Note: Descriptions are shown in the official language in which they were submitted.
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ANTIBODIES FOR THE DETECTION OF HLA-G
This invention was made with Government support under
5 Grant No. HD-82903, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
A central question in pregnancy is how the fetal-placental
unit avoids maternal immune rejection. Although fetal and materr,al cells
10 interact throughout pregnancy, the fetus typically remains a privileged
site, not subject to rejection. It is likely that the particular nature of the
cells at the fetal-maternal interface and their products help prevent
rejection of the fetus by the maternal immune system.
Implantation and placental development physically connect
15 the mammalian embryo to the maternal uterus. Establishing this
connection is essential for subsequent development. The initial
developmental events which occur in the embryo set aside unique
extraembryonic cellular lineages which are the precursors of the
placenta. The first differentiation event gives rise to trophoblasts, which
20 are specialized epithelial cells of the placenta that physically connect the
embryo and the uterus (See, Cross et~/. (1994), Science 266: 1508 for
a comprehensive review of the events surrounding implantation and
formation of the placenta).
After fertilization in the oviduct, a series of cell divisions
25 create a mass of totipotent cells (the morula). The first differentiation
event occurs after compaction of the morula, leading to formation of the
blastocyst. Cells of the trophoblast lineage are formed based upon their
position in t~e morula in a complex cascade of inter- and intra-cell
signaling events. In primates, implantation of the blastocyst occurs
30 shortly after the blastocyst hatches from the zona pellucida.
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The uterus is made receptive to implantation as a result of
events controlled largely by production of estrogen and progesterone
from the ovaries. During implantation, trophoblasts attach to the
receptive uterine epithelium initiating several changes in the
5 endometrium. Vascular changes occur, such as increased permeability
of uterine blood vessels, and inflammatory cells are recruited to the
implantation site. Proinflammatory cytokines are produced in the uterus
and several cellular changes occur. For example, the uterine epithelium
is lost and decidual cells undergo an epithelioid transition and proliferate,
10 producing a massively thickened uterine wall. The decidua also contains
abundant macrophages, Iymphocytes and other bone-marrow derived
cells with unusual properties such as reduced alloreactivity, and
responsiveness to stimulation by CD3 antibody.
After implantation in humans, distinct populations of
1 5 differentiated trophoblasts form. Proliferative cytotrophoblast stem cells
are anchored to basement membranes surrounding a stromal core in two
types of chorionic villi. In floating villi, cytotrophoblast stem cells
detach from the underlying basement membrane and fuse to form a
syncytium (a. polynucleate cell) which covers the villus and is in direct
20 contact with maternal blood. In anchoring villi, cytotrophoblast stem
cells differentiate by detaching from their basement membrane and
aggregating to form columns of mononuclear cells which attach to and
invade the uterine decidua (interstitial invasionJ and its arterial system
(endovascular invasion). Interstitial invasion puts cytotrophoblasts in
25 direct contact with the highly specialized subset of leukocytes that home
to the uterus during pregnancy. Endovascular invasion puts
cytotrophoblasts (like the syncytiotrophoblasts covering the anchoring
villi) in direct contact with maternal blood. Thus, antigen presentation
by trophoblasts at the maternal-fetal interface is an important
30 component of maternal immunological responses during pregnancy.
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MHC class I molecules and the peptides they present
regulate alloreactivity (Sherman, et al. ( 1 993) , Annu. Rev. Immunol. 1 1:
~ 385). Thus, one key to understanding maternal tolerance of the fetal
semi-allograft lies in studying trophoblast expression of class I
5 molecules. The molecule HLA-G, which is expressed by placental cells,
was cloned in a search for novel class I genes encoded by the human
MHC (Geraghty etal. (1987) Proc. Natl. Acad. Sci. U. S. A. 84: 9145).
The gene has an intron/exon organization identical to that of the class
la genes (HLA-A, -B and -C), and the HLA-G protein product has 86%
10 sequence identity to the class I consensus sequence (Parham et al.
(1988) Proc. Natl. Acad. Sci. U. S. A. 85: 4005). HLA-G has a lower
molecular mass (37-39 kDa) than class la molecules due to a stop codon
in exon 6 that results in the deletion of all but 6 amino acids in the
cytoplasmic tail (Shimizu et a/. (1988) Proc. Natl. Acad. Sci. U. S. A.
15 85: 227). With regard to the 5' flanking region of the gene, the HLA-G
promoter has elements (e.g, AP-1, NFKB) similar to sequences found in
class la genes, but lacks an interferon response element, suggesting
novel transcriptional regulatory mechanisms. The primary HLA-G RNA
transcript is also differentially spliced; in addition to the full length
20 mRNA, transcripts are produced that lack either exon two, both exons
two and three (Ishitani and Geraghty (1992) Proc. Natl. Acad. Sci. U. S.
A. 89: 3947), or exon four (Kirszenbaum et a/. (1994) Proc. Natl. Acad.
Sci. U. S. A, 91: 4209). To what extent these alternatively spliced
mRNAs are translated is unclear. Recently, a soluble form of HLA-G
25 encoded by an mRNA containing intron 4 was described (Fujii et al.
(1994) J. Immunol. 153: 5516).
Whereas HLA-A, -B and -C are highly polymorphic, HLA-G
appears to exhibit relatively less polymorphism. Immunoprecipitation of
HLA-G from 13 individuals and a human choriocarcinoma (malignant
30 trophoblast) cell line showed identical two-dimensional electrophoretic
profiles, suggesting reduced polymorphism at this locus. Genomic and
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cDNA sequehce data also indicate that HLA-G has reiatively limited
polymorphism. However, a recent study suggests that at least in some
populations (i e., African Americans), HLA-G exhibits substantial
polymorphism (van der Ven and Ober (1994) J. Immunol. 153: 5628).
Whether HLA-G is complexed with endogenous trophoblast peptides and
how this repertoire is affected by its degree of polymorphism remains to
be determined.
HLA-G is not generally expressed in non-pregnant adults,
making it a suitable marker for the diagnosis of pregnancy. The present
invention provides antigens for generating specific antibodies to both
soluble and membrane bound HLA-G, as well as exemplar antibodies.
In addition, HLA-G levels in the maternal blood are indicative of the vigor
of cytotrophoblast invasion and the corresponding health of the
placental-maternal interface.
Because HLA-G is not generally expressed in adults, it is an
ideal marker for diagnosing and monitoring pregnancy and for detecting
cytotrophoblasts from biological fluids. However, designing and
obtaining suitable antibodies to HLA-G was not previously feasible, due
to the high similarity of HLA-G to class la molecules which are
expressed in adults. This invention overcomes these problems by
providing specific epitopes for generating HLA-G-specific antibodies,
exemplar antibodies, and methods for their use.
SUMMARY OF THE INVENTION
The present invention provides polypeptides which can be
used to elicit antibodies which specifically bind to HLA-G. These
antibodies are used, for example, to monitor soluble HLA-G levels in
maternal blood. Soluble HLA-G levels in the maternal blood can be used
to diagnose pregnancy, or to monitor the health of the fetal maternal
interface.
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Accordingly, this invention provides purified polypeptides,
comprising a sequence of at least 5 contiguous amino acids selected
from an amino acid sequence consisting essentially of amino acid
residues 61 to 83 of the a1 domain of the human HLA-G protein,
5 wherein said polypeptide, when presented as an immunogen, elicits the
production of an antibody which specifically binds to HLA-G, and
wherein said peptide does not bind to antisera raised against HLA-G
which has been fully immunosorbed with the peptide of Seq. Id. No. 1.
An exemplar polypeptide is the polypeptide which consists or essentially
10 of the sequence EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. ~o. 1).
These polypeptides are useful as components of an immunogenic
composition. In one class of embodiments, the peptide(s) are covalently
linked to additional polypeptides such as an immunogenic carrier (e.g.,
keyhole limpet hemocyanin).
The present invention provides nucleic acids encoding the
polypeptides described above which comprise a sequence of at least 5
contiguous amino acids selected from an amino acid sequence
consisting essentially of amino acid residues 61 to 83 of the a1 domain
of the human HLA-G protein, wherein said polypeptide, when presented
20 as an immunogen, elicits the production of an antibody which
specifically binds to HLA-G, and wherein said peptide does not bind to
antisera raised against HLA-G which has been fully immunosorbed with
the peptide of Seq. Id. No. 1. For instance, in one preferred
embodiment, the present invention encodes a polypeptide substantially
25 identical to the sequence EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No.
1 ) .
Antibodies which specifically bind to a subsequence in the
a1 domain of HLA-G, and methods for making these antibodies are
provided. In one class of embodiments, the antibodies specifically bind
30 to subsequences of the amino acid sequence
EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ) . Exemplar antibodies
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include 1 B8 and 3F6, described herein. Monoclonal as well as
polyclonal antibodies are provided.
Recombinant cells which include nucleic acids encoding the
polypeptides and antibodies described above are also provided.
5 Exemplar cell lines include 1 B8 and 3F6.
A variety of detection formats are also appropriate,
including ELISA, RIA, western blot and other immunoassays.
The invention further provides for kits comprising the
various elements described above.
