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WO2009117785A1 - Procédé de typage tissulaire hla - Google Patents

Procédé de typage tissulaire hla Download PDF

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Publication number
WO2009117785A1
WO2009117785A1 PCT/AU2009/000382 AU2009000382W WO2009117785A1 WO 2009117785 A1 WO2009117785 A1 WO 2009117785A1 AU 2009000382 W AU2009000382 W AU 2009000382W WO 2009117785 A1 WO2009117785 A1 WO 2009117785A1
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hla
subject
polypeptide
class
amino acid
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PCT/AU2009/000382
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English (en)
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Matthew William Clemson
Wallace John Bridge
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Newsouth Innovations Pty Limited
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Priority claimed from AU2008901498A external-priority patent/AU2008901498A0/en
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Publication of WO2009117785A1 publication Critical patent/WO2009117785A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the present invention relates to methods for characterising the expression of HLA polypeptides from the cells, tissues or organs of an individual by obtaining an at least partial HLA polypeptide sequence from a biological sample from the individual.
  • HLA Human Leukocyte Antigens
  • MHC major histocompatibility complex
  • HLA polypeptides fall into two principal classes, the HLA class I and the HLA class II polypeptides.
  • HLA class I polypeptides are expressed on virtually all nucleated cells of the body, whilst HLA class II polypeptides are only expressed by cells of the immune system that can act as antigen presenting cells.
  • Each HLA class I protein is made up of two polypeptide chains: a heavy chain transmembrane polypeptide usually encoded by either the HLA-A, HLA-B or HLA-C genes, and a light chain beta-2 microglobulin (which is encoded on chromosome 15).
  • the major HLA class I proteins are co-dominantly expressed, so that an individual who is heterozygous at all three loci will express six different HLA class I complexes on the surface of most nucleated cells.
  • Each of the three HLA class I polypeptides are involved in immune responses, which allow for the recognition of "self and "non-self.
  • HLA class I molecules are involved in the presentation of endogenous antigens to cytotoxic T- cells, and provide a mechanism for T-cell mediated killing of viral-infected or tumour antigen-expressing cells. HLA class I molecules are also involved in protecting cells from natural killer (NK) cell mediated cytotoxicity by interacting with HLA-specific inhibitory receptors present on NK cells. The engagement of these receptors by autologous HLA class I molecules blocks the ability of NK cells to attack cells which would otherwise be recognized as foreign due to "missing self recognition.
  • NK natural killer
  • Each HLA class II protein is a heterodimer made up of two transmembrane polypeptides: an alpha chain and a beta chain, which are encoded by each of the HLA- DR, HLA-DQ and HLA-DP genes.
  • the alpha subunit is encoded by A or Al locus and the beta subunit is encoded by one B locus (Bl for DP and DQ, or Bl, B3, B4 or B5 locus for DR). All individuals possess a DRBl gene.
  • an HLA-DP complex may be encoded by the HLA-DPAl and HLA-DPBl loci
  • an HLA-DQ complex may be encoded by the HLA-DQAl and HLA-DQBl loci.
  • HLA-DR complex is encoded by the HLA-DRAl locus and by one of the HLA-DRBl, HLA-DRB3, HLA- DRB4 or HLA-DRB5 loci, hi humans, each chromosome 6 codes for four different HLA class II molecules (two different HLA-DR, one HLA-DQ and one HLA-DP molecule) such that a heterozygous individual will express eight different HLA class II molecules.
  • HLA class II proteins are only expressed on the surface of specialized antigen-presenting cells, such as B-lymphocytes, macrophages and dendritic cells.
  • HLA class II antigens are involved in the presentation of exogenous antigens, such as bacterial or viral antigens to T-helper cells, which in turn activate the production of specific B-cells and the production of antibodies which allow the neutralization of the antigen.
  • the HLA loci are amongst the most polymorphic coding sequences in the human genome. More than ten allele groups at each locus have been identified for the majority of the HLA loci discussed above, with approximately 1900 class I alleles and approximately 800 class II alleles being identified. Currently over 600 human HLA B alleles and over 390 alleles for the HLA DRBl loci alone have been identified. At least 1500 class I and 718 class II proteins and 110 null alleles (genetically encoded HLA alleles that are not expressed as protein) have been identified.
  • Histocompatibility that is the property of having the same, or mostly the same, alleles of the major histocompatibility complex is of vital importance in determining the probability of whether an individual will acutely reject a transplanted tissue, or whether a transplanted haematopoietic tissue will cause a graft-versus-host disease (GvHD).
