Note: Descriptions are shown in the official language in which they were submitted.
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~Or~;T~' b~ OY c~a~ ~
~D B~ 'uv~
FIFTr, ~F ~ L.~v ~ LlON
The present invention in~olves a nethod cf
separating and L.~ ing speci~ically-taryeted colls
or ~ol~ r structures fro~ ~ixed cell or mclP~l~r
structure populations usins anliho~ies.
REFERENCES
The fall~w~ns references are cited in the
applicaticn and hereby incorparated by reference in
their entirety.
1) ~.S. Patent No. 4,228,237;
2) ~.S. Patent No. 4,253,996;
3) ~.S. Patent No. 4,276,206;
4) U.S. Patent No. 4,2g8,685;
5) ~.S. Patent No. 4,468,470;
6) U.S. Patent Na. 4,496,654;
7) ~.S. Patent No. 4,814,434;
8) U.S. Patent No. 5,078,673;
9) ~.S. Patent Na. 5,116,724;
10) ~.S. Patent No. 5,139,933;
11) ~.S. Patent No. 5,215,927;
12) International Patent Application No. wa 87/04628;
13) International Patent Application No. WO 92/16841;
14) El~opean Patent Application Nc. 0 825 474;
15) Basch et al., (1983) "Cell Separation Using
Positive r~ no~elective Tp~hni~ues~ J. rmmt~n~l .
~etho~s 56:26g-80;
SIJ~S 111 UTE SHEET (RULE 26)
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16) Berenson et al., (1986) "Positive Selection of
Viable Cell Populations using Avidin-biotin
Immunoadsorption" J. Immunol. Methods 91:11-19;
17) Berman and Basch, (1980) "Amplification of the
S biotin-avidin immunofluorescence technique" J.
Tm~lrnol. Methods 3 6: 3 3 5 - 3 3 8;
18) Clark et al., (1982) "Positive and Negative
Selection of Cells by Hapten Modified Antibodies"
J. Immunol. Nethods 51:167-170;
19) Forsgren et al. (1977) J. Immunol. 99:19;
20) Goetzel and Metzger, (1970) "Affinity Labelling of
a Mouse Myeloma Protein which binds Nitrophenyl
Ligands. Kinetics of Labelllng and Isolation of a
Labeled Peptide" ~iochemlstry 9:1267-1278;
21) Hoffman et al. (1977) Proc. Natl. Acad. Sci. USA
7~:2697-2700;
22) Jasiewicz et al., "Selective Retrieval of Biotin-
labeled Cells using Immobilized Avidin" ~xp. Cell
Res. 100:213-19;
23) O'Shannessy, D.J. and Hofmann, W.L. (1987)
Biotech. Appl. Biochem. 9: 488;
24) Prud'homme et al., (1984) "Immune Dysfunction in
Dia~etes-prone BB Rats" J. Exp.Med. 159:463-78;
25) Tizard, Ed, Immunology: An Introduction, (1984)
Saunders College Publishing;
26) Vogt, R.F. et al., (1987) J. Immunol. Methods
101:43;
27) Scott, (1976) "Antifluorescein Affinity Columns"
~. Experimental Med. 1~: 69-78;
28) Weetall H. (1976) Meth. in Enzymol. ~:134-148;
and
29) Wormmeester et al., (1984) "A Simple Method for
Immunoselective Cell Separation with the Avidin-
~iotin System" J. Immunol. Methods 67: 389-94.
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BACKGROUND OF THE I ~ ENTION
There is a need to recover certain cell types or
certain molecular s~ructures from mixed populations of
cells or molecular structures.
Immunoselection is a generic term which
encompasses a variety of techniques for the separation
of cells or molecuiar structures bearing function
related or lineage specific antigenic determinants.
The specificity of the selection process is conferred
by antibody or antibody-like molecules, haptens or
lectins which interact with the specific cell surface
or molecular structure antigenic determinants.
In recent years the biotin-avidin system has been
applied to a variety of analytical and preparative
procedures and has found wide spread use in
immunoselection techniques for cell separation.
In general, two types of immunoselection
techniques are available.. "Negative selection"
involves the removal of a specific subpopulation of
cells from a heterogenous mixture of cell types. Using
this method of separation, one can obtain an enriched
but not pure preparation of remaining cells since all
cell types in the original mixture that are negative
for the selection antigen will be recovered. The
second method, "positive selection", involves the
specific targeting and recovery of cells expressing the
desired specificity from a heterogenous population of
contaminating cells. Positive selection tP~hniques can
provide a highly enriched or even pure population of
the desired antigen-positive cells, in contrast to
negative selection methods in which all cell types that
are target antigen-negative will be recovered.
~ iotinylated antibodies against rat thymocytes
have been used to remove such thymocytes from a cell
mixture by interaction with avidin covalently coupled
to nylon meshes but removal of the thymocyte cells from
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the avidin was not attempted (Jasiewicz et al., ~xp.
Cell Res. 100:213-19, 1976).