DEFINITIONS
Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although any
16 methods and materials similar or equivalent to those described herein
can be used in the practice or testing of the present invention, the
preferred methods and materials are described. For purposes of the
present invention, the following terms are defined below.
The term "antibody" refers to a polypeptide substantially
20 encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof. The recognized immunoglobulin genes include the
kappa, lambda, alpha, gamma, delta, epsilon and mu constant region
genes, as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
25 classified as gamma, mu, alpha, delta, or epsilon, which in turn define
the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplar immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical pairs
of polypeptide chains, each pair having one "light" (about 25 kD) and
30 one "heavy" chain (about 50-70 kD). The N-terminus of each chain
defines a variable region of about 100 to 110 or more amino acids
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primarily responsible for antigen recognition. The terms variable light
chain (V,) and variable heavy chain (VH) refer to these light and heavy
chains respectively.
Antibodies exist e.g., as intact immunoglobulins or as a
b 5 number of well characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce F(ab)'2 a
dimer of Fab which itself is a light chain joined to VH CH1 by a disulfide
bond. The F(ab)'2 may be reduced under mild conditions to break the
disulfide linkage in the hinge region thereby converting the F(ab)'2 dimer
into an Fab' monomer. The Fab' monomer is essentially an Fab with
part of the hinge region (see, Fundament~31 Immunology, Third Edition,
W.E. Paul, ed., Raven Press, N.Y. (1993), which is incorporated herein
by reference, for a more detailed description of other antibody
fragments). While various antibody fragments are defined in terms of
the digestion of an intact antibody, one of skill will appreciate that such
Fab' fragments may be synthesized de novo either chemically or by
utilizing recombinant DNA methodology. Thus, the term antibody, as
used herein, also includes antibody fragments either produced by the
modification of whole antibodies or those synthesized de novo using
recombinant DNA methodologies.
The term "biological sample or fluid" refers to material
derived from a living organism, including, e.g., blood, cervicovaginal
secretions, amniotic fluid, cord blood, urine, tissues, bones and cells.
The term "blood sample" as used herein includes whole
blood or derivatives of whole blood well known to those of skill in the
art. Thus a blood sample includes the various fractionated forms of
blood such as plasma or serum and whole or fractionated blood which
additionally comprises various diluents as may be added to facilitate
storage or processing in a particular assay. Such diluents are well
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known to those of skill in the art and include various buffers,
anticoagulants, preservatives and the like.
.The term "HLA-G" refers to human leukocyte antigen G and
unless otherwise stated includes both the soluble and insoluble forms.
5 The term may in appropriate context refer to either the antigen or the
genetic locus.
The term "immunoassay" is an assay that utilizes an
antibody to specifically bind an analyte. The immunoassay is
characterized by the use of specific binding properties of a particular
10 antibody to isolate, target, or quantify the analyte.
The terms "isolated" "purified" or "biologically pure" refer
to material which is substantially or essentially free from components
which normally accompany it as found in its native state.
The term "nucleic acid" refers to a deoxyribonucleotide or
15 ribonucleotide polymer in either single- or double-stranded form, and
unless otherwise limited, encompasses known analogues of natural
nucleotides that can function in a similar manner as naturally occurring
nucleotides.
The term "nucleic acid probe" refers to a molecule which
20 binds to a specific sequence or subsequence of a nucleic acid. A probe
is preferably a nucleic acid which binds through complementary base
pairing to the full sequence or to a subsequence of a target nucleic acid.
It will be understood by one of skill in the art that probes may bind
target sequences lacking complete complementarity with the probe
25 sequence depending upon the stringency of the hybridization conditions.
The probes are preferably directly labelled as with isotopes,
chromophores, lumiphores, chromogens, or indirectly labelled such as
with biotin to which a streptavidin complex may later bind. By assaying
for the presence or absence of the probe, one can detect the presence
30 or absence of the select sequence or subsequence.
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The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. The
terms apply to amino acid polymers in which one or more amino acid
residues is an artificial chemical analogue of a corresponding naturally
5 occurring amino acid, as well as to naturally occurring amino acid
polymers.
A "label" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include 32p, fluorescent dyes, electron-dense
10 reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
dioxigenin, or haptens and proteins for which antisera or monoclonal
antibodies are available (e.g., the peptide
EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ) can be made
detectible, e.g., by incorporating a radiolabel into the peptide, and used
15 to detect antibodies specifically reactive with the peptide).
A "labeled nucleic acid probe" is a nucleic acid probe that
is bound, either covalently, through a linker, or through ionic, van der
Waals or hydrogen bonds to a label such that the presence of the probe
may be detected by detecting the presence of the label bound to the
20 probe.
The term "recombinant" when used with reference to a cell
indicates that the cell contains nucleic acid with an origin exogenous to
the cell. Thus, for example, recombinant cells replicate and/or express
genes that are not found within the native (non-recombinant) form of the
25 cell.
The term "identical" in the context of two nucleic acid or
polypeptide sequences refers to the residues in the two sequences
which are the same when aligned for maximum correspondence.
Optimal alignment of sequences for comparison can be conducted, e.g.,
30 by the local homology algorithm of Smith and Waterman Adv. Appl.
Math. 2: 482 t 198 1) , by the homology alignment algorithm of
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Needleman and Wunsch J Mo/. Biol 48:443 (1970), by the search for
similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)
85: 2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFlT, FASTA, and TFASTA in the Wisconsin Genetics
5 Software Package, Genetics Computer Group, 575 Science Dr.,
Madison, Wl), or by inspection. These references are incorporated
herein by reference.
The term "substantial identity" or "substantial similarity" in
the context of a polypeptide indicates that a polypeptide comprises a
10 sequence with at least 80% sequence identity to a reference sequence,
or preferably 90%, or more preferably 95% sequence identity to the
reference sequence, over a comparison window of about 20 amino acid
residues. An indication thattwo polypeptide sequences are substantially
identical is that one peptide is immunologically reactive with antibodies
15 raised against the second peptide. Thus, a polypeptide is substantially
identical to a second polypeptide where the two peptides differ only by
a conservative substitution.
An indication that two nucleic acid sequences are
substantially identical is that the polypeptide which the first nucleic acid
20 encodes is immunologically cross reactive with the polypeptide encoded
by the second nucleic acid.
Another indication that two nucleic acid sequences are
substantially.identical is that the two molecules hybridize to each other
under stringent conditions. Stringent conditions are sequence
25 dependent and will be different with different environmental parameters.
Generally, stringent conditions are selected to be about 5~ C to 20~C
lower than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength and pH. The Tm is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence hybridizes
30 to a perfectly matched probe. However, nucleic acids which do not
hybridize to each other under stringent conditions are still substantially
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identical if the polypeptides which they encode are substantially
identical. This may occur, e g, when a copy of a nucleic acid is created
using the maximum codon degeneracy permitted by the genetic code.
The phrases "specifically binds to" or "specifically
5 hybridizes to" or "specifically immunoreactive with", when referring to
an antibody refers to a binding reaction which is determinative of the
presence of the protein in the presence of a heterogeneous population
of proteins and other biologics. Thus, under designated immunoassay
conditions, the specified antibodies bind preferentially to a particular
10 protein and do not bind in a significant amount to other proteins present
in the sample. Specific binding to a protein under such conditions
requires an antibody that is selected for its specificity for a particular
protein. For example, antibodies can be raised to the peptide
EEETRNTKAI:IAQTDRMNLQTLRG (Seq. Id. No. 1 ) which specifically bind
15 to proteins comprising the sequence EEETRNTKAHAQTDRMNLQTLRG
(Seq. Id. No. 1 ) (such as HLA-G) and not to other proteins present in a
blood sample. A variety of immunoassay formats are appropriate for
selecting antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
20 select monoclonal antibodies specifically immunoreactive with a protein.
See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold
Spring Harbor Publications, New York, for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity.
Note Regarding Nomenclature: A particular antibody and the cell
which produces the antibody are often referred to by the same
designation. For instance, the monoclonal antibody 1 B8 is produced by
the immortalized cell line 1 B8.
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DETAILED DESCRIPTION
Human placental trophoblasts lie at the maternal-fetal
interface, mediating maternal tolerance of the fetus. Central to this
mediation is their unusual MHC class I expression; they suppress class
la production while expressing HLA-G, a class Ib molecule.
A synthetic peptide corresponding to the region from amino
acids 61 to 83 of the a1 domain of the HLA-G protein was used to
produce monoclonal antibodies which specifically bound HLA-G. The
epitope has the advantage of being a linear epitope rather than a
conformational epitope. It is available to the antibody without
denaturation. It is highly antigenic and produces high titer, high avidity
antibodies that detect all forms of HLA-G. Prior to the present invention,
there was no way of knowing whether the synthetic peptide would be
specific to HLA-G, or whether the conformation of native HLA-G would
permit antibody binding to this region. Thus, prior to the present
invention, there was no way to know whether antibodies to the
synthetic peptide could be generated, or whether any antibodies which
were generated would bind to HLA-G. The present invention
demonstrates that antibodies are generated to the peptide corresponding
to the region from amino acids 61 to 83 of the a~1 domain of HLA-G, and
that these antibodies specifically bind to HLA-G.