  • GvHD graft-versus-host disease
  • HLA class I and II expressed by the potential donor and host are matched as closely as possible. Furthermore, certain HLA alleles are associated with an increased risk of autoimmune disorders and other disease states, and so HLA typing is of use in rapidly distinguishing individuals at risk or not at risk of developing certain diseases. It is noteworthy that only a small part of the amino acid sequence of each HLA protein is involved in self- recognition. For the purposes of transplantation, genetic polymorphisms which produce changes to the HLA protein primary structure (amino acid sequence), especially within the ⁇ l and ⁇ 2 domains of the class I HLA and both ⁇ and ⁇ chains of class II HLA, are involved in self recognition, affect self-recognition and antigen presentation.
  • amino acid sequence amino acid sequence
  • HLA matching may have no effect on the clinical outcome of a transplant pairing.
  • HCT hematopoietic cell transplantation
  • HCT hematopoietic cell transplantation
  • DNA molecular typing techniques which rely on the use of specific polynucleotide probes and primers to allow the amplification of the DNA which expresses the HLA, generally followed by restriction enzyme digestion and fragment analysis to identify the specific alleles which are expressed.
  • the DNA molecular typing techniques allow a more refined description of alleles, leading to an alternative nomenclature where each allele is assigned an identifier in the form "HLA-X*Y", where X is the HLA class letter and Y is an identifier of at least two and more usually four numerals for the allele.
  • Marsh et al. (2005) provides an overview of the nomenclature of serologically and genetically defined HLA antigens.
  • DNA molecular typing techniques are relatively slow to provide results due to the requirement for repeated polynucleotide amplification reactions, and these techniques expend a large plurality of HLA class-, type- and/or allele-specific oligonucleotide primers and probes for each screening test.
  • Analysis in DNA molecular typing techniques is complex, because heterozygous individuals may express two different bases at multiple positions along an HLA gene, requiring complex analysis to predict the two separate sequences.
  • a method of characterising Human Leukocyte Antigen (HLA) expression of a subject comprising enriching a biological sample from the subject for an HLA polypeptide from the subject, obtaining an at least partial amino acid sequence of the HLA polypeptide from the subject, and comparing the at least partial amino acid sequence of the HLA polypeptide from the subject to a library of known amino acid sequences of HLA polypeptides and assigning the at least partial amino acid sequence of the HLA polypeptide from the subject to one or more known HLA polypeptides.
  • HLA Human Leukocyte Antigen
  • the biological sample from the subject is a blood sample.
  • the step of enriching a biological sample from the subject for an HLA polypeptide comprises an immuno-purification step.
  • the immuno-purification step comprises the use of an HLA class I specific antibody and/or the use of an HLA class II specific antibody.
  • the subject is a potential donor of cells, tissue or an organ, hi other embodiments the subject is a potential recipient of donated cells, tissue or an organ.
  • the subject is suspected of, or at risk of, or with a predisposition towards a disease or condition associated with the expression of one or more specific HLA alleles.
  • the disease or condition associated with the expression of one or more specific HLA alleles may be an autoimmune disease.
  • the disease or condition associated with the expression of one or more specific HLA alleles may be selected from rheumatoid arthritis, lupus like disease, type I diabetes, Behcet's disease, HLA-B27-Associated Cardiac Disease, ankylosing spondylitis, Systemic Lupus Erythematosus, multiple sclerosis, Sjogren's syndrome, myasthenia gravis, viral infection such as HIV infection or hepatitis infection, and pemphigus vulgaris.
  • HLA Human Leukocyte Antigen
  • Figure 3 is a photograph of a Western blot analysis of irnmunoaffmity purified soluble HLA Class I antigens from human serum. This figure follows the immunoaffmity purification of HLA class I antigens from the serum of two individuals (identified as AC and LO). Lanes 1 and 9 contained protein molecular weight standards. Lanes 3 and 6 contained serum from subjects AC and LO respectively. Lanes 4 and 7 contained the immunoaffinity column flow through material from subjects AC and LO respectively. Lanes 5 and 8 contained the eluted HLA-enriched fraction from subjects AC and LO respectively. This figure illustrates that prior to immunoaffinity purification and subsequent concentration, the soluble HLA present in the serum samples was below the detection limits of the assay.
  • Figure 4 provides the amino acid sequence of the HLA class I, A-I alpha chain precursor (allele HLA-A*0101), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject HM.