T cells have been removed from spleen cell
preparations by reaction with biotinylated monoclonal
antibody directed against T cell antigen followed by
panning on avidin coated plates but the T cells were
not removed from the plates (Prud'homme et al., J.
Exp.Med. 159:463-78, 1984~. U.S. Patent No. 4,298,685
also describes a negative selection process using
biotin-avidin.
Biotinylated monoclonal antibodies have been used
in combination with avidin coated sheep erythrocytes to
form rosettes of selected cells which are then
separated on density gradients (Wormmeester et al., J.
Immunol. Methods 67:389-94, 1984). This procedure
reportedly allows for both positive and negative
selection of cell populations. However, positively-
selected cells are coated with sheep erythrocytes which
could affect their activity.
All of the described methods rely upon the direct
interaction of biotin with avidin or streptavidin bound
to a solid support matrix during the immunoselection
process to achieve cell separation, whether by a
process of positive or negative selection. However,
because of the very high affinity of the biotin-avidin
interaction, the process is essentially irreversible
and it is difficult to recover intact, functional cell
populations away from the solid support matrix.
Various attempts have been made to circumvent this
difficulty including the use of biotin analo~ues with
lower affinity for avidin (Basch et al., ~. Tm?~?nol.
~ethods 56:269-80, 1983) or the use of avidin-Sepharose
matrices with reduced affinity for biotin (U.S. Patent
Nos. 4,Z53,996 and 4,276,206). These methods
reportedly yield an operable avidin/biotin based
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positive selection procedure. However, the efficiency
of isolating the desired components is variable.
Additional antibody layers on targeted cells have
also been used such that the linkage to the cell can be
mechanically disrupted at the lower affinity antigen-
antibody interaction site (Berenson et al., J. Tmm~nol.
Methods 91~ l9, i986; U.S. Patent No. 5,215,g27;
International Patent No. WO 87t04628). This process,
however, does not allow for the specific elution of the
selected, retained cell population since any
contaminating cells non-specifically adsorbed to or
mechanically trapped by the avidin-derivatized solid
phase may also be released by the mechanical agitation
elution process. Further the mechanical agitation
process may damage the cells.
International Patent Application Publication No.
WO 92/16841 discloses a non-immune, reversible binding
displacement system for the detection of compounds in a
solution. Specifically, the application discloses the
attachment of a releasable ligand, a binding partner
for the releasable ligand, an analyte of interest, an
analytically detectable (reporter) group and at least
one binding partner for the analyte to an insoluble
phase. A displacer ligand is added to the solution
which displaces the releasable ligand along with some
portion of the reporter-labeled complex which may or
may not contain the analyte of interest so as to detect
the presence of the analyte. This method discloses the
use of biotin analogues with lower affinity for avidin
as the releasable ligand.
Both positive and negative immunoselection methods
for cell or molecular structure separation would be
improved by a procedure which allowed for a high degree
of specificity and selectivity in targeting of cells
and provided for an efficient means of recovering those
targeted cells from the solid support under conditions
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which minimized damage to the cellular structure and/or
molecular integrity.
SUMMARY OF THE INVENTION
The present invention involves a method of
separating and recovering specifically-targeted cells
or structures from mixed cell or molecular structure
populations under conditions which minimize damage to
the cellular structure and/or the molecular structure
and integrity. The method is based upon the specific
interaction of a label and an antibody directed against
the label and the ability of a competing ligand to
compete with the interaction between the label and the
antibody. The method employs a labelled binding
molecule specific for a target molecule or epitope on a
cell or structure, an antibody attached to a solid
support which reacts with the label on the binding
molecule to select the specific cell or structure and a
competitor to the label-antibody interaction to release
the cell or structure from the solid support.
One aspect of the invention is a method of
selection and recovery of components from a mixed
population wherein the desired components are attached
to a labeled molecule which labeled molecule is bound
to an anti-label antibody attached to a solid support
and wherein the label is avidin, streptavidin or biotin
comprising:
(a) reacting said solid support-antibody-label-
component complexes with competitor wherein the
competitor comprises biotin if the label is avidin or
streptavidin and the competitor comprises avidin or
streptavidin if the label is biotin, under conditions
to allow the component-binding molecule-label complexes
to be released from the solid support; and
(b) recovering the component-label complexes.
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Another aspect of the invention is a method of
selection and recovery of components from a mixed
population comprising:
(a) reacting a suspension of the components with a
suspension of labelled binding molecules which binding
molecules are complementary to the selected components
wherein the label is selected from the group consisting
of avidin, streptavidin and biotin, under conditions
which allow the labelled binding molecules to bind to
certain components and form component-binding molecule-
label complexes;
(b) removing the excess labelled binding
molecules;
(c) contacting said component-~inding molecule-
label complexes with a solid support comprising anti-
label antibodies attached to said support wherein the
antibodies are capable of binding to the label, under
conditions to allow the component-binding molecule-
label complexes to bind to the antibodies to form solid
support-antibody-label-binding molecule-component
complexes;
(d) recovering the solid support-antibody-label-
binding molecule-component complexes;
(e) reacting said recovered solid support-
antibody-label-binding molecule-component complexes
with competitor wherein the competitor comprises biotin
if the label is avidin or streptavidin and the
competitor comprises avidin or streptavidin if the
label is biotin, under conditions to allow the
component-binding molecule-label complexes to be
released from the solid support; and
(f) recovering the component-binding molecule-
label complexes.