Antibody specificity was demonstrated by immunoaffinity
purification of HLA-G from choriocarcinoma cells. These antibodies
were incubated with tissue sections of the maternal-fetai interface
containing cytotrophoblasts in all stages of differentiation, demonstrating
that HLA-G is expressed only by cytotrophoblasts which invade the
uterus. In vitro studies presented herein show that when early-gestation
cytotrophoblast stem cells are cultured they differentiate rapidly along
the invasive pathway, upregulating HLA-G production.
Cytotrophoblasts from term placentas, which have reduced
invasive capacity in vitro, also had decreased ability to upregulate HLA-G
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protein expression in vitro. High levels of HLA-G mRNA were detected
in cytotrophoblasts isolated from first and second trimester placentas
~ that had high invasive capacity. In comparison, term cells contained a
greatly reduced level of HLA-G mRNA. Taken together, these results
5 show that HLA-G production is a component of cytotrophoblast
differentiation along the invasive pathway.
This invention provides methods and compositions for the
generation and use of antibodies which specifically recognize HLA-G.
These antibodies were used to measure trophoblast HLA-G expression
10 in vivo and in vitro. The results showed that HLA-G production is
upregulated as an integral part of cytotrophoblast differentiation.
General Tech"i.~./es
Cloning, PCR, LCR, TAS, 3SR, And QB Amplification
The present invention can be used in conjunction with other
techniques such as PCR, TAS, 3SR, QB amplification and cloning, to
amplify a any nucleic acid in a biological sample. The nucleic acids of
the present invention encode the region of the a1 domain of HLA-G
which is specific to HLA-G, i.e, that region of the o1 domain which
20 exhibits the least similarity to other class I molecules. In preferred
embodiments, the flanking regions of nucleic acid which encodes amino
acid residues 61 to 83 of the a1 domain of the HLA-G protein can be
used as a primer binding site for amplification. The products can be
used in combination with conventional expression systems to generate
25 the peptide of Seq. ID. No. 1 for purposes of obtaining antigen for
antibody production.
More particularly, the nucleic acids of the present invention
may be cloned, or amplified by in vifro methods, such as the polymerase
chain reaction (PCR), the ligase chain reaction (LCR), the transcription-
30 based amplification system (TAS), the self-sustained sequence
replication system (3SR) and the Q,l~ replicase amplification system (QB) .
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14
A wide variety of in vitro amplification methodologies, cloning and
expression systems are known to persons of skill. Examples of these
techniques and instructions sufficient to direct persons of skill through
many cloning exercises may be found in Berger and Kimmel, Guide fo
Molecular Cloning Techniques, Methods in Enzymology 152 Academic
Press, Inc., San Diego, CA (Berger); Sambrook ef a/. (1989) Molecular
Cloning - A Laboratory M~nual (2nd ed.) Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); Current
Protocols in Molecular Biology, F.M. Ausubel et al., eds. (Current
Protocols, a joint venture between Greene Publishing Associates, Inc.
and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion et
al., U.S. patent number 5,017,478; and Carr, European Patent No.
0,246,864. Examples of techniques sufficient to direct persons of skill
through in vitro amplification methods may be found in Berger,
Sambrook, and Ausubel, as well as Mullis et al ., (1987) U . S. Patent No .
4,683,202; PCR Protocols A Guide to Methods and Applications (Innis
et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim
& Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH
Research (1991) 3, 81-94; (Kwoh, et al. (1989) Proc. N~tl. Acad. Sci.
USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87,
1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al.,
(1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,
291 -294; Wu and Wallace, (1989) Gene 4, 560, and Barringer et
al ., (1990) Gene 89, 117.
Synthetic or chemical means to produce the peptides of
Seq. ID. No.1 are also well known. Automatic peptide synthesizers are
available commercially from a variety of sources.
Antibodies to HLA-G
Antibodies may be raised to the polypeptides of the present
invention including individual, allelic, strain, or species variants, and
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fragments thereof, both in their naturally occurring (full-length) forms
and in recornbinant forms. Additionally, antibodies can be raised to
- these polypeptides in either their native configurations or in non-native
configurations. Anti-idiotypic antibodies may also be used. Many
5 methods of making antibodies are known to persons of skill. The
following discussion is presented as a general overview of the
techniques available; however, one of skill will recognize that many
variations upon the following methods are known.
a. Antibody Production
A number of immunogens may be used to produce
antibodies specifically reactive with HLA-G polypeptides. Recombinant
or synthetic polypeptides comprising the region from amino acid residues
61 to 83 of the a1 domain of the human HLA-G protein
15 (EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1)) are the preferred
polypeptide immunogen for the production of monoclonal or polyclonal
antibodies. However, the entire HLA-G moiety, or a subsequence
thereof (especially the a1 domain of HLA-G; see, Geraghty etal, (1987)
Proc, Natl, Acad. Sci, USA 84: 9145) may be used as an antigen, and
20 the resulting antibodies may be screened for HLA-G specificity in the
various competitive and non-competitive binding assays described
herein, particularly using antibodies generated against the peptide
EEETRNTKAHAQTDRMNLQTLRG (Seq. Id No 1 ) as competitors.
Naturally occurring polypeptides may also be used either in pure or
25 impure form.
Polypeptides are expressed in eukaryotic or prokaryotic
cells or chemically synthesized and purified using standard techniques.
The polypeptide, or a synthetic version thereof, is injected into an animal
capable of producing antibodies Either monoclonal or polyclonal
30 antibodies may be generated for subsequent use in immunoassays to
measure the presence and quantity of the polypeptide.
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Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a purified
polypeptide, a polypeptide coupled to an appropriate carrier (e.g,
keyhole limpet hemanocyanin), or a polypeptide incorporated into an
5 immunization vector such as a recombinant vaccinia virus (see, U.S.
Patent No. 4,722,848) is mixed with an adjuvant and animals are
immunized with the mixture. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the polypeptide of interest. When
10 appropriately high titers of antibody to the immunogen are obtained,
blood is collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to the
polypeptide is performed where desired. See, e.g., Coligan ( 1 991 )
Current Protocols in Immunology Wiley/Greene, NY; and Harlow and
1 5 Lane ( 1 989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,
NY, which are incorporated herein by reference, and the examples
below.
Antibodies, including binding fragments and single chain
recombinant versions thereof, against predetermined fragments of
20 polypeptides can be raised by immunization of animals with conjugates
of the fragments with carrier proteins as described above. Typically, the
immunogen of interest is a peptide of at least about 3 amino acids, and
more typically the peptide is 5 amino acids in length or greater. The
peptides are typically coupled to a carrier protein, or are recombinantly
25 expressed in an immunization vector. Antigenic determinants on
peptides to which antibodies bind are typically 3 to 10 amino acids in
length.
Monoclonal antibodies are prepared from cells secreting the
desired antibody. These antibodies can be screened for binding to
30 normal or modified polypeptides, or screened for agonistic or
antagonistic activity, e.g., activity mediated through membrane-bound
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HLA-G. Specific monoclonal and polyclonal antibodies will usually bind
with a KD of at least about 500 ,uM, and most preferably at least about
1 ~M or better.
In some instances, it is desirable to prepare monoclonal
~; antibodies from various mammalian hosts, such as mice, rodents,
primates, etc. Description of techniques for preparing such monoclonal
antibodies may be found in, e.g., Stites et ~/. (eds.) B~s;c and Clinic~l
Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and
references cited therein; Harlow and Lane, Supr~; Goding ( 1 986)
1 0 MonoclonalAntibodies: Principles and Practice (2d ed . ) Academic Press,
New York, NY; and Kohler and Milstein (1975) Nature 256: 4~5-497.
Summarized briefly, this method involves injecting an animal with an
immunogen. The animal is then sacrificed and cells are taken from its
spleen, which are then fused with myeloma cells. The result is a hybrid
15 cell or "hybridoma" that is capable of reproducing in vitro. The
population of hybridomas is then screened to isolate individual clones,
each of which secrete a single antibody species to the immunogen. In
this manner, the individual antibody species obtained are the products
of immortalized and cloned single B cells from the immune animal
20 generated in response to a specific site recognized on the immunogenic
substance.
Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses, or
other methods known in the art. Colonies arising from single
25 immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells is enhanced by various
techniques, including injection into the peritoneal cavity of a vertebrate
host. The polypeptides and antibodies of the present invention are used
30 with or without modification, and include chimeric antibodies such as
humanized murine antibodies.
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Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors. See, Huse et al.
(1989) "Generation of a Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage Lambda, " Science 246: 1275-1281;
and Ward, eta/. (1989) Nature 341: 544-546.
Frequently, the polypeptides and antibodies will be labeled
by joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in both
the scientific and patent literature. Suitable labels include
radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent
moieties, chemiluminescent moieties, magnetic particles, and the like.
Patents teaching the use of such labels include U.S. Patent Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;
and 4,366,241. Also, recombinant immunoglobulins may be produced.
See, Cabilly, U.S. Patent No. 4,816,567; and Queen etal. (1989) Proc.
