U.S. patent application number 14/658382 was filed with the patent office on 2015-09-17 for methods of depleting antibodies directed towards major histocompatibility complex.
This patent application is currently assigned to OXFORD RADCLIFFE HOSPITAL NHS ("ORH") TRUST OF THE JOHN RADCLIFFE HOSPITAL. The applicant listed for this patent is GUY'S & ST. THOMAS' HOSPITAL NHS TRUST ("GST"), KING'S COLLEGE LONDON, OXFORD RADCLIFFE HOSPITAL NHS ("ORH") TRUST OF THE JOHN RADCLIFFE HOSPITAL. Invention is credited to Martin BARNARDO, Michael Bunce, Andrea Harmer, Robert Vaughan, Kenneth Welsh.
Application Number | 20150260622 14/658382 |
Document ID | / |
Family ID | 38173801 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150260622 |
Kind Code |
A1 |
BARNARDO; Martin ; et
al. |
September 17, 2015 |
METHODS OF DEPLETING ANTIBODIES DIRECTED TOWARDS MAJOR
HISTOCOMPATIBILITY COMPLEX
Abstract
The invention provides a method of depleting anti-MHC antibodies
in a sample comprising contacting said sample with one or more
recombinant MHC molecules or functionally equivalent variants,
derivatives or fragments thereof and removing at least the
recombinant MHC molecules to which antibodies to said recombinant
MHC molecules contained within the sample have bound. This method
allows the depletion of one or more specific MHC particularly HLA
allele antibodies from a sample.
Inventors: |
BARNARDO; Martin; (Oxford,
GB) ; Harmer; Andrea; (Sheffield, GB) ; Bunce;
Michael; (Bromborough, GB) ; Vaughan; Robert;
(London, GB) ; Welsh; Kenneth; (London,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OXFORD RADCLIFFE HOSPITAL NHS ("ORH") TRUST OF THE JOHN RADCLIFFE
HOSPITAL
KING'S COLLEGE LONDON
GUY'S & ST. THOMAS' HOSPITAL NHS TRUST ("GST") |
Oxford
London
London |
|
GB
GB
GB |
|
|
Assignee: |
OXFORD RADCLIFFE HOSPITAL NHS
("ORH") TRUST OF THE JOHN RADCLIFFE HOSPITAL
Oxford
GB
KING'S COLLEGE LONDON
London
GB
GUY'S & ST. THOMAS' HOSPITAL NHS TRUST ("GST")
London
GB
|
Family ID: |
38173801 |
Appl. No.: |
14/658382 |
Filed: |
March 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14046673 |
Oct 4, 2013 |
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14658382 |
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11380062 |
Apr 25, 2006 |
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14046673 |
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10623802 |
Jul 22, 2003 |
8088586 |
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11380062 |
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09809029 |
Mar 16, 2001 |
8609436 |
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10623802 |
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60190027 |
Mar 17, 2000 |
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Current U.S.
Class: |
436/177 |
Current CPC
Class: |
G01N 33/56977 20130101;
Y10S 436/825 20130101; G01N 1/34 20130101; A61K 39/39525 20130101;
G01N 33/543 20130101; Y10T 436/25375 20150115; Y10S 435/962
20130101 |
International
Class: |
G01N 1/34 20060101
G01N001/34 |
Claims
1. A method of depleting anti-major histocompatibility complex
(anti-MHC) antibodies in a sample, said method comprising
contacting the sample with a peptide that is epitopic to said
anti-MHC antibodies to create an anti-MHC/peptide complex and
removing said complex from said sample, wherein said removal of
said complex depletes said sample of said anti-MHC antibodies.
2. The method of claim 1, wherein said epitopic peptide is linked
to a solid support.
3. The method of claim 2, wherein said sample is a serum
sample.
4. The method of claim 3, wherein said anti-MHC antibodies are
anti-human leukocyte antigen (anti-HLA) antibodies.
5. The method of claim 4, wherein said epitopic peptide is a Class
I human leukocyte antigen (HLA) molecule.
6. The method of claim 4, wherein said epitopic peptide comprises a
monomer of a Class I human leukocyte antigen (HLA) molecule.
7. The method of claim 6, wherein said epitopic peptide comprises a
class II heavy chain HLA monomer, a class II beta-2-microglobulin
HLA monomer and a folding peptide.
8. The method of claim 7, wherein said folding peptide comprises an
amino acid sequence at least 80% identical to the amino acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:7 and SEQ ID
NO:8.
9. The method of claim 4, wherein said epitopic peptide is a Class
II human leukocyte antigen (HLA) molecule.
10. The method of claim 4, wherein said epitopic peptide comprises
a monomer of a Class II human leukocyte antigen (HLA) molecule.
11. The method of claim 10, wherein said epitopic peptide comprises
a class II heavy chain HLA monomer, a class II beta-2-microglobulin
HLA monomer and a folding peptide.
12. The method of claim 11, wherein said folding peptide comprises
an amino acid sequence at least 80% identical to the amino acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:7 and SEQ ID
NO:8.
Description
[0001] The present invention relates to a method of depleting a
sample of anti Major Histo-Compatibility Complex (MHC) antibodies,
particularly anti-human leucocyte antigen (HLA) reactive
antibodies.
[0002] MHC antigens are glycoproteins expressed on the surface of
cells, such as platelets, macrophages and lymphocytes.
Functionally, these molecules play important roles in the
presentation of foreign antigenic peptides to cytotoxic T cells.
MHC molecules are divided into two types, Class I and Class II,
based on their structure and function. MHC Class I genes encode
glycoproteins which are expressed on the surface of almost all
nucleated cells of the body. MHC Class I molecules are involved in
activating cytotoxic T cells. MHC Class II genes encode
glycoproteins expressed primarily on antigen presenting cells
(macrophages, dendritic cells, B cells) where they present the
processed antigen (e.g. viral or other foreign antigens) to the T
helper cells. These antigens form one group of the so-called
histocompatibility antigens.
[0003] MHC molecules in humans are referred to as Human Leucocyte
Antigen (HLA) molecules. Typing of the numerous HLA molecules
present in humans has shown that individuals possess a particular
`signature` of HLA molecules present on their cells. HLA molecules
are coded for in the human genome by a series of four gene loci.
HLA Class I molecules are coded for by the A, B, C, E, F and G
regions whereas the HLA Class II molecules are coded for by the DR,
DQ, DP, DO and DM regions. The loci constituting the HLA molecules
are highly polymorphic, and many alternate forms of the gene or
alleles exist at each locus.
[0004] In normal immune responses self-MHC, e.g. HLA molecules are
recognised by T cell receptors in vivo. The T cell receptors see
the foreign antigen expressed as a small peptide in the context of
a MHC Class I or Class II molecule. This leads to production of
appropriate antibody responses to the foreign antigen or
destruction of the presenting cell depending on the presenting and
T cells which are involved.
[0005] The immune system is however also able to identify and mount
a challenge to non-self, i.e. foreign MHC molecules. Thus, when
presented with a non-host MHC molecule, the immune system will
react to destroy cells carrying the non-host MHC by normal
immunological means, i.e. produce antibodies, activate the
complement system etc. This is obviously undesirable when the cells
carrying the non-host MHC, e.g. HLA are purposively introduced, for
example, foreign cells or tissue, e.g. in a transplanted organ and
presents a bar to the introduction of such cells.
[0006] Placing a `foreign` (i.e. non-host) MHC molecule into an
individual may result in the individual producing anti-MHC
antibodies which will bind specifically to that MHC. Individuals
may raise anti-MHC antibodies, and become "sensitized", if they are
exposed to a foreign MHC, i.e. during pregnancy, by blood
transfusion, or by receiving an organ donation.