Another aspect of the invention is a kit for the
selection and recovery of components from a mixed
population, comprising:
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(a) labelled anti-component antibody wherein the
label comprises avidin, streptavidin or biotin;
(b) anti-label antibody bound to a solid support;
and
(c) competitor, wherein the competitor is biotin
if the label is avidin or streptavidin and the
competitor is avidin or streptavidin if the label is
biotin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 illustrates the ability of avidin or
streptavidin to inhibit the interaction between biotin
and an antibody directed against biotin.
FIGURE 2 illustrates the inhibition of the
interaction between biotin and monoclonal anti-biotin
antibody obtained with different concentrations of
avidin at different temperatures.
FIGURE 3 illustrates the inhibition of the
interaction between biotin and monoclonal anti-biotin
antibody obtained with different concentrations of
streptavidin at different temperatures.
FIGURE 4 illustrates a schematic diagram of a
preferred embodiment of the immunoselection technique
of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Briefly, the present invention involves a method
of separating specifically-targeted cell or molecular
structures from mixed cells or molecular structure
populations under mild conditions which minimize damage
to cellular structures and/or molecular integrity. The
method is based upon the differing binding affinities
of an anti-label antibody to a labeled component, and
that of a competing molecule to the label. The method
employs a labelled binding molecule specific for a
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__9__
target molecule or epitope on a cell or structure, an
antibody which reacts with the label on the binding
molecule and a competing molecule as an inhibitor of
the binding molecule-label-antibody interaction.
Definitions
Before discussing the methodology of the present
invention, the following terms will be defined.
The general term "immunoselection" as applied to
cell separation descri~es a number of related
techniques for separation of cells bearing function
related or lineage specific antigenic determinants.
The specificity of the selection process for cell
separation can be conferred by antibody or antibody-
like molecules, haptens or lectins which interact with
specific cell surface antigens or markers.
The "components" can be cells, peptides, sub-
cellular particles, oligosaccharides, or nucleic acids.
The "cells" can be bacterial cells, animal cells
or vegetable cells and cells obtained by genetic
recombination. The term also applies to other
microorganisms and viruses.
The "mixed pDpulation" is any group of components
containing a number of different component types. For
example, the mixed population can be cells of bone
marrow, blood, lymph nodes, spleen, liver or other
tissues and organs. Alternatively, the mixed
population can be a mixture of nucleic acids.
The "target molecule" or "epitope" is a molecule
present on the cell which can be recognized by or bound
with the 'Ibinding molecule". The 'Itarget molecule"
being selected will depend on the cell population to be
selected for, or against. The target molecule may be
antigen (binds to antibody); antibody (binds to
antigen); glycoconjugate (binds to lectin); lectin
(binds to glycoconjugate); substrates, cofactors,
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inhibitors, etc. (binds with enzymes); hormones,
effectors, toxins, etc. (binds with receptors);
~rit;~;rl~, amino acids, sugars, etc. (binds with
transport proteins); hydrophobic sites (lipids or fatty
acids); membranes (liposomes) or nucleic acids or genes
recognized by DNA/RNA probes. In one embodiment, the
"target molecule" is the CD4 antigen which is bound by
the anti-CD4 antibody.
The cells and/or molecular structures can be
selected from the mixed population under conditions
which minimize damage to the cellular structure and/or
molecular integrity by the methods of this invention by
exploiting differences in the specific affinity of
certain molecules for each other. The specific binding
affinity of the label to the competitor must be a high
specific binding affinity in the order of at least
about K, =1o~ In a preferred embodiment, the label
and competitor are biotin and avidin or streptavidin
respectively which have a binding affinity of K~ = 1ol5
~'. Alternatively the label may be avidin or
streptavidin and the competitor may be biotin. The
binding affinity of the antibody-label reaction is a
weaker specific affinity than the affinity between the
label and the competitor. Antibody-antigen reactions
have such a weaker affinity interaction characteristic
of typically K, =1O5 to 10l~ ~. See Immunolo~y: An
Introduction (1984), Editor I.R. Tizard, Saunders
College Publishing. This allows the competitor to
compete with the binding of the labelled binding
molecule to the anti-label antibody, releasing the
component.
The binding affinity of the competitor to the
label must be greater than the binding affinity of the
anti-label antibody to the label. It is contemplated
that the greater the ratio in binding affinity of the
competitor to the label as compared to the binding
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affinity of the anti-label antibody to the label, the
faster the reaction. Preferably the ratio of the
binding affinity of the competitor for the label to the
affinity of the anti-label antibody to the label is at
least about 105 ~, more preferably the binding affinity
is at least about 106 ~'. One skilled in the art could
readily determine the binding affinities of the
molecules.