Nat'lAcad. Sci. USA 86: 10029-10033.
The antibodies of this invention can also be used for affinity
chromatography to isolate HLA-G polypeptides. Columns can be
prepared, e.g., with the antibodies linked to a solid support, e.g.,
particles, such as agarose, Sephadex, or the like, where a cell Iysate is
passed through the column, washed, and treated with increasing
concentrations of a mild denaturant, whereby purified HLA-G
polypeptides are released.
The antibodies can be used to screen expression libraries
for particular expression products such as HLA-G or for histology studies
to locate HLA-G expressing cells. Usually the antibodies in such a
procedure will be labeled with a moiety allowing easy detection of
presence of antigen by antibody binding.
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19
b. Immuno~ss~ys
Concentration of HLA-G in a biological sample can be
~ measured by a variety of immunoassay methods. For a review of
immunological and immunoassay procedures in general, see Stites and
5 Terr (eds.) 199 1 Basic and Clinica/ /mmuno/ogy (7th ed.) . Furthermore,
the immunoassays of the present invention can be performed in any of
several configurations, e.g., those reviewed in Maggio (ed.) ( 1980)
Enzyme Immunoass~y CRC Press, Boca Raton, Florida; Tijan (1985)
" Practice and Theory of Enzyme Immunoassays, " Laborafory Techniques
10 in Biochemistryand Mo/ecu/arBio/ogy, Elsevier Science Publishers B.V.,
Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) /mmunoassay:
A Practical Guide Academic Press, Orlando, FL; Price and Newman
(eds.) ( 1 991 ) Princip/es and Practice of Immunoassays Stockton Press,
NY; and Ngo (ed.) (1988) Non-isotopic /mmunoassays Plenum Press,
15 NY.
Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the capture
agent and the analyte. The labeling agent may itself be one of the
moieties comprising the antibody/analyte complex. Thus, the labeling
20 agent may be a labeled HLA-G peptide or a labeled anti-HLA-G antibody.
Alternatively, the labeling agent may be a third moiety, such as another
antibody, that specifically binds to the antibody/HLA-G complex, or to
a modified capture group (e.g., biotin) which is covalently linked to the
HLA-G peptide or anti-HLA-G antibody.
2~ In a preferred embodiment, the labeling agent is an antibody
that specifically binds to the capture agent (anti-HLA-G). Such agents
are well known to those of skill in the art, and most typically comprise
labeled antibodies that specifically bind antibodies of the particular
animal species from which the capture agent is derived. Thus, for
30 example, where the capture agent is a mouse derived anti-human HLA-G
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antibody, the label agent may be a goat anti-mouse IgG, i. e., an
antibody specific to the constant region of the mouse antibody.
Other proteins capable of specifically binding
immunoglobulin constant regions, such as streptococcal protein A or
protein G may also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They exhibit a
strong non-immunogenic reactivity with immunoglobulin constant
regions from a variety of species. See, generally Kronval, et a/., J.
Immunol., 1 1 1 :1 401-1406 (1973), and Akerstrom, et al., J. /mmunol.,
135:2589-2542 (1985).
Throughout the assays, incubation and/or washing steps
may be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from about
5 minutes to about 24 hours. However, the incubation time will depend
15 upon the assay format, analyte, volume of solution, concentrations, and
the like. Usually, the assays will be carried out at ambient temperature,
although they can be conducted over a range of temperatures, such as
5~C to 45~C.
(i) Non-Competitive Assay Formats
Immunoassays for detecting HLA-G may be either
competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of captured analyte (in this case HLA-G) is
directly measured. In one preferred "sandwich" assay, for example, the
25 capture agent (anti-HLA-G antibodies) can be bound directly to a solid
substrate where they are immobilized. These immobilized antibodies
then capture HLA-G present in the test sample. The HLA-G thus
immobilized is then bound by a labeling agent, such as a second HLA-G
antibody bearing a label. Alternatively, the second HLA-G antibody may
30 lack a label, but it may, in turn, be bound by a labeled third antibody
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specific to antibodies of the species from which the second antibody is
derived.
Sandwich assays for detecting and/or quanlil~Lir,g HLA-G
may be constructed. As described above, immobilized anti-HLA-G
5 specifically binds to HLA-G present in the sample via the epitope defined
by Seq. ID. No. 1. Then the second labeled anti-HLA-G (not necessarily
HLA-G specific) binds to the bound HLA-G molecule. Free labeled
antibody is then washed away and the remaining bound labeled anti-
HLA-G is detected (e g., using a gamma detector where the label is
1 0 radioactive).
(ii) Competitive Assay Formats
In competitive assays, the amount of analyte (HLA-G)
present in the sample is measured indirectly by measuring the amount
15 of an added (exogenous) analyte displaced (or competed away) from a
capture agent (anti HLA-G antibody) by the analyte present in the
sample. In one competitive assay, a known amount of analyte is added
to the sample and the sample is contacted with a capture agent, in this
case an antibody that specifically binds the analyte. The amount of
20 analyte bound to the antibody is inversely proportional to the
concentration of analyte present in the sample.
In a particularly preferred embodiment, the capture agent
is immobilized on a solid substrate. The amount of HLA-G bound to the
capture agent may be determined either by measuring the amount of
25 HLA-G present in an HLA-G/antibody complex, or alternatively by
measuring the amount of remaining uncomplexed HLA-G. The amount
of HLA-G may be detected by providing a labeled HLA-G.
A hapten inhibition assay is another preferred competitive
assay. In this assay a known analyte, in this case HLA-G is immobilized
30 on a solid substrate. A known amount of anti-HLA-G antibody is added
to the sample, and the sample is then contacted with the immobilized
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HLA-G. In this case, the amount of anti-HLA-G antibody bound to the
immobilized HLA-G is proportional to the amount of HLA-G present in
the sample. Again the amount of immobilized antibody may be
measured by detecting either the immobilized fraction of antibody or the
5 fraction of the antibody that remains in solution. Detection may be
direct where the antibody is labeled or indirect by the subsequent
addition of a labeled moiety that specifically binds to the antibody as
described above.
10 Assays for HLA-G Pepfides
HLA-G peptides may be defined by their binding properties
to antibodies and antisera. As described in the definitions section
herein, one indication that two peptides are substantially similar is that
they both specifically bind to the same antibody or antibodies.
15 Conversely, if peptides are substantially similar, one peptide is not
specifically bound by an antibody which does not specifically bind the
second peptide. Thus, the peptides of the present invention can be
defined by their binding properties in the various immunoassays
described herein. For instance, antisera raised against HLA-G which is
20 immunosorbed with the peptide of Seq. Id. No.
(EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 )) until the antisera no
longer binds to the peptide of Seq. Id. No. 1 will not react with a peptide
which is substantially identical to the peptide of Seq. Id. No. 1. Antisera
which has been immunosorbed with the peptide of Seq. Id. No. 1 until
25 it no longer binds specifically to the peptide of Seq. Id. No. 1 is said to
be "fully immunosorbed."
In order to produce antisera for use in these immunoassays,
HLA-G is used to immunize mice from an inbred strain, such as BALB/c,
using a standard adjuvant, such as Freund's adjuvant, and a standard
30 mouse immunization protocol (see Harlow and Lane, supra and the
procedures described herein). Alternatively, a synthetic peptide derived
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from HLA-G and conjugated to a carrier protein or a peptide vector
containing the sequence can be used an immunogen. Polyclonal sera
- are collected and titered against the immunogen protein in an
immunoassay as described herein, for example, a solid phase
5 immunoassay with the immunogen (e.g., HLA-G synthetic peptide)
immobilized on a solid support. Polyclonal antisera with a titer of 1O4 or
greater are selected and tested for their ability to bind HLA-G, using a
competitive binding immunoassay as described above, e.g., using
generic competitors which are unrelated to HLA-G (e.g., bovine serum
10 albumin). Antisera which bind HLA-G are then selected for
characterization of a peptide of interest, i.e., one which may be
substantially identical to the peptide of SEQ Id. No. 1.
Immunoassays in the competitive binding format are then
used for cross-reactivity determinations between the peptide of Seq. Id.
15 No. 1 and a target peptide. For example, the protein of Seq Id No. 1
can be immobilized to a solid support and used to isolate antibodies
which specifically bind to the peptide of Seq. Id. No. 1 from the
antisera. Thç ability of a target protein to bind the antibodies isolated
from the pooled antisera, and to the pooled antisera which has been
20 stripped of antibodies which bind to the peptide of Seq. Id. No. 1 is then
compared to that observed for the peptide of Seq. Id. No. 1. Where the
target polypeptide and the peptide of sequence Id. No. 1 have an affinity
for both the immunoabsorbed polyclonal antisera (the antisera which
does not specifically bind to the peptide of Seq. Id. No. 1) and the
25 antibodies isolated during the immunoabsorbtion process (the antisera
which does bind the peptide of Seq. Id. No. 1 ) which is the same within
the experimental error of the system, plus or minus 10%, the
polypeptides are substantially identical. The experimental error of the
system is monitored by using identical polypeptides as controls from one
30 experiment to the next, i. e., identical polypeptides have the same
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24
antibody binding properties, and any discrepancies reflect the
experimental error in the system.