[0007] Pre-sensitization to MHC via transfusion, transplantation,
or pregnancy can cause rapid rejection of transplanted tissue or
poor platelet survival after transfusion. Therefore, testing for
anti-MHC antibodies prior to tissue or organ transplantation is of
great importance, as the presence in the recipient of anti-MHC
antibodies which bind to donor MHC molecules (donor specific
crossmatch) is predictive of a high risk of rejection of the
transplanted tissue or organ. Thus, prior to transplantation, the
donor tissue is typed for MHC molecules, and the recipient is typed
for anti-MHC antibodies. Screening of potential transplant
recipients for anti-HLA antibodies is an essential part of the
pre-transplant monitoring carried out by tissue-typing
laboratories.
[0008] Ideally, organs would be transplanted that are an ideal HLA
match to the recipient. However, in view of the high number of HLA
genes involved, up to hundred or more alleles for each, a perfect
match is very difficult to obtain.
[0009] Post-transplant monitoring is also valuable to assess the
level of anti-MHC antibodies which are being generated and hence
the continued success or likely rejection of a transplant.
[0010] Anti-HLA antibody testing methods are known in the art, and
include the screening of the blood or serum from the potential
recipient against a panel of cells which are considered to present
a representative selection of HLA antigens. This procedure can take
up to 6 weeks. Such screening determines the panel reactivity (PR)
for each sample, and gives an estimate of the degree of
sensitisation against the panel of cells used for testing and can
be related to the chance of a donor being suitable. The panel size
may range from 25 to 100 different cells, and the larger the panels
(50 to 100 different cells), the more reliable the results.
[0011] A more specific method is
complement-dependent-lymphocytotoxicity (CDC) testing. In this
method, the serum or blood of the potential recipient is tested
against a panel of lymphocytes (or more specifically, the donor's
lymphocytes) and the mixture is further incubated with complement
factors. In order to measure the level of cytotoxicity, the viable
and non-viable cells are distinguished using a dye. Therefore, this
method is not without drawbacks, as the discrimination between
living and dead cells can be subject to human error.
[0012] Another well known method is based on flow cytometry which
is a method that allows the analysis of a large number of
individual cells in a short time. The use of flow cytometric
crossmatching of recipient serum against donor lymphocytes has
shown it to be a more sensitive method of antibody detection than
conventional cytotoxic crossmatch. Further, FACs (fluorescent
activated cell sorting) screening of pooled cells can accurately
and rapidly detect anti-HLA antibodies. It has been shown (Harmer
at al., Transpl. Int. (1993) 6: 277-280) that FACS can detect IgG
antibodies which have not been detected by conventional screening
methods.
[0013] However, these prior art methods are constrained due to the
presence of molecules other than HLA on the whole cells, and
therefore other `non-HLA` antibodies are detected. The recent use
of ELISA (enzyme-linked-immunosorbent assay) employing purified
class I and class II antigens from platelets and cell lines
partially overcomes this problem.
[0014] In U.S. Pat. No. 5,948,627 (Lee at al.) the use of a
plurality of microbeads presenting multiple purified HLA antigens
from a cell population in order to test for anti-HLA antibodies is
discussed.
[0015] Therefore, there is still a need for an assay for anti-MHC
antibodies that detects single specific antibodies in a sample, in
a quick and reliable manner. This will enable the dissection of
component specificities and the detection of antibodies to rare
MHC, e.g. HLA alleles in sera from highly sensitized patients. In
brief, an assay is required that will precisely define the anti-MHC
antibodies present in a sample, quickly, simply and
reproducibly.
[0016] Surprisingly, it has now been found that individual
recombinant HLA or HLA-type molecules can be used for specifically
detecting anti-HLA antibodies. The recombinant HLA monomers have
been found by the inventors to be bound by anti-HLA antibodies in a
sample. Thus, recombinant MHC monomers can be utilized in the
detection, identification and removal of anti-MHC antibodies. These
recombinant MHC or MHC-type monomers, functioning as anti-MHC
antibody antigens, have the advantage that the identity of the MHC
is known, and that the sample can therefore be tested for anti-MHC
antibodies for each individual recombinant MHC or MHC-type. The
advantages of a more specific assay method for detecting anti-MHC,
e.g. anti-HLA antibodies are self evident and include the
advantages of being more convenient, less time consuming and labour
intensive to develop and therefore less costly.
[0017] Significantly, the use of recombinant MHC or MHC type
monomers avoids the need for purifying MHC molecules from a cell
population presenting said MHC and is far simpler and quicker than
CDC-type methods. The benefits of the method of the invention
include the ability to quantify anti-MHC-antibodies present in a
sample, in a highly reproducible manner. Uniquely, this assay
ensures that the only target present is a specific, individual, MHC
antigen. The method of the invention reduces assay time per sample
from several weeks, in the case of CDC, to less than 3 hours. Also,
as cells are not required for the assay, this circumvents the
recently evolved problem of lack of availability of CDC target
cells.
[0018] Thus, in a first aspect the present invention provides a
method of detecting the presence of anti-MHC antibodies in a sample
comprising contacting said sample with one or more recombinant MHC
molecules or functionally equivalent variants, derivatives or
fragments thereof and detecting the binding or absence of binding
of antibodies to said recombinant MHC molecules, variants,
derivatives or fragments thereof. This method allows the detection
and/or identification of one or more specific MHC antigen
antibodies.
[0019] As used herein "detecting" refers to a qualitative,
quantitative, or semi-quantitative assessment of the formation of
an antibody:MHC molecule complex in the sense of obtaining an
absolute value for the amount of complex formed in the sample or an
index, ratio, percentage or similar qualitative or
semi-quantitative indication. Thus "binding" denotes the formation
of a specific and selective interaction between said 2 components.
Appropriate means of detection are as described hereinafter and
include detection of labels or signalling means associated with
said complexes such as the use of antibodies to one of the
components, in which the antibodies themselves carry a label or
signalling means which can be detected by appropriate techniques.
Alternatively the MHC molecules may be designed to inherently carry
a label/signalling means e.g. by the use of radiolabelled amino
acids in their construction, or added post-synthesis.
[0020] The signalling means may in general be any moiety capable of
direct or indirect detection, e.g. by virtue of its enzymatic
properties, radiation emission, scattering, absorption or magnetic
properties or may cooperate with or bind to a complimentary agent
to produce a detectable effect, e.g. interact with an enzyme to
produce a signal, gas evolution, light emission, colour change,
turbidity, precipitation etc. Such moieties are well known within
the field of diagnostic assays. Alternatively the signalling means
may be or allow association with a label such as radiolabels,
chemical labels (for example chromophores or fluorophores such as
dyes e.g. fluoroscein and rhodamine) or reagents of high electron
density e.g. ferritin, haemocyamin or colloidal gold.
[0021] "Identification" refers to the identification of one or more
particular MHC allele antibodies based of the specific MHC molecule
to which they bind. "Absence of binding" as used herein refers to
absence of detectable binding above control levels. Binding is
considered to occur and hence signify the presence of a particular
MHC allele when levels above a determined threshold relative to
control levels are observed.