In a preferred embodiment, the high affinity
constant (K, = 1015 ~l) between the vitamin biotin and
the egg-white protein avidin or the bacterial protein
from Streptomyces avidln~i, streptavidin is exploited.
The "binding molecule" is a molecule which is
capable of recognizing and binding with the target
lS molecule present on the surface of the desired cell or
within the desired molecule. The binding molecule used
will depend on the target molecule on the cell or
structure to be selected. The binding molecule may be
antibody (recognizing antigen target); antigen
(recognizing antibody target); glycoconjugate
(recognizing lectin); lectin (recognizing
glycoconjugate); enzymes (binding substrates,
cofactors, inhibitors, etc.); receptors (hormones,
effectors, toxins, etc.); transport proteins (binding
vitamins, amino acids, sugars, etc.); lipids or fatty
acids (hydrophobic sites); liposomes (membranes) or
DNA/RNA probes recognizing nucleic acids or genes. In
one embodiment the binding molecule is anti-CD4
antibody which recognizes CD4 antigen on the cell
surface.
A binding molecule which has high specific binding
activity for a target molecule is said to be
complementary to the target molecule.
The binding molecule is tagged with a "label". In
a preferred embodiment, the label is either biotin,
avidin or streptavidin. Where the label is biotin, the
,. . . .
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competitor is avidin or streptavidin. On the other
hand, where the label is avidin or streptavidin, the
competitor is biotin.
The binding molecule is labelled by methods known
in the art. The biotin molecule is linked to amino or
carbohydrate residues located on the binding molecule.
Biotin and methods of biotinylation are known. See for
example Hof~man et al. (1977) Proc. Natl . Acad . Sci .
USA 7~:2697-2700 or Berman and Basch, (1980)
"Amplification of the biotin-avidin immunofluorescence
technique~' J. Immunol. Meth. 36:335-338, both of which
are incorporated herein by reference in their entirety.
Alternatively, the binding molecule may be indirectly
labelled by reacting it with a second biotinylated
reagent, selected because it has a specificity for the
binding molecule. For example the second reagent may
be a biotinylated antibody to the binding molecule.
Similarly, avidin or streptavidin may be linked to the
binding molecule by methods known in the art. The
parameters of such linkage are substantially as
described for biotin above.
The "anti-label antibody" is an antibody which is
able to detect and bind to the label. The antibodies
used in the methods of this invention may be polyclonal
or monoclonal antibodies. Preferably the binding
affinity of the antibody to the label is a K, of from
105 ~ to 101~ ~', more preferably the affinity is a K, of
at least about 106 ~' to 10~
The preparation of polyclonal antibodies is known
in the art and many polyclonal antibodies are
commercially availabie. The basic process for making
polyclonal antibodies involves injecting an animal with
an immunogenic substance. In one embodiment, the
substance is biotin, avidin, or streptavidin.
Generally the biotin, avidin or streptavidin is
attached to an antigenic carrier so as to elicit an
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i_mune response. After a suitable time for antibody
production, antibodies are collected and isolated from
the animal.
~ The preparation of monoclonal antibodies is known
in the art and the monoclonal antibodies described in
this invention are publicly available. The basic
process for making~ monoclonal antibodies involves
injecting an animal, usually a mouse with an
immunogenic substance. After suitable time for
antibody production to the immunogen, the mouse is
sacrificed. Cells are removed from the spleen and
fused with myeloma cells. Hybridoma cells resulting
from this fusion are able to reproduce in vltro, and
each expresses genetic information for one specific
antibody. The antibodies produced from one hybridoma
clone thus will recognize a single antigenic
determinant of the i_munogen.
Cells cultured from individual hybridoma cells are
screened for production of antibodies to a determinant
on the target antigen. Those hybridomas positive for
the target antigen are further screened to identify
those having the appropriate affinity. The monoclonal
antibodies used in the present invention will have an
affinity to the target antigen of at least 105-101~ ~'
and preferably at least 106-10~ Ml. Monoclonal
antibodies displaying all of these characteristics are
then screened using actual assay conditions to
determine if the assay condition alters the antibody
binding characteristics or affinity, and to screen out
those with cross-reactivity to possible contaminating
antigens.
In a preferred Pmho~;mDnt the anti-label antibody
is either an anti-biotin antibody, an anti-avidin
antibody or anti-streptavidin antibody. Preferably,
the anti-biotin antibody is monoclonal BN-34 (Sigma,
St. Louis, M0). For example, the anti-avidin antibody
,
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is polyclonal antibody A5170 (Sigma, St. Louis, M0) and
the anti-streptavidin antibody is antibody S6390
(Sisma, St. Louis, M0).
The "competitor" or "competing molecule" is a
molecule which is able to compete with the anti-label
antibody for binding with the labelled binding
molecule. In a preferred embodiment, where the label
is biotin and the anti-label antibody is anti-biotin
antibody, the competitor is preferably avidin or
streptavidin. Where the label is avidin and the anti-
label antibody is anti-avidin antibody, the competitor
is preferably biotin. Finally, where the label is
streptavidin and the anti-label antibody is anti-
streptavidin antibody, the competitor is preferably
biotin.