Assays for ~;oll~h/Q HLA-G
A. Sample Co/lection ~nd Processing
Soluble HLA-G is preferably quantified in a biological sample
derived from a patient. Particularly preferred biological samples include
blood and cervicovaginal secretions. U.S. Patent No. 5,096,830
describes cervicovaginal secretions as diagnostic assay sampies, and
provides means for taking such samples.
In a preferred embodiment, HLA-G is quantified in whole blood or
blood derivatives such as blood plasma or blood serum. Blood samples
are isolated from a patient according to standard methods well known
to those of skill in the art, most typically by venipuncture.
The sample may be pretreated as necessary by dilution in
an appropriate buffer solution or concentrated, if desired. Any of a
number of standard aqueous buffer solutions, employing one of a variety
of buffers, such as phosphate, Tris, or the like, at physiological pH can
be used.
B. Quantific~tion of HLA-G.
HLA-G may be detected and quantified by any of a number
of means well known to those of skill in the art. These include analytic
biochemical methods such as electrophoresis, capillary electrophoresis,
high performance liquid chromatography (HPLC), thin layer
chromatography (TLC), hyperdiffusion chromatography, and the like, and
various immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassays (RIA), enzyme-linked immunosorbent assays
(ELlSAs), immunofluorescent assays, and the like.
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Where levels of soluble HLA-G in the biological sample
(e.g., sera) are low (e.g., 1-100 nM) radio immunoassays (RlAs) and
~ capillary electrophoresis are preferred methods of monitoring the level
of soluble HLA-G. In one class of embodiments, the biological sample
5 is enriched for soluble HLA-G, e g., by removing non-HLA-G components
prior to performing the assay. It is of~en desirable to fractionate the
biological sample prior to performing the above-described techniques to
increase the sensitivity of any of the assays above for the detection of
soluble HLA-G. For instance, an initial rough separation of soluble HLA-
10 G from other biological components can be performed by appropriatecentrifugation, filtration, column chromatography, or isotonic washing
of the biological sample. In addition, certain non-HLA-G components of
the biologica~ sample can be specifically removed from the sample using
the techniques described herein, where capture agents specific for non-
15 HLA-G components are used to remove those non-HLA-G components
from the sample. For instance, erythrocytes can be specifically removed
from a biological sample comprising blood by, e.g, immunoadsorbtion or
affinity chromatography.
C. Reduction of Non-Specific Binding
One of skill in the art will appreciate that it is often
desirable to reduce non-specific binding in immunoassays and analyte
purification. Where the assay involves an antigen, antibody, or other
capture agent immobilized on a solid substrate, it is desirable to minimize
the amount of non-specific binding to the substrate. Means of reducing
such non-specific binding are well known to those of skill in the art.
Typically, this involves coating the substrate with a proteinaceous
composition. In particular, protein compositions such as bovine serum
albumin (BSA), nonfat powdered milk, and gelatin are widely used.
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26
D. OtherAssayFormats
Western blot analysis can also be used to detect and
quantify the presence of HLA-G in the sample. The technique generally
comprises separating sample proteins by gel electrophoresis on the basis
5 of molecular weight, transferring the separated proteins to a suitable
solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized
nylon filter), and incubating the sample with the antibodies that
specifically bind HLA-G. The anti-HLA-G antibodies specifically bind to
HLA-G on the solid support. These antibodies may be directly labeled
10 or alternatively may be subsequently detected using labeled antibodies
(e.g., labeled sheep anti-mouse antibodies where the antibody to HLA-G
is a murine antibody) that specifically bind to the anti-HLA-G.
Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
15 antibodies) and release encapsulated reagents or markers. The released
chemicals are then detected according to standard techniques (see,
Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)), which is
incorporated herein by reference.
E. Labels
The particular label or detectable group used in the assay
is not a critical aspect of the invention, so long as it does not
significantly interfere with the specific binding of the antibody used in
the assay. The detectable group can be any material having a
detectable physical or chemical property. Such detectable labels have
been well-developed in the field of immunoassays and, in general, most
labels useful in such methods can be applied to the present invention.
Thus, a label is any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Useful labels in the present invention include magnetic
beads (e.g. DynabeadsTM), fluorescent dyes (e.g., fluorescein
CA 02213620 1997-08-22
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isothiocyanate, texas red, rhodamine, and the like), radiolabels (e.g., 3H,
1251, 35S, 14C, or 32p)l enzymes (e.g., horseradish peroxidase, alkaline
~ phosphatase and others commonly used in an E~ISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g. polystyrene,
5 polypropylene, latex, etc.) beads.
The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in the
art. As indicated above, a wide variety of labels may be used, with the
choice of label depending on sensitivity required, ease of conjugation of
10 the compound, stability requirements, available instrumentation, and
disposal provisions.
Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to the
molecule. The ligand then binds to an anti-ligand (e.g., streptavidin)
15 molecule which is either inherently detectable or covalently bound to a
signal system, such as a detectable enzyme, a fluorescent compound,
or a chemiluminescent compound. A number of ligands and anti-ligands
can be used. Where a ligand has a natural anti-ligand, for example,
biotin, thyroxine, and cortisol, it can be used in conjunction with the
20 labeled, naturally occurring anti-ligands. Alternatively, any haptenic or
antigenic compound can be used in combination with an antibody.
The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be hydrolases,
25 particularly phosphatases, esterases and glycosidases, or
oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, etc. Chemiluminescent compounds include
luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review
30 of various labelling or signal producing systems which may be used, see,
U.S. Patent No. 4,391,904, which is incorporated herein by reference.
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28
Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive label,
means for detection include a scintillation counter or photographic film
as in autoradiography. Where the label is a fluorescent label, it may be
detected by exciting the fluorochrome with the appropriate wavelength
of light and detecting the resulting fluorescence, e.g., by microscopy,
visual inspection, via photographic film, by the use of electronic
detectors such as charge coupled devices (CCDs) or photomultipliers
and the like. Similarly, enzymatic labels may be detected by providing
10 the appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally, simple colorimetric labels may be detected
simply by observing the color associated with the label. Thus, in various
dipstick assays, conjugated gold often appears pink, while various
conjugated beads appear the color of the bead.
Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to detect
the presence of the target antibodies. In this case, antigen-coated
particles are agglutinated by samples comprising the target antibodies.
In this format, none of the components need be labeled and the
20 presence of the target antibody is detected by simple visual inspection.
F. Substr~tes
As mentioned above, depending upon the assay, various
components, including the antigen, target antibody, or anti-idiotypic
25 antibody, may be bound to a solid surface. Many methods for
immobilizing biomolecules to a variety of solid surfaces are known in the
art. For instance, the solid surface may be a membrane (e.g.,
nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, or
polystyrene), a test tube (glass or plastic), a dipstick (e.g. glass, PVC,
30 polypropylene, polystyrene, latex, and the like), a microcentrifuge tube,
or a glass, silica, plastic, metallic or polymer bead. The desired
CA 022l3620 l997-08-22
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29
component may be covalently bound, or noncovalently attached through
nonspecific bonding.
~ A wide variety of organic and inorganic polymers, bothnatural and synthetic may be empioyed as the material for the solid
5 surface. Illustrative polymers include polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene
difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose
acetate, nitrocellulose, and the like. Other materials which may be
10 employed, include paper, glasses, ceramics, metals, me~alloids,
semiconductive materials, cements or the like. In addition, are included
substances that form gels, such as proteins (e.g., gelatins),
lipopolysaccharides, silicates, agarose and polyacrylamides can be used.
Polymers which form several aqueous phases, such as dextrans,
15 polyalkylene glycols or surfactants, such as phospholipids, long chain
( 12-24 carbon atoms) alkyl ammonium salts and the like are also
suitable. Where the solid surface is porous, various pore sizes may be
employed depending upon the nature of the system.
In preparing the surface, a plurality of different materials
20 may be employed, e.g., as laminates, to obtain various properties. For
example, protein coatings, such as gelatin can be used to avoid non-
specific binding, simplify covalent conjugation, enhance signal detection
or the like.
If covalent bonding between a compound and the surface
25 is desired, the surface will usually be polyfunctional or be capable of
being polyfunctionalized. Functional groups which may be present on
the surface and used for linking can include carboxylic acids, aldehydes,
amino groups, cyano groups, ethylenic groups, hydroxyl groups,
mercapto groups and the like. The manner of linking a wide variety of
30 compounds to various surfaces is well known and is amply illustrated in
the literature. See, for example, Immobilized Enzymes, Ichiro Chibata,
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Halsted Press, New York, 1978, and Cuatrecasas, J. Bio/. Chem. 245
3059 (1970) which are incorporated herein by reference.
In addition to covalent bonding, various methods for
noncovalently binding an assay component can be used. Noncovalent
binding is typically nonspecific absorption of a compound to the surface.
Typically, the surface is blocked with a second compound to prevent
nonspecific binding of labeled assay components. Alternatively, the
surface is designed such that it nonspecifically binds one component but
does not significantly bind another. For example, a surface bearing a
10 lectin such as Concanavalin A will bind a carbohydrate containing
compound but not a labeled protein that lacks glycosylation. Various
solid surfaces for use in noncovalent attachment of assay components
are reviewed in U.S. Patent Nos. 4,447,576 and 4,254,082, which are
incorporated herein by reference.