[0022] As used herein "recombinant" refers to a molecule or part
thereof (preferably the MHC heavy chain) that has been produced in
one or more cells via genetic engineering of that cell. Thus, the
cell has been genetically modified relative to its naturally
occurring form and has been provided, by any suitable means, with
all the genetic information necessary to produce, or produce
greater quantities of, the molecule of interest. Preferably the
cell is a prokaryotic cell (e.g., bacterial cells, e.g. E. coli
derivatives) or a simple eukaryotic cell (e.g. yeast, plant or
insect cells). It is possible to produce recombinant molecules in
higher organisms, i.e. mammals, but it will be understood by those
skilled in the art that such methods are more labourious and time
consuming than recombinant molecule production in unicellular
organisms.
[0023] Preferably cells are modified by introduction of a vector
(e.g. a plasmid) which may replicate separately to the genome of
the cell or may be integrated into that genome. In the case of
expression of class I molecules the vector comprises for example a
nucleic acid molecule encoding all or part of the MHC heavy chain
operatively linked to elements suitably controlling its
replication, transcription and/or translation. Additionally, when
preparing a class I MHC molecule the same or a further genetically
modified cell may be utilised which expresses
.beta..sub.2-microglobulin. When preparing a class II MHC molecule
the vector comprises the .alpha. and/or .beta. chain under suitable
regulatory control. In essence, the component of the MHC molecule
that dictates the variability and hence polymorphism of MHC alleles
is expressed recombinantly, whereas non-variant molecules, e.g.
.beta..sub.2-microglobulin may be purified, although preferably all
components are expressed recombinantly.
[0024] The recombinant MHC or MHC-type molecule used in the method
of the invention can be either class I or class II, but must be
sufficiently antigenic in order to be bound by anti-MHC antibodies.
Preferred Class I molecules are A, B and C, although other possible
molecules are E, F, G, MICA, MICB and CDI. Preferred Class II
molecules are DRB1/3/4/5, DQA1, DQB1, DPA1 and DPB1. The
recombinant MHC or MHC-type molecule therefore has to present the
extracellular polymorphic residue (i.e. the residue that is altered
in each HLA type) for binding by the anti-MHC antibody.
[0025] As used herein an MHC molecule for use in the method of the
invention, is preferably an HLA molecule and includes functionally
equivalent variants, derivatives or fragments thereof. The MHC
molecule and its variants, derivatives or fragments may comprise
more than one component which together form a complex with
antigenic properties, e.g. in the case of Class I molecules, the
heavy chain, .beta..sub.2-microglobulin and a peptide. Thus the
sequence of naturally occurring MHC molecules may be modified
providing areas which make up at least one unique epitopic site
(which may be provided by one or more of the components of the MHC
molecule), particularly regions of variability which allow
presentation of at least one unique epitopic region which typifies
a particular MHC allele and which results in the production of
specific MHC antibodies directed to that region are maintained such
that the said specific antibodies are still capable of binding to
that region. As will be appreciated this requires maintenance of
not only residues at the epitopic site, but also key skeletal
residues to achieve correct folding of the MHC molecule to form the
epitopic site. However, the use of misfolded MHC molecules in the
method of the invention is also envisaged, wherein the epitopic
site remains available for antibody binding.
[0026] Thus derivatized MHC molecules are also contemplated
providing they exhibit the same function, i.e. allow appropriate
presentation of one or more of the epitopic sites of the MHC allele
of interest. Thus functionally equivalent variants, derivatives or
fragments refer to MHC molecules related to or derived from
naturally occurring MHC molecules wherein the amino acid sequence
of one or more components of said MHC molecules (e.g. the class I
heavy chain, class II.alpha. and/or .beta. chain) has been modified
by single or multiple amino acid (e.g. at 1 to 50, e.g. 10 to 30,
preferably 1 to 5 bases) substitution, addition and/or deletion but
which nonetheless retains functional activity.
[0027] Within the meaning of "addition" variants are included amino
and/or carboxyl terminal fusion proteins or polypeptides,
comprising an additional or replacement protein or polypeptide
fused to the polypeptide sequence e.g. a portion of the MHC
molecule (e.g. the MHC class I heavy chain transmembrane/cytosolic
tail) is preferably supplemented or replaced with a means for
immobilisation, e.g. a peptide which allows biotinylation of said
molecule.
[0028] Derivatives may be prepared by post-synthesis modification
or by modification during synthesis e.g. using modified residues or
modified encoding nucleic acid material e.g. modified by
site-directed mutagenesis. Derivatives preferably include those in
which conservative amino acid substitutions are employed so as not
to significantly affect structure and function at that residue.
Derivatized or modified amino acids may also be used, e.g.
methylated residues, where compatible with recombinant
expression.
[0029] Preferably, the derivatives and variants are closely related
to one or more components of the naturally occurring MHC molecules,
e.g. are encoded by nucleic acid molecules with more than 70%,
preferably more than 80, 90 or 95% sequence identity to naturally
occurring sequences or exhibit such sequence identity to the
functional portions of these sequences, e.g. to naturally occurring
allelic geographical or allotypic variants e.g. to sequences as
described in the MGT website (http://www.ebi.ac.uk/img/hla).
Preferably said derivatives or variants exhibit the above-stated
sequence identity to sequences identified in "Nomenclature for
factors of the HLA system, 1998, Tissue Antigens 1999: 53,
407-446". Such derivatives or variants may be for example, as
described in (http://www.ebi.ac.uk/img/hla) or Table 4.
Alternatively stated, the encoded polypeptides may exhibit more
than 70%, preferably more than 80, 90 or 95% sequence identity or
exhibit such sequence identity to the functional portions of these
sequences, e.g. to naturally occurring allelic geographical or
allotypic variants e.g. to sequences as described in the IMGT
website (http://www/ebi.ac.uk/img/hla). Preferably said derivatives
or variants exhibit the above-stated sequence identity to sequences
disclosed in "Nomenclature for factors of the HLA system, 1998,
Tissue Antigens 1999: 53, 407-446". Such derivatives or variants
may be for example, as described in (http://www.ebi.ac.uk/img/hla)
or Table 4.
[0030] "Sequence identity" as referred to herein in connection with
nucleotide sequences refers to the value obtained when assessed
using ClustalW (Thompson et al., 1994, Nucl. Acids Res., 22, p
4673-4680) with the following parameters:
Pairwise alignment parameters--Method: accurate, Matrix: IUB, Gap
open penalty: 15.00, Gap extension penalty: 6.66; Multiple
alignment parameters--Matrix: IUB, Gap open penalty: 15.00, %
identity for delay: 30, Negative matrix: no, Gap extension penalty:
6.66, DNA transitions weighting: 0.5.
[0031] In connection with amino acid sequences, "sequence identity"
refers to sequences which have the stated value when assessed using
ClustalW (Thompson et al., 1994, supra) with the following
parameters:
Pairwise alignment parameters--Method: accurate, Matrix: PAM, Gap
open penalty: 10.00, Gap extension penalty: 0.10; Multiple
alignment parameters--Matrix: PAM, Gap open penalty: 10.00, %
identity for delay: 30, Penalize end gaps: on, Gap separation
distance: 0, Negative matrix: no, Gap extension penalty: 0.20,
Residue-specific gap penalties: on, Hydrophilic gap penalties: on,
Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular
residue is intended to include identical residues which have simply
been derivatized.
[0032] Functionally equivalent fragments according to the invention
may be made by truncation, e.g. by removal of regions of components
of the MHC molecule not crucial to formation of one or more
epitopes on said recombinant MHC molecule. Thus fragments may, as
with MHC molecules comprise more than one component. Such fragments
may be derived from naturally occurring molecules as described
above or from the functionally equivalent variants or derivatives
thereof. Preferably the fragments are between 50 and 500 residues,
e.g. 100 and 250 residues in length.