The "solid phase" is any solid support to which
the anti-label antibody may be bound. For example, the
solid support may be polyacrylamide beads, magnetic
beads, polystyrene, polyurethane, agarose, collagen,
gelatin, Sepharose, Sephadex, Sepharon, nylon, rayon or
glass. Additionally, the solid phase may be animal
erythrocytes in which case the isolated cells could be
separated from the other cells by the rosetting
procedure, known in the art. The solid support can
take various forms, including fibers, mesh, microtiter
plates or tubing and can be housed in other flow-
through devices such as extracorporeal cartridges in
systems for continuously removing selected components.
The components are "recovered" when they are
substantially isolated from the mixed population.
Further the desired components are no longer attached
to the solid phase. The percentage of isolation will
vary depending on the components to be isolated.
Preferably, the recovered components comprise at least
about 70% of the total components in the recovered
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population, more preferably the recovered components
comprise at least about 80% of the total components.
MethodoloqY
The present invention is based upon the
interaction between the label and antibody directed
against the label and the inhibition of this
interaction by the competitor. In a preferred
embodiment, the label is biotin which is attached to
the binding molecule and the anti-label antibody is
anti-biotin antibody and the competitor is avidin or
streptavidin. In another preferred embodiment, the
invention may be practiced by employing an avidin-
labeled binding molecule and antibody directed against
avidin (commercially available from Sigma, St. Louis
MO) and biotin as the competitor to inhibit the
interaction between the avidin and the anti-avidin
antibody.
Briefly, the mixed component suspension is
incubated with labelled binding molecules under
conditions which allow the binding molecule to bind to
the specific desired components. Excess labelled
binding molecules are removed. The component suspension
is then placed in contact with a solid support to which
anti-label antibody has been bound under conditions
which allow the anti-label antibody to bind to the
remaining binding molecules. Unabsorbed components are
washed away from the solid support. The recovery of
component populations which react with the labelled
binding molecule are amplified using the methods of
this invention. The solid support with the absorbed
components is placed in contact with a solution having
the competing molecule. The competitor competes with
the anti-label antibody for binding to the labeled
binding molecule and the specific desired components
,
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are released from the solid support into the solution
and can be recovered.
The methods of this invention are now discussed in
more detail.
The desired component is chosen and the binding
molecule and target molecule selected to allow the
desired component to be selected from the general mixed
population. As discussed above, the binding molecule
and the target molecule can vary depending on the
component desired. In one embodiment, the desired cell
type is CD4 positive lymphocytes; the binding molecule
is anti-CD4 antibody to which a label such as biotin
has been covalently attached; and the target molecule
is CD4.
It is contemplated that the desired component may
also be labelled with a reporter molecule. Any
reporter group which is analytically detectable and
compatible with the selection assay of the present
invention can be used in the methods of this invention.
Examples of such reporter groups include but are not
limited to: enzymes, fluorescent dyes, phosphorescent
dyes, radioisotopes, and electron dense markers.
Enzymes are preferred reporters. The reporter group
can be attached using known methods.
The solid support is prepared such that it is
capable of binding the anti-label antibody in a manner
such that the anti-label antibody is oriented so that
the "active site" or Fab portion of the anti-label
antibody molecule is available to bind to the label-on
the binding molecule. Methods to so bind antibodies to
solid supports are well known in the art including the
covalent and non-covalent attachment of the antibodies
to the support.
In one embodiment anti-mouse IgG (Fc specific)
antibodies are used to attach the monoclonal anti-
biotin antibodies to the glass bead solid support. The
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anti-mouse IgG (Fc specific) antibodies are attached to
the glass beads by physical adsorption. The excess
anti-mouse IgG antibodies are removed and the glass
beads are contacted with mouse anti-biotin antibody.
The anti-mouse IgG antibody binds to the Fc portion of
the mouse anti-biotin antibody such that the Fab
portion of the mouse anti-biotin antibody is free.
Other materials such as protein A or protein G
which also bind to the Fc region of antibody may be
employed. Protein A is first attached to the solid
support and the antibodies of choice are then bound to
the Protein A (Forsgren et al. (1977) J. Immunol .
99:19). The method of attachment of Protein A to the
solid support may proceed by any one of several process
available through the literature (Weetall H. (1976)
Meth . in Enzymol . ~4 :134-148). Protein A attaches to
the Fc portion of IgG subclass antibodies, thus
extending and presenting the Fab portion of these
antibodies. The resulting correct orientation of the
antibodies and extension away from the particles leads
to a very effective interaction between the bound
antibodies and their target.
Chemical procedures such as hydrazide coupling
chemictry (Bio-~ad) which would specifically attach
antibody directly to the solid support via the Fc~
region of the molecule may also be used and provide for
covalent attachment. See O'Shannessy, D.J. and
Hofmann, W.L. (1987) Biotech. Appl. Biochem. 9:488.
