules from gDNA
that fully satisfies the objectives and advantages set forth herein above.
Although the invention has been described in conjunction with the specific
drawings, experimentation, results and language set forth herein above, it is
evident that many alternatives, modifications, and variations will be apparent
to those skilled in the art. Accordingly, it is intended to embrace all such
alternatives, modifications and variations that fall within the spirit and
broad
scope of the invention.
All of the numerical and quantitative measurements set forth in this
application (including in the examples and in the claims) are approximations.
The invention illustratively disclosed or claimed herein suitably may be
m2
CA 02514872 2005-07-04
WO 03/057852 PCT/US03/00243
practiced in the absence of any element which is not specifically disclosed or
claimed herein. Thus, the invention may comprise, consist of, or consist
essentially of the elements disclosed or claimed herein.
The following claims are entitled to the broadest possible scope
consistent with this application. The claims shall not necessarily be limited
to the preferred embodiments or to the embodiments shown in the
examples.
113
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WO 03/057852 PCT/US03/00243
REFERENCES
The following references, to the extent that they provide exemplary
procedural or other details supplementary to those set forth herein, are
specifically incorporated herein in their entirety by this reference.
Cresswell, P., M.J. Turner, and J.L. Strominger, Papain-solubilized HL-A
antigens from cultured human lymphocytes contain two peptide fragments.
Proc Natl Acad Sci U S A, 1973. 70(5): p. 1603-7.
Tanigaki, N. and D. Pressman, The basic structure and the antigenic
characteristics of HL-A antigens. Transplant Rev, 1974. 21(0): p. 15-34.
Tanigaki, N., et al., Common antigenic structures of HL-A antigens. II. Small
fragments derived from papain-solubilized HL-A antigen molecules.
Immunology, 1974. 26(1): p. 155-68.
Prilliman, K., et al., Large-scale production of class I bound peptides:
assigning a signature to HLA-B*1501. Immunogenetics, 1997. 45(6): p.
379-85.
Prilliman, K.R., et al., HLA-B15 peptide ligands are preferentially anchored
at
their C termini. J Immunol, 1999. 162(12): p. 7277-84.
Prilliman, K.R., et al., Peptide motif of the class I molecule HLA-B*1503.
Immunogenetics, 1999. 49(2): p. 144-6.
Cresswell, P., et al., Papain-solubilized HL-A antigens. Chromatographic and
electrophoretic studies of the two subunits from different specificities. J
Biol
Chem, 1974. 249(9): p. 2828-32.
Peterson, P.A., L. Rask, and J.B. Lindblom, Highly purified papain-solubilized
HL-A antigens contain beta2-microglobulin. Proc Natl Acad Sci U S A, 1974.
71(1): p. 35-9.
Collins, E.J., et al., The three-dimensional structure of a class I major
histocompatibility complex molecule missing the alpha 3 domain of the
heavy chain. Proc Natl Acad Sci U S A, 1995. 92(4): p. 1218-21.
114
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WO 03/057852 PCT/US03/00243
Bjorkman, P.J. and P. Parham, Structure, function, and diversity of class I
major histocompatibility complex molecules. Annu Rev Biochem, 1990. 59:
p. 253-88.
Laemmli, U. K et al., Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature,1970, 227, p. 680-685.
ms
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SEQUENCE LISTING
<110> HILDEBRAND, WILLIAM H.
BUCHLI, RICO
<120> PURIFICATION AND CHARACTERTZATION OF HLA PROTEINS
<130> 620521-8/JP/347,906
<140> PCT/US03/00243
<141> 2003-01-02
<150> 10/022,066
<151> 2001-12-18
- <150> 60/347,906
<151> 2002-O1-02
<160> 19 .
<170> PatentIn Ver. 2.1
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