[0033] Preferably said MHC molecules are, or are derived from,
humans, domestic animals or livestock, such as cats, dogs, horses,
donkeys, sheep, cows, goats, rabbits, rats, mice, monkeys or apes,
although preferably the MHC molecules are HLA molecules, i.e.
derived from humans. Preferably said MHC molecules (or derivatives,
variants or fragments thereof) are class I molecules. The invention
does however also extend to class II MHC molecules. Especially
preferably the MHC molecules are in monomeric form. Said monomeric
form however refers to the minimum number of components necessary
to display one or more epitopes corresponding to an epitope of a
naturally occurring MHC allele, e.g. a heavy chain,
.beta.-microglobulin and an associated peptide. However, for class
II MHC molecules, less components may be necessary to form the
epitope. Therefore, it may not be necessary to include the
associated peptide, for example, for class II molecules. The method
of the invention employs recombinant MHC or MHC-type monomers in
order to detect anti-MHC antibodies. A number of recombinant HLA or
HLA-type molecules are known and described in the literature. As
mentioned above, any of these or their fragments, may be used
according to the present invention as indeed may any molecule, or
fragment thereof, exhibiting the properties and characteristics of
an HLA molecule ("HLA-type") as described further below.
[0034] Tetramers of human lymphocyte antigen A2 complexed with two
differed HIV-derived peptides or a peptide derived from influenza A
matrix protein are described in Altman et al., 1996, Science, 274,
94-96. In this paper, recombinant multimeric peptide-MHO complexes
are synthesized in order to bind to T cells. Tetramers of
recombinant HLA-2 complexed with any one of the three peptides
mentioned above were used. However, in the method of the invention
it is envisaged that monomers of recombinant HLA or HLA-type
molecules will preferably be used. The tetrameric complexes of
recombinant HLA class I are further described in Ogg et al., J.
Exp. Med., 1998, 188, 1203 to 1208 the disclosure of which is
herein incorporated by reference. In this paper, the HLA heavy
chain was expressed in Escherichia Coli.
[0035] In a preferred embodiment of the invention, the recombinant
MHC is synthesised in a prokaryotic expression system (see Example
1). It will therefore be understood by those skilled in the art
that the MHC molecule will be synthesised in an un-glycosylated
form, as prokaryotic cells do not have the capacity to glycosylate
proteins. Glycosylated sites are known to play important roles in
ligand binding, and would therefore be thought to be a necessary
component of anti-MHC antibody binding to MHC molecules.
Surprisingly, the inventors have discovered that the lack of
glycosylation of the recombinant HLA or HLA-type molecules is not
detrimental to anti-HLA antibody binding.
[0036] In the method of the invention, any suitable recombinant MHC
or MHC-type monomer may be used. It may be necessary to complex the
recombinant MHC with a peptide in order to present the recombinant
MHC/MHC-type monomer for anti-MHC antibody binding, and this forms
a preferred aspect of the invention especially with respect to
class I MHC molecules, fragments or derivatives thereof. Any
suitable peptide may be used in the complex, but it will be
understood that, in order to prevent antibodies in the sample
specific for the complexing protein binding to the complexing
protein, it is preferable to use a peptide against which most
individuals do not possess antibodies. Thus peptides may be used
which are ubiquitous or preferably to which the majority of a given
population or a particular individual has not been exposed.
[0037] The peptide utilized in the complex may be obtained from
natural sources or be synthetic. Naturally occurring peptides
include those which are endogenous in the cell in which the
recombinant MHC is synthesized. Such peptides may simply complex
with the MHC molecule during its synthesis. As discussed
previously, the expression system used to synthesize the
recombinant MHC may be a prokaryotic system. However, when using a
eukaryotic expression system, if an endogenous, expression-system
derived peptide is to be used in the complex, the expression system
used preferably involves cells from a distinct organism relative to
the organism, or sample thereof, to which said recombinant MHC will
be applied, e.g. the expression system involves non-human cells
when human antibodies are to be depleted.
[0038] Synthetic peptides may also be utilized in the MHC-peptide
complexes. Such synthetic peptides may be designed and synthesized
in order to fit the specific recombinant MHC molecule involved.
Preferably such peptides, are between 5 and 20 residues, e.g.
between 8 and 15 residues. Most preferably the synthetic peptide is
9 residues in length. Generally, the peptides accepted by MHC class
II molecules may be more variable in length. The synthetic peptide
is preferably generated on the basis of structural and/or spatial
and/or charge predictions of the peptide-binding groove of the MHC
molecule in order to obtain the best fit. Such generated synthetic
peptides are preferred in generating MHC molecules for use in the
invention.
[0039] Such synthetic peptides may be generated by any suitable
method, including conventional known synthesis techniques.
Non-naturally occurring amino acids may be used or the peptide
derivatized during or after synthesis providing this does not
affect the epitopic properties of the MHC molecule into which it
binds and/or does not involve an epitope which would be recognised
by antibodies in the organism, or a sample thereof, to which said
MHC molecule will be applied.
[0040] One example of suitable peptides which might be used
include, but are not limited to, peptides of viral origin such as
from the HIV (Human Immunodeficiency Virus) HCV (hepatitis C virus)
or influenza viruses or particular strains of the latter to which
most individuals have not been exposed. For example, the peptide
may be HIV or HCV derived. In such cases it will therefore be
necessary to ascertain whether the individual is HIV, HCV or
influenza positive prior to analysis of anti-MHC antibodies. Thus,
a recombinant MHC or MHC-type monomer complexed to HIV, HCV or
influenza virus derived peptides form a further preferred aspect of
the invention. Preferably said peptide is between 5 and 20
residues, e.g. between 8 and 15 residues, e.g. as described in
Example 1.
[0041] In order to detect the binding of anti-MHC antibodies to the
recombinant MHC molecules, it is preferred to attach the monomers
to a solid support, via any suitable linkage. As used herein
"linkage" refers to any interaction between the MHC molecule and
the solid support, enabling them to be associated. Such interaction
may involve physical association such as covalent binding and may
also involve so-called "weak" interactions such as hydrogen bonds,
Van der Waals forces and ionic interactions. Alternatively, the MHC
molecules may be provided with means for attachment to a solid
support. Such means may constitute or comprise, for example one
partner of an affinity binding pair, e.g. biotin, binding to the
corresponding binding partner of the affinity binding pair, i.e.
streptavidin, provided on the solid support. Alternative binding
pairs include antibodies:antigens and DNA:DNA binding proteins. The
HLA molecules may inherently comprise one of said partners, e.g. an
epitope or may be synthesized to contain said partner.
[0042] It forms a preferred aspect of the invention that the
recombinant MHC or MHC-type molecule is synthesized to contain an
enzymatic biotinylation site, such as Bir A. The monomer may then
be biotinylated using Bir A, biotin, ATP and Mg.sup.2+. The biotin
enables the MHC monomer to bind to streptavidin-coated solid
support, for example a solid support such as glass, plastic, tissue
culture plastic, a matrix such as sepharose, solid supports such as
iron or other metals, a solid particle such as for example a
magnetic or non-magnetic bead.
[0043] Where appropriate binding partners or the MHC molecules may
be attached to the solid support by any convenient means e.g.
attachment by methods well known in the art such as attachment
through hydroxyl, carboxyl, aldehyde or amine groups which may be
provided by treating the solid support to provide suitable surface
coatings.