The solid support with the anti-label antibody
attached may be treated with a blocking solution to
reduce non-specific binding of components to the solid
support by methods known in the art. See Vogt, R.F.,
et al. (1987). J. Immunol. Methods 101:43.
The binding molecule is labelled by methods known
in the art. The biotin molecule is linked to amino or
carbohydrate residues located on the binding molecule.
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Biotin and methods of biotinylation are known. See for
example Hoffman et al. (1977) Proc. Natl. Acad. Sc1.
USA 7~:2697-2700 or Berman and Basch, (1980)
"Amplification of the biotin-avidin immunofluorescence
technique" J. Immunol . Meth. 36:335-338, both of which
are incorporated herein by reference in their entirety.
Where the binding molecule is an antibody, biotin may
be linked to antibody in a ratio from 1:1 to 1:100
(antibody:biotin). Preferably, biotin may be linked to
an antibody in a ratio of 1:1 to 1:30. Similarly,
avidin or streptavidin may be linked to the binding
molecule by methods known in the art. The parameters
of such linkage are substantially as described for
biotin above including the use of a ratio of antibody:
avidin of 1:1 to 1:100 and preferably 1:1 to 1:30.
The population from which the components are to be
selected, in the form of a solution, is placed in
contact with the labelled binding molecule complex and
incubated for a sufficient period of time and under
conditions sufficient to allow the components having
the selected target molecule to bind to the binding
molecule. The length of time can vary from 20 minutes
to 1 hour, more preferably from 40 minutes to 1 hour.
The concentration of the components will depend on the
type of binding molecule employed in the invention and
the system in use. As an example, however, for CD4 the
concentration of components would be on the order of 5
~g of labelled antibody per 50 x 106 cells. A
sufficient concentration of labelled binding molecule
is added to the components such that the molecules are
in excess. One skilled in the art could readily
determine the conditions necessary to ensure that
sufficient components having the target molecule are
bound to the labelled binding molecule. After a
sufficient period of incubation, the excess labelled
binding molecule is removed. One skilled in the art
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would know how to remove the excess labelled binding
molecules. For example, if the components are ce~ls,
the cells may be centrifuged away from the excess
labelled binding molecules. Alternatively, the eYçPCF
labelled binding molecules may be removed by passage
through a size gradient or gel.
The labelled components are then placed into
contact with the solid support-anti-label antibody
complex and incubated for a sufficient period of time
and under conditions sufficient to allow the component-
binding molecule-label complexes to bind to the anti-
label antibody on the solid support. The length of
time can vary from 10 minutes to 60 minutes, more
preferably from 10 minutes to 15 minutes. The
conditions are preferably neutral pH and isotonic salt.
After a sufficient period of time the component
solution is removed from the solid support.
Those unabsorbed components will be components
which lack the target molecule. The components
absorbed to the solid support will be components having
the target molecule. The solid support may be washed a
number of times to remove the non-specifically bound
molecules which lack the target molecule.
The solid support is then placed in contact
with a solution having the competitor. The competitor
is preferably added in a solution wherein the
competitor is in a molar excess to ensure that most of
the components are released from the solid support.
The solution is preferably of a salt concentration and
pH which is compatible with the components (i.e. it
does not result in denaturation of the components).
The length of time of the incu~ation is sufficient to
allow most of the components to be released from the
solid support. The length of time will vary with the
type of label and competitor used and the specific
binding affinity of the label and the competitor. One
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skilled in the art given this disclosure could readily
determine the conditions required to ensure sufficient
release of the components from the solid support.
Preferably, the time of incubation is from 20 minutes
to 1 hour, more preferably, the amount of time is 40
minutes to 1 hour. The temperature of the reaction will
be that temperature which is compatible with
maintaining the viability of the components to be
selected. Preferably the temperature is from 20~C to
42~C, more preferably from 25~C to 37~C. By the
methods of this invention, the components are removed
from the solid support without significant agitation.
Significant agitation means any agitation other than
the small agitation caused by the movement of
containers from one position to another.
Since the competitor has a greater affinity for
the component-binding molecule complexes than the anti-
label antibody on the support, it allows the release of
the components. The components released from the solid
support are recovered. It is contemplated that the
components may ~e washed with buffer to remove any
excess competitor.
It is further contemplated that if the binding
molecule is an antibody, the labelled binding molecule
2~ may be removed from the desired component by methods
known in the art. For example chaotropic agents and
extremes of p~ may be used. Alternatively, methods
compatible with the components may be used. For
example, if the components are cells, they may be
incubated in culture for a sufficient period of time to
allow the anti-component la~eled antibodies to be shed
into the solution.
It is anticipated that this invention can be
applied to the specific targeting, separation or
quantitation of cells, viruses, yeast and molds,
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bacteria and other microorqanisms and structures as
well as their component parts.