G. Comparison of HLA-G Levels to a Sample Population
In one class of preferred embodiments, the assays of the
present invention are used to quantify average soluble HLA-G levels in
a reference population of pregnant women for comparison to, e.g., a
20 particular pregnant patient. Any of the techniques described herein for
quantitating an analyte are used to quantify HLA-G levels in biological
samples from a particular population such that an average level of HLA-
G in, e.g., the maternal blood is derived for the population. The
population is selected such that all members of the population are in the
same term of their pregnancy, are all apparently healthy with apparently
normal pregnancies, and such that all members of the population are of
a similar genetic background (i.e., all members of a single racial sub-
type, e.g., Northern Europeans). This population is termed a "reference
population. " Individual patients with the same general genetic
30 background and in the same gestational age of their pregnancy are then
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compared to the reference population to determine whether their HLA-G
levels are normal (i.e., the same as the reference population~.
.
De~r"~;"ation of HLA-G Leve/s for Moniforing r~:y~ y
Because adults do not express HLA-G, the presence of
HLA-G, e.g, in the blood indicates that a patient is pregnant. The
presence of HLA-G in maternal blood may be determined by the methods
described above. The level of HLA-G in the maternal blood may be a
general indicator of the health of the fetal-maternal interface. Because
HLA-G is expressed largely by invasive trophoblasts, an elevated or
reduced level of HLA-G in the maternal blood of a patient compared to
a reference population could indicate that trophoblast invasion is
proceeding abnormally.
The present invention also provides kits for the diagnosis
of pregnancy and disease states related to abnormal levels of circulating
HLA-G levels. The kits preferably include an antibody that specifically
binds to HLA-G. The antibody may be free or immobilized on a solid
support as described above. The kit may also contain, e.g., instructional
materials teaching the use of the antibody in an assay for the detection
of pregnancy, appropriate diluents, chemical reagents and the like.
Localization of HLA-G E~ ressi~"
The interpretation of localization studies to determine the
temporal and spatial pattern of placental HLA-G expression was
previously complicated by the use of antibodies and probes that react
with other class I molecules. Earlier immunolocalization studies
describing placental MHC class I expression were performed with
monoclonal antibodies that do not distinguish between class la and Ib
molecules. More recently, investigators localized HLA-G mRNA within
the placenta by in situ hybridization. All of these studies detected HLA-
G mRNA in extravillus cytotrophoblasts. Two of these investigators
CA 02213620 1997-08-22
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detected HLA-G mRNA in early-gestation villus cytotrophoblasts and
villus mesenchyme (Yelavarthi, et al. (1991) J Immunol. 146: 2847;
and Chumbley, etal. (1993) Hum. Immunol. 37: 17). In contrast, others
reported that the transcript is present only in syncytiotrophoblasts (Lata
5 (1992) J. Exp. Med. 175: 1027).
In agreement with the in situ hybridization data, the
immunolocalization studies presented herein (see, e.g., the Examples
section below) show that extravillus cytotrophoblasts express HLA-G
protein. However, no reactivity with anti-HLA-G antibodies in villus
1 0 cytotrophoblasts, syncytiotrophoblasts (except for weak brush border
staining in the first trimester) or elements of the villus core were
observed. The discrepancy between the presence of the HLA-G mRNA
and the HLA-G proteins in specific tissues indicates that HLA-G
transcripts are not translated, or alternatively, are spliced and result in
1~ a form of the protein not recognized by certain antibodies. However, the
amino acid sequence to which these antibodies were raised is not
deleted in any of the alternatively spliced mRNAs which have been
described .
It is also possible that nucleic acid probes used in previous
20 studies resulted in artifacts from cross reaction of the probes with class
la mRNAs present in the villus core. Supporting this possibility, northern
hybridization using probes corresponding to the 450 bp Pvu ll fragment
from the 3' untranslated region of the HLA-G cDNA cross-reacted with
class la mRNAs under all but the most stringent conditions (See,
25 Examples below).
Purified Firsf and Second Term cytotrophoblasts Up-Regulafe HLA-G
Expression in vitro
To study dynamic aspects of cytotrophoblast differentiation
30 along the invasive pathway, an in vitro model of this process was used.
Cytotrophoblast stem cells, isolated from early-gestation placentas,
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wa g6~l6~4 PCT~US96/03765
rapidly invade extracellular matrices on which they are plated. During
this process they switch their repertoire of integrin extracellular matrix
~ receptors, (as do cytotrophoblasts invading the uterus in vivo) and
upregulate 92 kDa type IV collagenase synthesis/ activation ~Librach et
5 a/. (1994) J Biol. Chem. 269:17125). Perturbing either adhesion
molecule or proteinase function inhibits invasion, suggesting that these
molecules play key roles in acquisition of cytotrophoblast invasiveness.
Expression of these molecules is controlled, at least in part, at the
translational level; however, upregulation of proteinase and integrin
10 expression in vitro is paralleled by an upregulation in production of the
corresponding mRNAs. Cytotrophoblast proteinase and integrin
expression in vitro also varies dramatically as gestation proceeds. Term
cytotrophoblasts, which have lost their invasive capacity, produce little
of the 92 kDa type IV collagenase, fail to undergo integrin switching and
15 contain no detectable mRNA encoding these molecules.
As with proteinases and integrins, first and second
trimester cytotrophoblasts upregulated HLA-G expression in culture.
Immunolocaiization studies performed on tissue sections showed that
villus cytotrophoblasts do not react with anti-HLA-G antibodies. Thus,
20 as would be expected, only a small percentage of isolated villus cells
produce HLA-G immediately after plating. After 12 h in culture, 62% of
first trimester cytotrophoblasts expressed HLA-G. However, it is clear
that cytotrophoblast HLA-G expression is regulated differently from that
of integrins and proteinases. HLA-G mRNA levels remained constant
25 throughout the culture period. There are two possible explanations for
this phenomenon. First, cytotrophoblast stem cells may contain high
levels of HLA-G mRNA; isolating them does not change these levels, but
promotes translation. Second, HLA-G mRNA production could be
induced to maximal levels during the isolation procedure. In either case,
30 it is clear that the time course of cytotrophoblast HLA-G mRNA
production in vifro differs from that of proteinases and adhesion
CA 022l3620 l997-08-22
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34
molecules. Surprisingly, a significant percentage (42%) of term cells,
which no longer express antigens involved in invasion, upregulated HLA-
G expression in culture even though they contained much lower HLA-G
mRNA levels than early gestation cells.
Together these observations reflect the fact that the
placenta performs many unique functions, some of which change
dramatically during pregnancy. Enhanced metalloproteinase expression
and integrin switching are characteristics of the early gestation
cytotrophoblasts that mediate uterine invasion. Downregulating the
expression of these genes is one mechanism that controls
cytotrophoblast invasiveness. In contrast, extravillus cytotrophoblasts
of all gestational ages retain the ability to upregulate HLA-G expression
in vitro. These observations are consistent with the fact that the cells
need only transiently express invasive characteristics, while consistently
avoiding maternal immune surveillance.
EXAMPLES
The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily recognize
a variety of noncritical parameters which can be changed or modified to
yield essentially similar results.
Examp/e 1: Characterization of the anti-HLA-G monoclonal
antibodies
Monoclonal antibodies to HLA-G were generated by
immunizing mice with a synthetic peptide corresponding to amino acids
61-83 of the a~1 domain of HLA-G (EEETRNTKAHAQTDRMNLQTLRG
(Seq. Id. No. 1)).
The peptide was coupled to maleimide-activated keyhole
limpet hemocyanin (KLH; Imject; Pierce Chemical Co., Rockford, IL) via
a C-terminal cysteine (added for this purpose) according to the
CA 02213620 1997-08-22
W O96131604 PCT~US96/0376S
manufacturer's instructions. Two-month-old female BALB/c mice
(Charles River) were given i.p. injections of 100 ,ug peptide-KLH
conjugate emulsified in Freund's complete adjuvant and were boosted
after 14 days with 50,ug antigen in incomplete adjuvant~ Test bleeds
5 were obtained 10 days later from tail veins, and sera were screened by
ELISA (described below) for reactivity against the peptide immunogen.
Mice exhibiting the best humoral responses were given a final
intravenous boost (50 ~g) and sacrificed three days later. Spleen cells
were isolated and fused with SP2/0 myeloma cells according to
10 published procedures (Kohler and Milstein (1975) Nature 256: 495; and
Harlow and Lane (1988), Supra). Cultures were selected in HAT
medium (UCSF Cell Culture Faciiity) and cloned by limiting dilution.