[0044] The solid support may be any of the well known supports or
matrices which are currently widely used or proposed for
immobilisation, separation etc. As mentioned above, these may take
the form of particles, sheets, dip-sticks, gels, filters,
membranes, fibres, capillaries, or microtitre strips, tubes, plates
or wells etc.
[0045] Conveniently the support may comprise glass, silica, latex
or a polymeric material such as for example nitrocellulose,
agarose, alginate, teflon, latex, polystyrene or nylon. Preferred
are materials presenting a high surface area for binding. Such
supports will generally have an irregular surface and may be for
example be porous or particulate eg. particles, fibres, webs,
sinters or sieves. Particulate materials e.g. beads are generally
preferred due to their greater binding capacity, particularly
polymeric beads.
[0046] Conveniently, a particulate solid support used according to
the invention will comprise spherical beads. In an alternative
preferred feature the solid support is nitrocellulose e.g. a strip.
In a further alternative preferred feature the solid support is an
ELISA plate.
[0047] Non-magnetic polymer beads suitable for use in the method of
the invention are available from Dynal Particles AS (LillestrOm,
Norway) as well as from Qiagen, Pharmacia and Serotec.
[0048] However, to aid manipulation and separation, magnetic beads
are preferred. The well-known magnetic particles sold by Dynal
Biotech AS (Oslo, Norway) under the trade mark DYNABEADS, are
particularly suited to use in the present invention.
[0049] The attachment of the recombinant MHC or MHC-type molecule
to a solid phase allows easy manipulation of the monomer. Thus, the
attachment to some kind of solid phase can enable the separation of
the anti-MHC antibodies from the rest of the components in the
sample. This can be achieved for example by carrying out washing
steps, or if the components are attached to magnetic beads, using a
magnetic field to effect physical separation of the linked
component from the rest of the components in the mixture.
[0050] Therefore, in a preferred embodiment of the method of the
invention, the individual recombinant MHC or MHC-type monomer is
attached to a solid support, and is contacted with a sample from an
individual.
[0051] Alternative methods are however contemplated in which
detection of antibodies binding to MHC molecules is not achieved
using immobilized MHC molecules. Essentially, the assay described
herein relies on specific binding between recombinant MHC molecules
and antibodies thereto and this complex may be detected by any
convenient means e.g. by binding a further labelled molecule
specific for that complex or able to bind to and be used to remove
said complex from the sample or techniques which identify complex
formation per se, e.g. techniques which discriminate on the basis
of size.
[0052] The sample for use in the invention may be any suitable
sample that contains antibodies, and is preferably a body fluid
sample, i.e. blood, blood-derived samples, (e.g. plasma or serum),
saliva, interstitial fluid, lymphatic fluid or eluate from cells or
tissues. The sample may be used as collected or prepared or diluted
where appropriate. The sample is contacted with MHC molecules for a
time and under conditions appropriate for the formation of MHC
molecule:antibody complexes. The individual from whom the sample is
derived corresponds at least by genus, preferably by species, to
the MHC molecules to be used in the assay, and are preferably
human.
[0053] Where a solid support is used, after contact of the sample
with the solid support carrying the MHC molecules the solid support
is then washed or the physical separation of solid support and
sample is effected, in order to remove unbound antibodies and the
sample, leaving the relevant anti-MHC antibody bound to individual
recombinant MHC monomer attached to the solid support. In a
preferred embodiment of the invention, different individual
recombinant MHC or MHC-type molecules e.g. relating to one or more
epitopes of a naturally occurring allele are bound in individual
discrete wells-on a microtitre plate, to allow a `one well-one
antigen` format, which allows the testing of a sample for multiple
anti-MHC antibodies, delivering an accurate and precise picture of
which antibodies are present.
[0054] Any means known in the art can be used to detect the
antibody bound to the recombinant MHC or MHC-type molecules. When
sensitized to foreign MHC molecules, an individual produces
antibodies of both immunoglobulin G and M types (IgG and IgM) and
other isotypes such as Immunoglobulin A (IgA). The significance of
IgM and IgA antibodies directed against MHC in transplantation is
not completely understood. However, the presence of anti-MHC IgG
antibodies in a patient has been demonstrated to increase the risk
of graft rejection. Using appropriate recognition steps it is
possible to differentiate between anti-MHC IgG or IgA antibodies
and anti-MHC IgM antibodies bound to MHC molecules in accordance
with the invention. Both IgG and IgM may be detected in a single
assay of the invention, alternatively, the presence of IgG or IgM
can be independently determined.
[0055] The preferred method of detecting antibody bound to the
recombinant MHC monomer, is via ELISA (enzyme-linked immunosorbent
assay) and related methods, which involves the use of anti-IgG or
anti-IgM antibody conjugated to a signalling means, e.g. a label or
an enzyme (such as horseradish peroxidase). In the latter case,
upon addition of a known quantity of substrate for the conjugated
enzyme (such as p-nitrophenyl phosphate disodium), the presence and
quantity of bound antibody can be ascertained, as well known in the
art. As will be appreciated the source of the anti-IgG/IgM
antibodies are appropriately selected in line with the IgG/IgM to
be detected, e.g. in a human-derived sample anti-human anti-IgG or
anti-IgM antibodies are used, e.g. as may be generated by challenge
of e.g. rats, mice, or rabbits with human IgG/IgM.
[0056] However, any standard method of detecting antibodies may be
used in the method of the invention to detect anti-MHC antibodies
bound to MHC molecules optionally attached to a solid support, such
as the use of colloidal gold conjugated to protein A,
immunoelectron microscopy, flow cytometry or immunofluorescent
detection.
[0057] The method of the invention can primarily be used to detect
anti-MHC antibodies, particularly anti-HLA antibodies in the body
fluid sample of an individual prior to or after transplantation or
blood transfusion. The advantage of the method of the invention
over the prior art includes speed, reliability, accuracy and lack
of dependency on cells or cell derived products. Thus in further
aspects of the invention, the invention provides a method of
assessing MHC, preferably HLA compatibility, between 2 individuals
comprising the steps of detecting one or more anti-MHC antibodies
in a sample from said first individual according to the method
described hereinbefore and comparing the results thus obtained to
the MHC alleles present in said second individual.
[0058] In a preferred embodiment of the invention, the recombinant
MHC or MHC type molecules bound to the solid support are utilised
when treating blood derived products prior to analysis or
processing to produce a blood product--i.e. purified plasma, for
transfusion. The blood product may then be suitable for introducing
into a recipient. Thus in a further aspect the invention provides a
method of depleting a sample of MHC molecule antibodies comprising
at least the steps of contacting said sample with one or more
recombinant MHC molecules or functionally equivalent variants,
derivatives or fragments thereof, optionally attached to a solid
support and removing at least the recombinant MHC molecules to
which antibodies contained within the sample have bound. The
invention further extends to products thus formed. As referred to
herein, "depleting" refers to reduction, preferably complete
removal of said antibodies.
[0059] It will be understood that contacting a sample with
recombinant MHC molecule will remove antibodies which bind to the
MHC molecule. These antibodies may be free (circulating) antibodies
or may be attached to cell surfaces.
[0060] The MHC molecule antibodies may be attached to lymphocytes,
particularly .beta.-lymphocytes and mast cells, for example.
[0061] Thus, the method of depleting a sample of MHC molecule
antibodies will deplete free and cell-surface bound antibodies.