The methods of this invention can be used to
positively select cells for use in therapeutic
purposes. Immature populations of peripheral
lymphocytes can be collected by the methods of this
invention and grown in vitro until maturation in order
to PYr~ their population prior to autologous
reinfusion into the patient. For example, lymphokine
activated killer cells (LAK) for use in cancer therapy
or AIDS therapy, suppressor lymphocytes for treating
thyroid diseases, T4 lymphocytes for treating AIDS,
certain subpopulations af lymphocytes for multiple
sclerosis and certain macrophages for attacking
cholesterol plaques could be obtained by the methods of
this invention. The methods of this invention can also
be employed as a sensitive assay for detectinq cancer
remission or metastases, by monitoring the numbers of
tumor cells in a patient~s bloodstream or bone marrow.
Z0 In order to further illustrate the present
invention and advantages thereof, the following
specific examples are given, it being understood that
the same are intended only as illustrative and in
nowise limitative.
EXAMPLES
Example l: Inhibition of interaction between biotin
and an anti-biotin antibodY bY avidin or stre~tavidin
This example illustrates the ability of avidin or
streptavidin to inhibit the interaction between biotin
and an anti~ody directed against biotin.
Bovine serum albumin (BSA) (Sigma Chemical
Comr~y, St. Louis MO) was biotinylated with N-
hydroxysuccinimidobiotin (Pierce Chemical Company,
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Rockford, IL) using standard procedures (See Berman and
Basch (1980) J. Immunol . Methods 36: 33s-338) ) .
BSA was dialyzed against 0.1 M NaHCO3 (no azide) at
pH 8.2 to 8.6. The BSA concentration was adjusted to 1
mg/ml. N-hydroxysuccinimidobiotin (Pierce No. 20217)
was dissolved in dimethyl sulfoxide (DMSO) to a
concentration of 1 mg/ml. The bottle of N-
hydroxysuccinimidobiotin was warmed to room temperature
before weighing, to prevent condensation. 120 ~l of the
biotin succinimide ester mixture was added to 1 ml of
the dialyzed BSA, mixed immediately, and incubated at
room temperature for 4 hours in the dark. The mixture
was then dialyzed against phosphate buffered saline
(PBS) with azide overnight at room temperature to
remove uncoupled biotin.
Phosphate buffered saline (PBS) is 32g NaCl, 0.8g
KCl, 4.6g Na2HPO~, 0.8g KH2PO4 in 1 liter H~O (pH 7.2).
PBST is PBS with 0.05% Tween 20 (Sigma Chemical
Company, St. Louis, MO).
The resulting biotinylated BSA (8-BSA) was used to
coat the polyvinylchloride wells of enzyme immunoassay
plates (Fisher, Edmonton, AB). The B-BSA was bound to
each well by adding approximately 100 ~1 of a 1:4000
dilution of the B-BSA solution to each well. The
plates were incubated at 4~C temperature in a humid
atmosphere for 16 hours (approximately overnight). The
unbound B-BSA solution was removed by washing the wells
with PBST. The wells were then filled with Blocking
Buffer (20% fetal calf serum (FCS) ~GibcoBRL,
Burlington, Ontario, Canada] in PBS) and incubated at
37~C temperature for 20 minutes. The Bloc~ing Buffer
was removed.
The wells coated with B-BSA were then incu~ated
for 1 hour at 37~C with 100 ~ls (1:8000 dilution) of
mouse monoclonal anti-biotin antibody (monoclonal, BN-
34, Sigma, St. Louis, MO). The excess antibody was
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removed by washing the wells three times with PBST.
Duplicate wells were incubated at 37~C for 1 hour with
increasing concentrations from 0.01 to 100 ~g/ml of the
competitors: avidin, streptavidin (Boehringer Mannheim,
In~i~n~polis IN) or BSA, as a control, diluted in PBS.
The excess competitors were then removed by
washing the wells three times with PBST to remove the
competitor and any eluted anti-biotin antibody.
The relative amount of anti-biotin antibody
remaining bound to the solid phase B-BSA was
quantitated using horseradish peroxidase (HRP)
conjugated anti-mouse IgG (Sigma Chemical Company, St
Louis MO) in standard enzyme immunoassay (EIA) methods.
Specifically, goat anti-mouse IgG-HRP was diluted to
1/5000 in PBST + 1% BSA and 75 ~ls were added to each
well. The wells were then allowed to incubate for 30
minutes at 37~C temperature. The excess IgG-HRP was
removed by washing S times with PBST. Finally, 100 ~ls
of Substrate Buffer (lmg/ml 2',2'-Azino-bis(~-
ethylbenz-thiazoline-6-sulfonic acid), 0.003% H2O2, 44
mM Na~HPO4, and 28 mM citric acid in distilled water)
was added to each well, and allowed to incubate for 30
minutes at room temperature. Positive wells appear
green. The OD reading was determined at 405-490 nm.
Figure 1 shows that increasing concentrations of
either avidin or streptavidin were able to effectively
inhibit the interaction between biotin and the
monoclonal anti-biotin antibody. The amount of
antibody remaining bound to solid phase B-BSA as
detected by standard EIA methods decreased to very low
levels at concentrations of inhibitor approaching 10
~g/ml. Increasing concentrations of the BSA control
had no effect on the biotin-antibody interaction.