The HLA-G antibodies isolated using the above procedure
belonged to the IgM ciass. At least two factors probably contributed to
15 this outcome. First, the mice were immunized for a relatively short
period of time before the hybridomas were produced, and early antigenic
responses result primarily in IgM-class antibody production. Second, the
reporter antibodies used in the hybridoma screening react with IgM as
well as IgG. Using the above method, one can reproducibly obtain HLA-
20 G specific antibody producing hybridomas at at least 1% or greater.
Two methods to determine whether 1 B8 antibody reactedwith class la molecules (HLA-A, -B, -C) were used. Flow cytometry
experiments (flow cytometry techniques are described below) indicated
that peripheral blood leukocytes isolated from the blood of 12 different
25 individuals did not bind the antibody. In contrast, JEG-3 cells and a B-
lymphoblastoid HLA-null cell line (LCL 721.221 ) stably transfected with
a vector expressing HLA-G gave strong positive signals when stained
with 1 B8 or W6/32, an antibody that recognizes monomorphic
determinants of all class I heavy chain/,B2-microglobulin complexes
30 (Barnstable et al (1 978) Cell 14: 9). However, the parental
(untransfected) Iymphoblastoid cells reacted with neither antibody. An
CA 02213620 1997-08-22
W O96131604 PCTrUS96/03765
isotype-matched irrelevant IgM (Sigma) did not react with any of the
cells.
That the antibody does not react with class la molecules is
further supported by our immunolocalization data (discussed in detail in
5 the following sections). The placenta and placental bed contain many
cells that express class la antigens. For example, a hallmark of human
pregnancy is leukocyte infiltration of the decidua (Ferry et a/. (1990)
/mmuno/ogy 70: 446). Floating chorionic villi, composed entirely of fetal
cells, contain abundant stromal cells and macrophages (Hofbauer cells)
10 which also express class la proteins (Nakamura et a/ (1990) Hum.
Pathol. 21: 936). However, immunolocalization studies performed on
placental tissue from 26 individuals showed that none of these class la-
expressing cell populations stained with HLA-G antibodies; the 1 B8 and
3F6 mAbs reacted only with specific populations of cytotrophoblasts.
Example 2: Antibody screening
Hybridomas were screened for reactivity against the peptide
immunogen by antibody-capture ELISA according to standard methods
(see, Harlow and Lane, supra). Microtiter plates were coated with 50
20 ,ul of PBS containing 10,ug/ml of the synthetic peptide, washed three
times with PBS and blocked for 1 hour at room temperature with PBS
containing 0.02% (v/v) Tween 20,0.25% BSA (w/v) and 0.02% sodium
azide (w/v; blocking buffer). Plates were incubated with test fluids (test
sera or hybridoma supernatants) for 1 h at room temperature, followed
25 by rabbit anti-mouse IgG conjugated to alkaline phosphatase (Jackson
Immuno Research Labs., Inc., West Grove PA) diluted 1 :2000 in
blocking buffer. Reactivity was assessed by adding 50,ul p-nitrophenyl
phosphate substrate solution (3 mM PNPP, 0.05 M NaC03, 0.5 mM
MgCI2, pH 9.5) and measuring the absorbance at 405 nm with a
30 microplate reader (Molecular Devices Inc., Menlo Park, CA).
CA 02213620 1997-08-22
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37
Hybridomas were also screened for their ability to react
with cytotrophoblasts in tissue sections of early second trimester (16-18
~ week) placentas that contained anchoring villi. Double indirect
immunofluorescence was performed using hybridoma supernatants and
5 a rat monoclonal IgG against cytokeratin (7D3). The cytokeratin antigen
is present in differentiated epithelial cells, but not on connective tissue,
endothelium, muscle or blood cells.
Placental tissues were fixed for 30 min with 3 %
paraformaldehyde in calcium-containing PBS, pH 7.2. They were then
10 incubated in 10 mM glycine to quench unreacted aldehyde groups,
infiltrated with 15% sucrose, embedded in OCT (Miles Scientific,
Naperville, IL), and frozen in liquid nitrogen. Sections (5,um) were cut
using a Slee HR cryostat and collected on 22-mm2 coverslips. Before
staining, the sections were washed for 10 min each in PBS and in PBS
15 containing 0.2% BSA. Primary antibodies were applied to the tissue
sections as undiluted hybridoma supernatants for 1 h at room
temperature. After being washed, sections were incubated for 30 min
at room temperature with a 1:1 mixture of fluorescein-conjugated goat
anti-mouse IgG and rhodamine-conjugated goat anti-rat IgG (diluted
20 1:200; Jackson Immuno Research). The samples were mounted and
examined with a Zeiss epifluorescence-phase microscope and
photographed with Kodak Tri X film.
The class of the antibodies selected for further characterization
was determined using an ImmunoType Kit (Sigma, St. Louis, MO)
25 according to the manufacturer's instructions. Several antibodies were
isolated that bound HLA-G peptide using the ELISA and stained
cytotrophoblasts in the first trimester placenta. Two of these antibodies
(1 B8 and 3F6), both IgM class antibodies, were further characterized.
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Example 3: Affinity pLlrification of HLA-G
To characterize the specificity of the antibodies, we
constructed an affinity column using the 1 B8 mAb, through which we
passed an 35S-labeled JEG-3 detergent extract. This choriocarcinoma
cell line expresses HLA-G, but not class la molecules. After washing,
bound proteins were eluted and analyzed by 10% SDS-PAGE and
fluorography. A single 38 kDa protein, corresponding to the molecular
mass of the HLA-G a chain, was detected. ,~2-microglobulin (12 kDa)
would be expected to run off the bottom of the gel under the conditions
1 0 used.
The immunoaffinity column was constructed using the 1 B8
antibody as follows. First, the peptide immunogen was coupled to
thiopropyl-activated Sepharose 6B (Pharmacia, Piscataway, NJ)
according to the manufacturer's instructions. Antibody was then bound
to the matrix by passing 1 B8 ascites fluid, diluted 1:10 in PBS, through
the column and washing with 20 bed volumes of PBS. That the
antibody was specifically bound was demonstrated by eluting the
column with 100 mM glycine, pH 2.5. This fraction contained pure IgM
as assessed by silver staining of SDS-polyacrylamide gels.
The antibody column was used to purify HLA-G from 35S-
labeled JEG-3 choriocarcinoma cell extracts as follows. 1 x 10' JEG-3
cells were metabolically labeled by overnight incubation in methionine-
and cysteine-free DMEM (Gibco, Gaithersburg, MD) containing 0.5
mCi/ml 35S-protein labeling mix (EXPRE35S35S Protein Labeling Mix,
> 1000 Ci/mmol, New England Nuclear, Boston, MA). After labeling,
cells were washed once with cold PBS and Iysed with cold buffer
containing 50 mM Tris and 1% NP-40, pH 8Ø Lysates were cleared by
centrifugation at 16,000 x g for 10 min at 4~ C, and supernatants were
applied to the column. The column was washed with 20 bed volumes
30 of PBS and eluted with 100 mM glycine, pH 2.5. Eluates and flow-
-
CA 02213620 1997-08-22
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39
through fractions (800 cpm per sample) were separated by 10% SDS-
PAGE, processed for fluorography, and exposed to x-ray film at -80~C.
Example 4: Flow Gytometry
Peripheral blood Iymphocytes (PBLs) were prepared by
centrifugation of whole blood through Ficoll-Hypaque 1077 (Sigma).
Single-cell suspensions of JEG-3 were obtained by trypsinization of
monolayer cultures grown in Eagle's minimum essential medium (UCSF
Cell Culture Facility) supplemented with 10% fetal bovine serum
(Sigma). PBLs or JEG-3 cells were washed in PBS containing 1 mg/ml
BSA and incubated with either 1 B8 (hybridoma supernatant diluted 1 :10)
or W6/32 (1 ~g/ml) followed by fluorescein-conjugated anti-mouse IgM
or IgG, respectively (diluted 1 :100; Jackson Immuno Research). Cells
were then washed, fixed in 0.2% paraformaldehyde, and analyzed on a
FACScan cytometer (Becton Dickinson, Mountain View, CA).
Example 5: Immunofluorescent localization of HLA-G in the
placenta and placental bed; HLA-G is expressed by
cytotrophoblasts that differentiate along the invasive pathway in
vivo.
Immunohistochemical analysis using fluorescent detection
was carried out on frozen sections of placenta and placental bed
prepared from tissues Gbtained during the first, second and third
trimesters of pregnancy. Frozen sections were prepared from first,
second and third trimester human chorionic villi or placental bed biopsies
aspreviouslydescribed (Damskyeta/. (1992)J. Clin. Invest. 89: 210).
Double indirect immunofluorescence using 1 B8 or 3F6 (anti-HLA-G
antibodies) and 7D3 (anti-cytokeratin) was performed essentially as
described above. Reactivity of the 1 B8 and 3F6 antibodies was
detected using fluorescein-conjugated goat anti-mouse IgM, diluted
1:200 (dackson Immuno Research). Control experiments included
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incubation of tissues with primary or secondary antibodies alone, non-
immune mouse serum or normal mouse IgG.
Sections contained floating chorionic vilii and anchoring villi
(including cytotrophoblast cell columns), as well as decidualized
5 endometrium and myometrium. Thus, cytotrophoblast stem cells, as
well as differentiated trophoblasts (syncytiotrophoblasts and invasive
cytotrophoblasts), were evident. Sections were double-stained with an
anti-cytokeratin antibody (7D3), which in the placental bed is specific for
trophoblast cells, and either the 1 B8 or 3F6 anti-HLA-G mAbs (described
1 0 supra).