[0062] In the method of the invention, any number of recombinant
MHC or MHC type molecules may be used individually to assess the
anti-MHC antibodies present in the sample. The use of multiple
individual molecules is preferred, obtaining a complete picture on
the anti-MHC antibodies. Preferably 1 to 100, more preferably 1 to
75, more preferably 1 to 50 and most preferably 1 to 35 e.g. 10 to
75 or 15 to 30 recombinant MHC or MHC type Class I or II monomers
are used in the method of the invention. In a preferred embodiment,
at least 30, e.g. 30 to 35, e.g. 33 recombinant MHC or MHC type
class I monomers are used. Table 4 shows comprehensive coverage for
the method of the invention for anti-Class I HLA antibodies. The
invention further extends to a single solid support carrying more
than 2, preferably more than 10 different recombinant MHC molecules
or functionally equivalent variants, derivatives or fragments
thereof at discrete locations on said solid support which may be
processed separately according to the method of the invention to
allow separate detection and identification of antibodies directed
to each of said MHC molecules. Conveniently said support is in the
form of a filter or plate with discrete sites or wells and a
different MHC molecule is bound to each site or well and the
binding of antibodies at each of said sites or wells is
determined.
TABLE-US-00001 TABLE 4 Comprehensive allele coverage for future
monoLISA screening. This list comprises the alleles equivalent to
those which would be regarded as providing a broad coverage of HLA
class I specificities in a standard CDC test. Alleles in bold are
predicted to be the minimum required for monoLISA in the first
instance. A locus B locus C locus A*0101 B*0702 B*4402 Cw*0102
A*0201 B*0801 B*4501 Cw*0202 A*0301 B*1302 B*4601 Cw*0304 A*1101
B*1401 B*4701 Cw*0303 A*2301 B*1402 B*4801 Cw*0401 A*2402 B*1501
B*4901 Cw*0501 A*2501 B*1502 B*5001 Cw*0602 A*2601 B*1503 B*5101
Cw*0701 A*2902 B*1509 B*5201 Cw*0802 A*3001 B*1512 B*5301 Cw*1202
A*3101 B*1513 B*5401 Cw*1203 A*3201 B*1516 B*5501 Cw*1402 A*3301
B*1801 B*5601 Cw*1502 A*3401 B*2705 B*5701 Cw*1601 A*3601 B*3501
B*5801 Cw*1701 A*4301 B*3701 B*5901 Cw*1801 A*6601 B*3801 B*6701
A*6801 B*3901 B*7301 A*6901 B*4001 B*7801 A*7401 B*4002 B*8101
A*8001 B*4101 B*8201 B*4201
[0063] The invention also comprises kits for use in the methods of
the invention, which will normally include at least the following
components:
[0064] a) one or more recombinant MHC molecules or functionally
equivalent variants, derivatives or fragments thereof;
[0065] b) optionally a solid support, together with means for
attachment of the MHC molecules; and
[0066] c) a means for detecting anti-MHC-antibodies, preferably an
antibody which binds to the complex formed between said MHC
molecules and naturally occurring antibodies to said molecules,
e.g. anti-IgG or anti-IgM antibodies, preferably anti-human
anti-IgG or IgM antibodies.
[0067] The invention will now be described by way of non-limiting
examples with reference to the drawings in which:--
[0068] FIG. 1 shows the correlation between CDC and monoLISA
.delta.A for HLA-A2 reactivity, as a scatter plot of .delta.A
versus CDC reactivity. MonoLISA and CDC were performed on 85 sera
from renal dialysis or transplant patients and the corrected data
(.delta.A) are plotted. An arbitrary cut-off value of 0.5 was
applied. The experimental conditions are defined in Example 2;
[0069] FIG. 2 shows the correlation between CDC and monoLISA
.delta.A for HLA-B8 reactivity, as a scatter plot of .delta.A
versus CDC reactivity. The experimental conditions are as defined
in FIG. 1;
[0070] FIG. 3 shows the influence of presented peptide on
anti-HLA/monomer binding. Ten sera were tested in duplicate against
a panel of 4 recombinant HLA/peptide complexes made up of A*1101
monomers refolded around either HIV- or EBV-derived peptides. Mean
.delta.A values are shown here, on a plot of mean delta absorbance
versus serum 1D;
[0071] FIG. 4 shows the influence of glycosylation on anti-HLA
antibody binding to the monomer, as described in Example 4. Seven
sera were tested in duplicate against two monomer/peptide
combinations expressing the Bw6 motif (B*0702/EBV and B*0801/HCV).
These sera were from 5 patients with CDC-defined anti-Bw6
reactivity, one unsensitized male (MB) and one Bw4-reactive
individual (JA). Standardization between the two monomers was
carried out by measuring W6/32 reactivity. Mean .delta.A values for
the two different monomer/peptide complexes are shown on a plot of
mean delta absorbance v serum ID.
EXAMPLE 1
Materials and Methods
Recombinant HLA Molecules
[0072] Purified HLA heavy chain and 132 microglobulin were
synthesized using a prokaryotic expression system (pET; Novagen,
Milwaukee, Wis.). The heavy chain was modified by deletion of the
transmembrane/cytosolic tail and COOH-terminal addition of a 15
amino acid sequence containing the BirA enzymatic biotinylation
site. The heavy chain, .beta.2 microglobulin and peptide were
refolded by dilution. The 45-kD refolded product was isolated using
fast protein liquid chromatography and biotinylated on a single
lysine within the BirA substrate peptide, by BirA (Avidity, Denver,
Colo.) in the presence of biotin (Sigma Chemical Co., St. Louis,
Mo.), ATP (Sigma Chemical Co.) and Me (Sigma Chemical Co.). The
biotinylated product was separated from the free biotin by gel
filtration and ion exchange using FPLC. The monomers used had been
refolded with the peptides listed in table 1. The use of monomers
refolded with HIV- and HCV-derived peptides avoided the possible
confounding contribution of antibody specific for the peptide
rather than the alloantigen. In this manner, HLA-A2, HLA-A11,
HLA-B7 and HLA-B8 monomers (Class I HLA molecules) were
synthesized. Two different monomer/peptide combinations were used
as target antigens, Az/gag and B8/HCV.
TABLE-US-00002 TABLE 1 Details of recombinant monomer/peptide
combinations used. Monomer ID Allele Peptide sequence A2/gag (6)
A*0201 SLYNTVATL (9) B7/EBV B*0702 RPPIFIRRL (10) B8/HCV (8) B*0801
HSKKKKDEL (11) A11/nef A*1101 QVPLRPMTYK (12) All/pol A*1101
AIFQSSMTK (13) A11/EBV1 A*1101 IVTDFSVIK All/EBV2 A*1104 AVFDRKSVIK
(14)
Solid Support
[0073] 50 .mu.l of HLA monomer (at approximately 0.5
ng-.mu.l.sup.-1) was incubated in a streptavidin-coated 96 well
`combiplate` (Labsystems, Finland) at 22.degree. C. for 30 minutes.
All volumes were per well, and the monomers were conjugated to the
streptavidin-coated microtitre plates in a "one well, one antigen"
format. The plate was washed 4 times with 200 .mu.l wash buffer
(PBS with 0.05% Tween 20).
EXAMPLE 2
Materials and Methods
Serum Samples
[0074] Test sera were separated from the clotted blood of 100
transplanted and pre-transplant renal patients (test sera), and the
negative control for both assays was from an untransfused blood
group AB male. All patients were tested and were negative for HIV,
and sera from patients positive for HCV were excluded from this
study. All sera were stored at -20.degree. C. The sera were
selected for the presence of IgG of the following specificities as
determined by complement-dependent cytotoxicity (CDC): 26
anti-HLA-A2 sera, 28 anti-HLA B8 sera and 25 sera showing either no
alloreactivity (cytotoxic negativity), or alloreactivity to other
Class I molecules (irrelevant antibody). Four of the sera with
relevant IgG specificity contained antibodies with specificity for
both HLA-A2 and HLA-B8.