  • Figure 5 provides the amino acid sequence of the HLA class I, A-11 alpha chain precursor (allele HLA-A* 1101), overlayed on which in bold and underlined font are the amino acid sequences which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject HM.
  • Figure 8 provides the amino acid sequence of the HLA class I, Cw-7 alpha chain precursor (allele HLA-Cw*0701), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject HM.
  • Figure 9 provides the amino acid seqiience of the HLA class I, Cw-7 alpha chain precursor (allele HLA-Cw*0706), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject HM.
  • Figure 10 provides the amino acid sequence of the HLA class I, Cw-7 alpha chain precursor (allele HLA-Cw*0718), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject HM.
  • Figure 15 provides the amino acid sequence of the HLA class I, A-2 alpha chain precursor (allele HLA-A* 0201), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject MC.
  • Figure 16 provides the amino acid sequence of the HLA class I, B-7 alpha chain precursor (allele HLA-B*0702), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject MC.
  • Figure 18 provides the amino acid sequence of the HLA class I, A-I alpha chain precursor (allele HLA-A*0101), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject AC.
  • Figure 19 provides the amino acid sequence of the HLA class I, A-2 alpha chain precursor (allele HLA-A*0203), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject AC.
  • Figure 20 provides the amino acid sequence of the HLA class I, B-8 alpha chain precursor (allele HLA-B*0801), overlayed on which in bold and underlined font are the amino acid sequences, which were identified by mass spectroscopy analysis of the immunoaffinity purified soluble HLA from subject AC.
  • Figure 22 provides the known predicted amino acid sequence alignment between
  • HLA-B8 alpha chain precursor (allele HLA-B*0801) and HLA B-49 alpha chain precursor (allele HLA-B *4901), demonstrating the expected regions of homology between the expressed proteins at the HLA-B locus in individual HM.
  • "Consensus” shows amino acids which are the same for alleles, and amino acids which differ between these two proteins are listed in the rows HLA-B*0801 and HLA-BM901.
  • This figure demonstrates that the amino acid sequence of the expressed HLA-B 8 and HLA-B49 proteins should be identical in the region spanning amino acid positions 207 to 298. For simplicity, this region has been chosen to outline several ambiguities which remain following full molecular DNA sequence based typing.
  • Figure 23 provides the forward and reverse DNA sequence which was obtained during bi-directional sequencing of the HLA-B locus over exon 4 for subject HM.
  • Consensus shows nucleotides which are the same within both the forward and reverse DNA sequencing data.
  • b4f indicates DNA sequence obtained from the HLA-B locus, exon 4 forward sequencing primer.
  • b4r.l indicates DNA sequence obtained from the HLA-B locus, exon 4 reverse sequencing primer.
  • " - indicates regions of homology between the DNA sequence obtained in the forward and reverse sequencing reactions.
  • the sequence beyond nucleotide 257 indicates that the forward sequencing reaction proceeded beyond the limits of the reverse reaction in this region.
  • the DNA codes for ambiguous positions are listed as:
  • the method comprises enriching a biological sample from the subject for an HLA polypeptide, obtaining an at least partial amino acid sequence of the HLA polypeptide by subjecting the biological sample which is enriched for an HLA polypeptide to a mass spectrometry analysis, and comparing the at least partial amino acid sequence of the HLA polypeptide to a library of amino acid sequences of known HLA polypeptides and assigning the at least partial amino acid sequence of the HLA polypeptide to one or more known HLA polypeptides.
  • HLA polypeptides The direct sequencing of expressed HLA polypeptides from a subject negates the identification of null alleles that is crucial in DNA-based typing methods, and allows transcriptional (such as alternate mRNA splice variants) and translational (glycosylation) variations to be identified, which cannot be resolved using conventional genotyping techniques.
  • the term "Human Leukocyte Antigen” is intended to encompass HLA class I and HLA class II molecules. Within each of these classes of HLA molecules, there are "types" of HLA.
  • HLA class I molecules comprise the types HLA-A, -B, -C, -E, -F, -G, -H, -J, -K, -L, -N, -S, -X and -Z, with the type based on the identity of the heavy chain (the chain which is responsible for anchoring the HLA to the cell membrane) HLA class I polypeptide which is expressed.
  • the class of HLA class II molecules comprises the types HLA-DRA, HLA-DRBl to HLA-DRB9, HLA-DQAl, HLA-DQBl, HLA-DQA2, HLA-DQB2, HLA-DQB3, HLA-DOA, HLA_DOB, HLA_DMA, HLA-DMB, HLA-DPAl, HLA-DP A2, HLA-DP A3, HLA-DPBl, and HLA- DPB2, with the type based on the identity of the HLA class II alpha or beta chain polypeptide which is expressed.