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ExamDle 2: Effect of time and temPerature on the
inhibition of the biotin-antibodY interaction
The effect of time and temperature of incubation
~f the competitor on the biotin-antibody interaction
was examined at two concentration levels of avidin and
streptavidin by the method disclosed in Example 1. The
concentrations of avidin used were either 10 ~g/ml or
50 ~g/ml. The concentrations of streptavidin used were
either 1.0 ~g/ml or 5.0 ~g/ml. The period of
incubation of the avidin or streptavidin with the wells
coated with B-BSA and treated with anti-biotin antibody
varied from 0 to 60 minutes. The incubation was
conducted at either room temperature or at 37 ~C.
Figure 2 shows that there was no significant
difference between the inhibition o~tained with 10
~g/ml and 50 ~g/ml concentrations of avidin. However,
increasing the temperature of incubation with avidin
from room temperature to 37~C increased the initial
rate at which antibody was eluted from the B-BSA coated
wells. By 60 minutes the level of antibody remaining
in the wells was the same, regardless of the incubation
temperature or the concentration of avidin used.
The same experiment was performed with 1 ~g/ml and
5 ~g/ml of streptavidin (instead of avidin). The
results are shown in Figure 3. The 5 ~g/ml
concentration of streptavidin proved to be more
effective than the 1 ~g/ml concentration in competing
with the antibody-biotin interaction, both at room
temperature and at 37~C. For both concentrations of
streptavidin, increasing the temperature of incubation
also increased the rate at which antibody was eluted
from the solid phase B-BSA. After 60 minutes of
incubation, differences in concentration of the
streptavidin or the temperature of incubation were no
longer significant, similar to that observed with
avidin.
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It is anticipated that this invention can be
applied to the specific targeting, separation or
quantitation of cells, viruses, yeast and molds,
~acteria and other microorganisms and structures as
wells as their component parts.
ExamDle 3: SeParation of CD4 lYm~hocYtes from human
~eri~heral blood lYm~hocYte Dre~aration
In this Example, CD4 positive lymphocytes will be
separated from human peripheral blood lymphocyte
preparations. During this procedure, 5 ~gs (for SO x
106 cells) of anti-human CD4 antibody (Pharmingen, San
Diego, CA, product no. 301SlA) is biotinylated by the
method described in Example l. The biotinylated anti-
human CD4 antibody is inc~bated with a lymphocyte
preparation isolated from whole human blood using a
Histopaque density gradient (Sigma Chemical Company,
St. Louis, MO) for l hour at 4cC temperature. The
isolated cells are then washed with PBS twice to remove
excess unbound antibody and loaded onto the following
prepared column.
The column is composed of 2 ml glass beads, 80 to
lO0 mesh, to which goat IgG antibodies specific for the
Fc portion of mouse IgG molecules are physically
adsorbed by treating the ~eads with 0.15 mg/ml of goat
IgG antibodies for 24 to 48 hours at 4~C temperature.
The beads are washed 5 times with PBS.
The beads thus coated are then incubated with 20
~l of the mouse monoclonal anti-biotin IgG antibodies
(monoclonal, BN-34; Sigma, St. Louis, MO) per ml of
beads. The antibodies are attached to the glass beads
by interaction of their Fc portion with the anti-IgG
goat antibodies, leaving the anti-biotin combining
sites free to bind biotin. The beads are aqain washed
with PBS and loaded into a 2 ml disposable
polypropylene column (Isolab, Inc., Akron, OH).
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The treated cell population contains CD4 positive
cells coupled to biotin via their specific interaction
with the targeting biotinylated anti-CD4 antibody, as
described above. The 50 x 1o6 cells are loaded in 1 ml
onto the prepared column and are retained on the column
matrix via the interaction between the solid phase
anti-biotin antibody and the biotinylated CD4 antibody
on the cell surface. The column is washed with five to
six volumes of PBS + 2% fetal calf serum to remove
those cells lacking CD4. The positively selected CD4
cells are then eluted from the column by washing with
PPS containing 5 ~gtml of streptavidin.
This same process may also be used to deplete a
cell population of a specific cell type by the process
of negative selection.
The described example uses anti-mouse IgG (Fc
specific) antibodies to attach the monoclonal anti-
biotin antibodies to the solid support but other
materials such as protein A or protein G which also
bind to the Fc region of antibody may be employed.
Chemical procedures such as hydrazide coupling
chemistry (Bio-Rad) which would specifically attach
anti-biotin antibody directly to the solid support via
the Fc region of the molecule may also be used.
While the invention has been described in terms of
various preferred em~odiments, the skilled artisan will
apprec~ate that various modifications, substitutions,
omissions and changes may be made without departing
from the scope thereof. Accordingly, it is intended
that the scope of the present invention be limited
solely by the scope of the following claims, including
equivalents thereof.