ELISA Assay of Serum Samples--"MonoLISA"
[0075] The serum was diluted 1:20 in dilution buffer (wash buffer
with 5% skimmed milk powder, Tesco Ltd, UK), and then incubated at
22.degree. C. for 30 minutes, on the HLA-coated `Combiplate`,
prepared as described in Example 1. The plate was then washed 3
times with 200 .mu.l wash buffer (supra). Each well was incubated
for 1 hour at 22.degree. C. with 100 .mu.l of sheep anti-human IgG
antiserum conjugated to horseradish peroxidase (Serotec, UK)
diluted 1:10000 in dilution buffer and then washed 3 times as
above. One hundred microlitres of 1 mg-ml.sup.-1
orthophenylenediamine dihydrochloride (Sigma, UK) solution in
phosphate-citrate buffer with sodium perborate (Sigma, UK) was
added and incubated for 15 min. at 22.degree. C. in the dark. The
reaction was stopped with 100 .mu.l of 1N HCl and the absorbance
(A) evaluated at 490 nm with a reference wavelength of 630 nm using
a Dynatech MRX plate-reader.
[0076] Results were corrected (.delta.A) for non-specific binding
(background) by subtracting the absorbance of the blank well, which
contained serum but no antigen, (A.sub.no antigen) from that of the
test well (A.sub.test). Preliminary data (not shown) led to the
assigning of an arbitrary cut-off value for .delta.A of 0.5. To
standardize the concentration of different monomers, the above
method was carried out identically, except that the mouse
monoclonal, W6/32 (Serotec, UK) at a concentration of 1:1000 in
dilution buffer, was substituted for patient's serum. Consequently,
a rat anti-mouse IgG2a monoclonal conjugated to horseradish
peroxidase (Serotec, UK), also at 1:1000 in dilution buffer was
used in place of the anti-human conjugate. The monomer was bound at
a range of doubling dilutions, and the concentration giving a
.delta.A closest to a standard value (3 absorbance units) was
chosen as the working concentration for that monomer.
EXAMPLE 3
CDC-Defined Specificity and .delta.A Values
[0077] Table 2 shows the CDC-defined specificity and .delta.A for
each of the 85 sera after testing against the A2/gag and B8/HCV
monomers. These same data are presented graphically as categorical
scatter plots in FIGS. 1 and 2, respectively. Briefly, 4/85
CDC-A2-negative sera were positive with the A2 monomer whilst no
CDC-A2-positive sera were negative with A2 monomer. The remaining
81 sera were concordant between the two methods, with 34 double
positive sera and 47 double negatives. With the B8 monomer, there
were 4 CDC-B8-ve/monoLISA+ve sera and, again, no CDC+ve/monoLISA-ve
serum, with 27 double positive and 54 double negative sera. The
monoLISA test using the A2 monomer exhibited 100% sensitivity and
92% specificity compared to CDC. Similarly, the B8 monoLISA
attained 100% sensitivity and 93% specificity.
TABLE-US-00003 TABLE 2 Comparison of CDC-defined specificities with
monoLISA .delta.A values. CDC and monoLISA detection results for 85
sera tested against the HLA-2 and HLA-B8 monomers are shown here.
Sera that were negative by CDC but positive with the relevant
monomer are highlighted. No sera were positive by CDC but negative
by monoLISA. Cytotoxicity MonoLISA Specificities A2.sup.2 B8.sup.3
A2.sup.4 B8.sup.5 A2, 28 + - 3.92 0.43 A2, 9 + - 3.74 0 A1, 2, 9,
28 + - 3.73 0.16 A2, 3 + - 3.67 0.09 A2, 3, 9, 28 + - 3.66 -0.05
A2, 24, 28 + - 3.55 -0.1 A2, 28 + - 3.55 0.33 A2, 28 + - 3.17 0.06
A1, 2, 3, 11 (?24) + - 3.07 0.74 A1, 2, 28 + - 3.05 0.53 A2, B8 + +
2.89 2.16 A2, 28, classII + - 2.88 0.23 A2, 28, B7 + - 2.85 0.18
A2+ + - 2.75 -0.01 A2, 28, 10, 34 + - 2.64 0.24 A2, 9 + - 2.49 0.01
a2, a9 + - 2.43 0.02 A2, B8 + + 2.11 2.72 A2, 9, 28 + - 2.07 0.16
Weak A2 + - 1.84 0.19 A2 + - 1.82 0.37 A2, B8, B57, DR7, 53 + +
1.81 0.55 A2, B8, 44 + + 1.75 1.3 A2, Bw4, Aw4 + - 1.09 0.13 A2 + -
1.08 0.71 A2, 28 + - 1 0.02 A2, 28 + - 0.91 0.06 A2, B7 CREG + -
0.9 0.21 A2 + - 0.86 0.05 A11, weak A2 + - 0.79 0.01 A2, B7 + -
0.72 0.4 Weak A2 + - 0.63 0.09 A2, IgM B8 + - 0.53 -0.05 B8, DR3 -
+ -0.04 2.71 A1, B8 - + -0.26 2.32 B8 - + 0.92 2.23 B8 - + 0.02 2.1
Weak B8 - + 0.05 2.01 A1, A9, B8 - + 0.92 1.87 B8, 18, 35, 53, 5,
21, 61 - + 2.02 1.85 B8, 59 - + -0.03 1.78 B8, weak 14, 39 - +
-0.03 1.51 B7, 8, weak Cw7 - + -0.02 1.41 B8, weak 14, 16 - + -0.02
1.4 B8, DQ2 - + 0.08 1.39 Weak B8 - + 0.06 1.3 B8, 16 - + 0.22 1.26
B8 - + 0.04 1.25 B8, 64, 65 - + 0.29 1.18 B8 - + 0.2 1 A1, B5, 35,
8, Bw6 - + -0.13 1 B8 - + 0.12 0.97 Weak B8 - + -0.06 0.82 B8, weak
B65, DQ2 - + 0.11 0.7 B8, Cw7 - + -0.06 0.66 B8, DR52 - + 0.08 0.57
A2 IgM - - 0.04 0.66 DR 3, 13 - - 0.1 0.47 Negative - - 0.23 0.45
Negative - - 0.43 0.42 Aw4, Bw4 - - -0.07 0.4 A9, B5, 35, 53, 15,
17 - - 0.48 0.36 A2 IgM, B44, 40, 41 - - 0.41 0.31 B51, 52, 53, IgM
B8, Bw4 - - -0.03 0.3 Multi B (not B8) - - 0.01 0.27 A9, weak A10,
A11 - - 0.01 0.27 A1, 3, 9, 11 - - 0.01 0.25 B7, 27 - - 0.11 0.14
A10 - - 0.18 0.07 B57+ - - -0.04 0.07 AB serum (Neg control) - -
-0.13 0.07 A10, 34, 32, 40, 41, 12, 13 - - 0.63 0.06 Weak IgM autos
- - 0.04 0.05 B17+ autos - - 0.1 0.04 B7, DQ1 - - 0.26 0.03 Weak
IgM B8 - - 0.02 0.03 B12, A1 - - -0.03 -0.01 A1, Bw4+ - - -0.15
-0.03 Probable autos - - -0.02 -0.04 IgM B8, 17, 27 - - -0.08 -0.04
DR11, 13, 8, (3, 14), DR52 - - 0.37 -0.07 Probable autos - - -0.23
-0.1 (DR13) - - 0.01 -0.14 Negative - - -0.12 -0.17 IgM B8, 27 - -
0.27 -0.25 .sup.1 CDC-defined specificities .sup.2,3Presence or
absence of CDC A2 and B8 reactivity respectively .sup.4,5.delta.A
values obtained with A2 and B8 monomers, respectively
Discussion
[0078] This study demonstrates an excellent correlation between the
techniques of CDC and monoLISA. With both the A2 and B8 monomers
tested there were positive reactions which, had previously been
negative by CDC. These reactions, which tended to be relatively
weak, are to be expected in a system that is designed to detect IgG
isotype-binding and not just the presence of cytotoxic antibodies.