  • Human Leukocyte Antigen-like molecule is intended to include any one or more of TAPl and 2, PSMB8 and 9, MICA, MICB, MICC, MICD, and MICE. (Marsh et ah, 2005). These proteins are all involved in immune responsiveness, although in most cases the details of the mechanism(s) involving these molecules are yet to be elucidated.
  • the nature of this invention relates to methods for characterizing HLA expression, which in certain embodiments comprises identifying or distinguishing between different HLA classes, types and alleles. Variations to the sequences of members of different HLA classes, types or alleles present in the database, or the addition or deletion of members of different HLA classes, types or alleles present in the database may occur as new alleles are discovered or duplications or errors of records in the sequence database are identified.
  • the methods of characterizing HLA expression described herein relate to the identification of an HLA class, type or allele of one or more HLA polypeptide(s) expressed by a subject.
  • the number of series of contiguous amino acid sequences which may be used to attribute the sequence to a particular HLA class, or a particular HLA type, or a particular HLA allele, or to a group consisting of two or more HLA classes, types or alleles may vary depending on the size of the contiguous amino acid sequence(s) and the position(s) the sequences lie on the sequence of the complete HLA polypeptide.
  • a single contiguous amino acid sequence may be sufficient to characterize the HLA polypeptide where the contiguous amino acid sequence passes through a unique sequence which is characteristic of the particular HLA class, or the particular HLA type, or the particular HLA allele, or to a group consisting of two or more HLA classes, types or alleles.
  • the number of contiguous amino acid sequences may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more.
  • HLA-A*0201 NCBI accession no. NM_0021166
  • exons 2, 3 and 4 combined represent 822 nucleotides of DNA sequence, or 274 amino acids corresponding to positions 25 to 298 of the expressed HLA protein.
  • Most SBT strategies currently employed use only exons 2 and 3 for HLA class I typing. This would correspond (in the case of HLA- A2) to 546 nucleotides encoding 182 amino acids at positions 25 to 206 of the expressed HLA protein.
  • Elevated serum concentrations of soluble HLA antigens have been shown to correlate with rejection of heart, liver, kidney, kidney/pancreas and bone marrow allografts and has been suggested as a practical means of monitoring post-transplant alloreactivity. More specifically, the detection of donor derived soluble HLA in the serum of transplant recipients led to the observation that high and stable concentrations of donor soluble HLA may induce immune tolerance and prolonged, stable graft function.
  • HLA classification representing a low resolution of HLA of the subject.
  • the characterizing of HLA expression of a subject may be used in screening a subject, or cells, tissue or organs of a subject for suitability in cell, tissue or organ donation.
  • the characterizing may be used for screening a subject for the expression of specific HLA alleles associated with risk of, or with a predisposition towards a disease or condition.
  • the disease or condition associated with the expression of one or more specific HLA alleles may be an autoimmune disease.
  • the disease or condition associated with the expression of one or more specific HLA alleles may be selected from rheumatoid arthritis, lupus like disease, type I diabetes, Behcet's disease, HLA-B27-Associated Cardiac Disease, ankylosing spondylitis, Systemic Lupus Erythematosus, multiple sclerosis, Sjogren's syndrome, myasthenia gravis, susceptibility to viral infection such as HIV, or hepatitis virus and pemphigus vulgaris.
  • individuals who are HLA- B27 or HLA-B57 positive demonstrate "long-term non progression" in that HIV disease progression is much slower in these individuals.
  • the step of comparing the at least partial amino acid sequence of the HLA polypeptide to a library of known HLA polypeptide sequences is most conveniently carried out using bioinformatics software packages such as Mascot version 2.2.04, Matrix Science (David N. Perkins 1999).
  • a searchable database of known HLA polypeptide sequences may be found at ⁇ ftp://ftp.ebi.ac.uk/pub/databases/imgt/mhc/hla/> (Robinson, Waller et al 2003).
  • the HLA polypeptides are soluble HLA polypeptides.
  • Soluble forms of HLA include class I heavy chain polypeptides and class II alpha and beta chains of all the types discussed above.
  • the isolation of soluble HLA polypeptides have been described from the serum of healthy individuals, and in individuals with infections, tumours, following transplantation or blood transfusions (Aultman, Adamashvili et al 1999; Ghio, Contini et al. 1999; Munoz-Fernandez, Martin et al. 2001; Toussirot, Saas et al. 2006; Novikov, Egorova et al. 2007).