The 3F6 mAb gave an identical staining pattern to that of
1 B8. None of the components of floating villi, including undifferentiated
cytotrophoblasts anchored to the villus basement membrane and fetal
elements within the villus core, reacted with the anti-HLA-G antibodies.
In contrast, . invasive cytotrophoblasts within the cell columns of
anchoring villi stained brightly (Fig. 2, panel B). Antibody reactivity was
first detected in the distal part of the cell columns as the
cytotrophoblasts made contact with the uterine wall. Cytotrophoblasts
participating in interstitial invasion also stained brightly. During this
stage of pregnancy, when endovascular invasion peaks,
cytotrophoblasts within blood vessels also showed intense reactivity
with the anti-HLA-G antibodies. Incubation of tissues with primary or
secondary antibodies alone, non-immune mouse serum or normal mouse
IgG showed no reactivity.
Cytotrophoblast staining in first trimester samples was
nearly identical to that in second trimester tissue. None of the floating
villi components reacted with either antibody. The only exception was
occasional s.yncytial brush border staining. Third trimester tissue
exhibited the same pattern; floating villi (including the syncytial brush
border) did not stain whereas interstitial and endovascular
cytotrophoblasts reacted with the antibodies. However, by the third
CA 02213620 1997-08-22
W O 96~1604 P ~ AUS96/03765
trimester, cytotrophoblasts within uterine wall stained less brightly than
cells in a comparable location earlier in gestation.
Example 6: HLA-G is expressed by cytotrophob/asts th~t
differentiate along the invasive pathway in vitro
Highly purified early gestation cytotrophoblast stem cells
plated on Matrigel aggregate and acquire an invasive phenotype, as
shown by the expression of stage-specific antigens. This experimental
system was used to examine HLA-G production during the course of
cytotrophoblast differentiation along the invasive pathway in vifro~ Two
culture conditions were employed. In the first, early-gestation
cytotrophoblasts were plated on Matrigel plugs, which promotes the
formation of large aggregates. Staining sections of these aggregates
allowed determination of the spatial pattern of HLA-G expression as the
cells invaded the extracellular matrix substrate. Cells on the surface that
had not invaded the Matrigel did not stain, while those that had
penetrated the substrate reacted with the 1 B8 antibody.
Highly purified cytotrophoblasts were prepared from first,
second and third trimester chorionic villi as previously described (Fisher
eta/. (1989) J. Ce//Bio/. 109: 891; and Librach, eta/. (1991) J. Cel/
Bio/. 113: 437). Cells were plated on the laminin-rich extracellular
matrix preparation Matrigel (Collaborative Research, Bedford, MA), in
MEM (UCSF Cell Culture Facility) containing 2% Nutridoma (Boehringer
Mannheim Biochemicals, Indianapolis, IN). To promote the formation of
large aggregates, 2. 5 x 1O5 cells were plated on plugs of Matrigel
formed in capsules (6.5 mm diameter). After 3 days in culture, the
Matrigel plugs and cytotrophoblast aggregates were fixed in 3%
paraformaldehyde, sectioned and processed for immunostaining.
Sections of aggregates that had invaded Matrigel plugs were stained
essentially as described above, except that reactivity was detected using
CA 02213620 1997-08-22
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42
a Vectastain ABC kit tVector Laboratories, Burlingame, CA) according
to the manufacturer's instructions.
The second culture condition was used to determine the
time course of HLA-G protein production in vitro. Cytotrophoblasts
5 isolated from placentas of different gestational ages were cultured on a
thin layer of Matrigel. Under these conditions the cells differentiate
along the invasive pathway, as they do when cultured on Matrigel plugs
(Damsky etal. (1994) Development 120: 3657). However, they form
smaller aggregates, which permits determination of the staining pattern
10 of individual cells without sectioning. Cytotrophoblasts of all gestational
ages upregulated HLA-G production in culture. For example,
immediately after plating approximately 25% of first trimester cells
expressed HLA-G, but by 12 h in culture nearly 60% reacted with the
antibody. This level remained constant throughout the 48 h assay
15 period. With increasing gestational age there was a decrease in the
percentage of immunopositive cells at most time points. For example,
significantly fewer third than first trimester cells expressed HLA-G after
12 h (34 % vs. 63 %). The assays were performed as described below.
To determine the percentage of cells expressing HLA-G
20 after various times in culture, 2.5 x 1O5 cells were plated on coverslips
(22 mm2) coated with a thin layer of Matrigel (10,ul). After 0-48 h of
culture, the cells were fixed in 3% paraformaldehyde for 5 min at room
temperature,-washed with PBS, permeabilized with cold methanol for 5
min and processed for immunofluorescence exactly as described above.
Example 7: Norfhern hybridization shows that Cytofrophoblast
HLA-G mRNA production in vitro is gestationally regu/ated
Northern hybridization was used to quantify mRNA levels
in cultured first, second and third trimester cytotrophoblasts. High
30 levels of HLA-G mRNA were detected in first and second trimester
cytotrophoblasts immediately after isolation, and the level of mRNA
CA 022l3620 l997-08-22
W O96t31604 PCTAUS96/0376
43
expression did not change over time in culture. In comparison, term
cells contained a greatly reduced level of HLA-G mRNA throughout the
~ culture period. The northern analysis was performed as described
below.
Total RNA was extracted from cultured cytotrophoblasts
according to published methods (Chomczynski and Sacchi (1987) Anal.
Biochem. 162: 156). Briefly, 2-5 x 1O7 cells per sample were
homogenized in 500 ,ul guanidine buffer (4 M guanidine isothiocyanate,
25 mM sodium citrate, 0.5% sarcosyl), after which 50,ul 2 M sodium
acetate (pH 4), 500,ul water-saturated phenol and 100,ul chloroform
were added. After centrifugation, RNA was pelleted from the aqueous
phase by the addition of 500,ul isopropanol, extracted with 4 M LiCI,
and reprecipitated from a solution containing 10 mM Tris (pH 7.5), 1
mM EDTA, and 0.5% SDS. The pellets were then washed with 70%
ethanol, vacuum dried and dissolved in sterile water. The concentration
of RNA was determined by measuring the absorbance at 260 nm.
An HLA-G-specific cDNA probe was synthesized by random
priming of the 450 bp Pvu ll fragment from the 3' untranslated region
of HLA-G using 32P-CTP and the Klenow fragment of DNA polymerase
1 according to standard methods (Tabor et al. ( 1 993) In Current
Protocols in Molecular Biology, vol. 1 . K. Jannssen, ed. John Wiley and
Sons, New York p. 3Ø1). Probes had a specific activity of 2 x 109
dpm/,ug. Total RNA (1 OJU9) was separated by formaldehyde-agarose gel
electrophoresis, transferred to Nytran membranes (Schleicher and
Schuell, Keene, NH) and analyzed by Northern blot hybridization as
previously described (Lehrach eta/. (1977) Biochemistry 16: 4743; and
De etal. (1990) J. Biol. Chem 265: 15267)). In all experiments, gels
were stained with acridine orange prior to transfer to ensure integrity of
the RNA samples, and to confirm that equal amounts of RNA had been
loaded onto each lane. The final post-hybridization washes were carried
CA 02213620 1997-08-22
W O 96/31604 PCTrUS96/03765
out in 0.3 X SSC (150 mM NaCI, 15 mM sodium citrate, pH 7.4) and
0.1% SDS at 68~ C.
All publications and patent applications cited in this
5 specification are herein incorporated by reference for all purposes as if
each individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in
10 some detail by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary skill in the
art in light of the teachings of this invention that certain changes and
modifications may be made thereto without departing from the spirit or
scope of the appended claims.
CA 02213620 1997-08-22
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~U~N~ LISTING
(1) ~N~T. INFORMATION:
(i) APPLICANT: THE R~lS OF T~E UNIVERSITY OF CALIFORNIA
(ii) TITLE OF INv~l~loN: ANTIBODIES FOR THE V~ lON OF HLA-G
(iii) N~MBER OF ~U~N~S: 1
(iv) CO~.~PU~N~ ADDRESS:
(A) ADDRESSEE: R~hhin-~, Berliner & Carson
(B) STREET: 201 N. Figueroa Street, 5th Floor
(C) CITY: Los Angeles
(D) STATE: California
(E) COUNL~Y: US
(F) ZIP: 90012-2628
(v) COMPUTER ~n~RT.T~ FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(Vi) ~U~R~N1 APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) AllORN~Y/AGENT INFORMATION:
(A) NAME: Berliner, Robert
(B) REGISTRATION NUMBER: 20,121
(C) REFERENCE/DOCKET NUMBER: 5555-372
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (213) 977-1001
(B) TELEFA~: (213) 977-1003
(2) INFORMATION FOR SEQ ID NO:1:
u~N~ CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) sTR~n~nN~s not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:1:
Glu Glu Glu Thr Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met
1 5 10 15
Asn Leu Gln Thr Leu Arg Gly