This phenomenon has previously has been reported using the ELISA
method, PRA-STAT which detected HLA-specific IgG antibodies
relevant to transplant outcome that were not detected by CDC. More
importantly in this study, all the sera that showed positivity in
the CDC test were also positive using monoLISA. This is a good
demonstration that, at least using the sera tested, there was no
abrogation of the antibody/antigen interaction using recombinant
molecules in place of the natural ligand.
EXAMPLE 4
The Influence of Presented Peptide on Anti-HLA/Monomer Binding
[0079] To investigate whether the presence of different peptides
presented in the monomer would exert any effect on the strength of
antibody binding, 4 recombinant HLA/peptide complexes comprising
identical monomers but different peptide were used. This panel was
available for HLA-A* 1101 and included 2 HIV-derived peptides (nef
and pol) and 2 EBV-derived peptides (EBVI and EBV2; table 1). These
monomer/peptide combinations were applied to sera containing
antibodies against HLA-A11 as detected by CDC. To minimize the
interference of any possible specific humoral response directed at
the peptides, the peptides were derived from pathogens which were
either absent from the patient population (HLV) or ubiquitous
(EBV). Nine sera from sensitized patients were selected for the
presence of anti-A11 antibodies. A negative control serum was also
used for the absence of HLA-reactive immunoglobulin. These sera
were reacted in duplicate with each of the 4 different A11
monomer/peptide combinations. Each of the monomer/peptides was also
standardized with W6/32 in the same plates so that correction could
be made for slight variations in actual monomer concentrations.
Results
[0080] Each of the 10 sera gave grossly similar results with the
different monomer/peptide combinations, and all sera had
significantly higher .delta.A values than the negative control
(p<0.001, paired T). This demonstrates that bound peptide did
not significantly interfere with reactivity in the monoLISA test.
The results are depicted graphically as FIG. 3.
Discussion
[0081] The use of monomers with identical HLA antigens, but
different peptides within the groove offered an opportunity to
analyse the importance of the peptide in alloantibody responses.
Using a limited number of monomers of the same HLA specificity
(A11), but with different peptides, we have demonstrated that the
peptide does not have a large impact on alloantibody binding. This
is an important observation, as it suggests that single monomers
with irrelevant peptides within the groove can be used for antibody
screening in this assay.
EXAMPLE 5
The Influence of Glycosylation on Anti-HLA/Monomer Binding
[0082] Prokaryotic expressions systems such as Escherichia coli are
potentially restricted by their inability to glycosylate proteins.
As native HLA class I heavy chains have an N-linked carbohydrate
moiety at asparagine 86 it is possible that antibodies specific for
epitopes in the proximity of this residue may have differential
binding characteristics with recombinant monomers compared to
native protein. The Bw4 and Bw6 motifs, present on all expressed
HLA-B locus antigens constitute a well-defined operationally
dimorphic system. The residues responsible for these motifs are at
positions 77-83 on the class I heavy chain. This region of the
heavy chain sequence constitutes the most proximal known epitope to
the glycosylation site. Anti-Bw6 reactivity was thus used to
determine whether the absence of carbohydrate had a measurable
effect on the binding of alloantibodies. Antisera of the Bw6
specificity were tested with monoLISA using the two
monomers--B7/EBV and B8/HCV as Bw6 motif-bearing targets. Bw4
motif-bearing monomers were not available for testing. Antisera
from 5 patients with CDC-defined anti-Bw6 reactivity, one AB serum
and one Bw4-reactive serum were tested in duplicate against the two
monomer/peptide combinations. Standardization between the two
monomers was carried out by measuring W6/32 reactivity as above.
The sensitization details of the selected sera are given in table
3.
TABLE-US-00004 TABLE 3 Sensitization ID Specificity Route of known
HLA Transfusions MB Negative control None None IM Bw6 B7 tx.sup.1
None BC Bw6+ B7 & 8 tx B7 preg.sup.2 14 ABI Bw6 None 7 ABu Bw6
None 4 CP Bw6 B7 preg 7 JA Bw6 None 2 .sup.1tx, kidney transplant;
.sup.2preg, pregnancy-sensitization determined on the basis of the
HLA of the presumed father.
[0083] All the sera reacted above the arbitrary cut-off .delta.A of
0.5, and the negative controls both reacted at a lower level than
the no-monomer wells. Variation in binding levels between the
different sera was apparent, and with the exception of serum BC,
the results obtained with the two monomers followed the same trend.
These results show that the lack of a carbohydrate moiety did not
reduce the binding of antisera to a very close epitope, i.e. the
Bw6 motif. Indeed, the absorbance values obtained were mostly high
in the range of values seen using sera against other epitopes.
Discussion
[0084] The reactivity exhibited by the two Bw6 bearing monomers
suggests that the Bw6 motif can be detected using the monoLiSA
method. As the putative Bw6 motif is situated close to the
carbohydrate-bearing amino acid on HLA class I, this suggests that
lack of glycosylation of the monomers is not detrimental to
binding, and thus detection, of antibody. The possibility, however,
that the selected sera contained not only anti-Bw6 reactivity, but
also antibodies reacting with B7- and B8-specific motifs that are
spatially separate from the putative Bw6 region cannot be ruled
out. However, this is unlikely, given that similar strengths of
reactivity are demonstrated against both molecules with each
individual serum. The exception is serum BC. With this serum, the
monomers reacted differentially, the B8 eliciting approximately
double the reactivity produced with B7. This disparity in
reactivity can be explained, as the patient was sensitized with a
graft bearing both B7 and B8. A later serum from the same patient
showed a decrease in PRA that allowed the two specificities, B7 and
B8, to emerge from the broader Bw6 specificity (data not shown).
Presumably, the graft-presented B8 elicited a stronger response
than did B7, to which the patient was exposed on the graft and in
previous pregnancies.
Sequence CWU 1
1
819PRTArtificialderived from humans, domestic animals or livestock,
such as cats, dogs, horses, donkeys, sheep, cows, goats, rabbits,
rats, mice, monkeys or apes 1Gly Pro Ser Asn Asp Gln Glu Lys Arg1
529PRTHomo sapiens 2Ser Leu Tyr Asn Thr Val Ala Thr Leu1 539PRTHomo
sapiens 3Arg Pro Pro Ile Phe Ile Arg Arg Leu1 549PRTHomo sapiens
4His Ser Lys Lys Lys Lys Asp Glu Leu1 5510PRTHomo sapiens 5Gln Val
Pro Leu Arg Pro Met Thr Tyr Lys1 5 1069PRTHomo sapiens 6Ala Ile Phe
Gln Ser Ser Met Thr Lys1 579PRTHomo sapiens 7Ile Val Thr Asp Phe
Ser Val Ile Lys1 5810PRTHomo sapiens 8Ala Val Phe Asp Arg Lys Ser
Val Ile Lys1 5 10
* * * * *
References