  • the concentration of sHLA correlates to the presence of certain HLA allotypes (higher levels for HLA- A23, -24, -29 and -33, and lower for HLA- A2) and the concentration may change due to a number of disease states including cancer (McDonald and Adamashvili 1998).
  • soluble HLA polypeptides from blood serum may offer advantages in the relative ease of obtaining a biological sample which comprises the HLA polypeptides, and the relative ease by which the HLA polypeptides may be enriched and preferably substantially isolated from other expressed polypeptides.
  • the HLA polypeptide is a membrane-bound HLA polypeptide, such as an HLA polypeptide which is expressed on the surface of white blood cells or on cells present in a tissue biopsy or biological sample.
  • the method may further comprise the separation of the HLA polypeptide from the membrane. For example separation of the HLA polypeptide from the membrane may take place before or during the step of enriching the biological sample for the HLA polypeptide.
  • the characterising of the HLA expression of a subject may comprise the identification of one HLA type expressed by the subject. It may comprise the identification of multiple HLA types expressed by the subject. It may comprise the identification of all the HLA types expressed by the subject.
  • the characterising of the HLA expression of a subject may comprise the identification of one or more HLA alleles expressed by the subject.
  • the characterising of the HLA expression of a subject may comprise the identification that one or more alleles are not expressed by the subject.
  • the characterising of the HLA expression of a subject may comprise the identification that the subject expresses any one of a group of alleles.
  • the characterising of the HLA expression by a subject may comprise the characterising of the expression of an HLA class I polypeptide.
  • the characterising of the HLA expression of a subject may comprise the identification of the expression of an HLA class II polypeptide.
  • the characterising of HLA expression of a subject may comprise the identification of the expression of an HLA-like polypeptide as defined above.
  • the "subject" is a non-human animal or non-human animal tissues, cells or organs expressing the animal equivalent of HLA or a homologous MHC antigen, such as an animal of commercial, economic or research importance, or tissue or organs donated by or obtained from the subject.
  • HLA is intended to encompass the animal equivalent of HLA or a homologous MHC antigen.
  • a database of a broad range of non-human species whose MHC has been partially characterised is available at The Immuno Polymorphism MHC Database ⁇ http://www.ebi.ac.uk/ipd/mhc/>. This database is described in Ellis et al.
  • cell lines such as B-lymphoblastoid cell lines
  • transformed cell lines such as melanoma cell lines derived from primary human cell cultures also express soluble and membrane bound forms of HLA class I and/or II antigens that are the same as those from the host from which the cell line was derived.
  • the identification of sufficient sequence data to infer a HLA type may require an enrichment step prior to analysis.
  • the purpose of enriching for HLA polypeptide is to ensure that the HLA is detectable and that the amino acid sequence information from the HLA may be generated.
  • the term "enriching" may comprise increasing the concentration of the HLA polypeptide relative to the concentration of at least one non-HLA polypeptide present in the sample prior to the enrichment, for example the enrichment will reduce the proportion of at least one non- HLA polypeptide which may be present in amounts in the biological sample prior to enrichment which may otherwise mask or confound the presence of HLA sequences.
  • HLA protein For a typical mass spectrometry analysis, 100 fmol of protein would be trypsin digested and applied to the HPLC column for analysis.
  • the purity of the HLA protein will affect the amount of HLA sequence that is identified in a single analysis. As exemplified herein, less than 5 pmol of total peptide was loaded for mass spectrometry analysis and the proportion of the HLA-derived peptides to total peptides within the sample was approximately 10% w/w.
  • Variables such as the mass range of the instrument (50 - 4000 Da), the efficiency of the peptide separation, the ionization efficiency, and the collision induced dissociation efficiency may affect the quality of the data obtained.
  • anti HLA class II antibodies may recognise conserved epitopes presented on either the ⁇ or ⁇ chain. Since the ⁇ and ⁇ chains are normally associated with each other in vivo, immuno-purification of the ⁇ - chain of an intact soluble HLA may co-precipitate the ⁇ -chain and vice versa.
  • the monoclonal antibodies IVA12 (ATCC number HB-145) and LGII-612.14 are examples of monoclonal antibodies recognising conserved regions on HLA class II molecules. These antibodies were chosen for the purpose of simplifying the purification, since a single mAb can be used to capture all of the known polymorphic HLA polypeptides within the sample.
  • combinations of any one of HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-N, HLA-S, HLA-X and HLA- Z with any one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-N, HLA-S, HLA-X and HLA-Z may be used.
  • the combination is of two antibodies specific to two different HLAs.
  • the combination is of three antibodies specific to three different HLAs.
  • the combination is of an HLA-A specific antibody, an HLA-B specific antibody and an HLA-C specific antibody.
  • a combination of two or more antibodies specific to HLA-DRA, HLA-DRBl to HLA-DRB9, HLA-DQAl, HLA- DQBl, HLA-DQ A2, HLA-DQB2, HLA-DQB3, HLA-DOA, HLA_DOB, HLA_DMA, HLA-DMB, HLA-DPAl, HLA-DP A2, HLA-DP A3, HLA-DPBl, and HLA-DPB2 may be used.
  • the combination is of two antibodies specific to two different HLAs.
  • the combination is of three antibodies specific to three different HLAs.
  • the degree of enrichment desirable to optimise the resolution of particular HLA sequences from a biological sample will depend on the initial concentration of the HLA sequence in the biological sample, and the concentration and nature of other non-HLA proteins in the sample. Based on densitometry analysis of western blot data provided in the examples described herein, it may be estimated that the immunoaffmity purified HLA which was subsequently analysed by mass spectrometry was enriched at least about 1, 000-fold relative to the initial concentration of HLA present in serum.
  • an HLA polypeptide includes polypeptides of a single HLA type, as well as polypeptides of two or more HLA types; and so forth.
  • HLA specific antibodies are described herein, and are contemplated for use in the kits. In addition, other HLA-specific antibodies not expressly identified herein are contemplated.
  • the purification procedure was assessed via SDS-PAGE (NuPAGE 4-12% Bis-Tris gel, Invitrogen cat no NPO322BOX).
  • the antigen-loaded column was sequentially washed with 10 mL each of 0.1 M Tris-HCl (pH 7.8), 0.5 M NaCl, 1.0 M Tris-HCl (pH 7.8) and 0.1 M Tris-HCl (pH 7.8) to remove any non-specifically bound proteins.
  • the bound HLA proteins were eluted with 10 mL 0.05 M diethylamine (pH 7.8)
  • mAb HC-10 (IgG2a, a gift of Prof. Soldano Ferrone, Roswell Park Cancer Institute, Buffalo New York) recognizes a determinant expressed on all ⁇ -2- microglobulin-free HLA-B and -C heavy chains and on ⁇ -2-micro globulin-free HLA- A3, AlO, A28, A29, A30, A31, A32, and A33 heavy chains (Stam, Spits et al. 1986) and was visualised using an HRP-conjugated goat-anti mouse Ig and the peroxidase- tetramethylbenzidine reaction to produce a coloured label.
  • Lanes 1 and 9 are molecular weight markers (10 ⁇ L pre-stained standard proteins, Invitrogen, cat no LC5925); Lane 2 was blank; Lane 3 was 13 ⁇ L 4% v/v of serum from individual "AC”; Lane 4 was 13 ⁇ L of immunoaffmity column flowthrough from a sample from individual "AC”; Lane 5 was 13 ⁇ L of soluble HLA concentrate from individual "AC”; Lane 6 was 13 ⁇ L of 4% v/v of serum from individual "LO”; Lane 7 was 13 ⁇ L of immunoaffinity column flowthrough from a sample from individual "LO”; and Lane 8 was 13 ⁇ L of soluble HLA concentrate from individual "LO".
  • HLA class I histocompatibility antigen Cw-4 alpha chain 1C04_HUMAN 255 40969 6 12.8
  • HLA class I histocompatibility antigen A-I l alpha chain
  • HLA class I histocompatibility antigen B-15 alpha chain
  • the amino acid sequence which is recognised by the Mass Spectometry analysis lies between the periods.
  • the periods represent the position of the cleavage by the typsin.
  • the single amino acid residue on each side of the recognised sequence is part of the greater sequence which is required for typsin digestion.
  • the second stage fragmentation performed during the tandem mass spectrometry analysis selects the most abundant peptides from the first stage of mass spectrometry and then fragments the peptide to produce fragment ions. Identification of the mass/charge ratios of these ions and data collation allows a peptide sequence to be obtained.
  • the present analysis is based on probability scoring, and so increasing the enrichment of the sample or the individual isolation of HLA allospecific sequences may improve the amount of sequence data generated. For example, Western blotting to identify the location of HLA heavy chains separated in one-dimensional SDS polyacrylamide gel electrophoresis will allow the appropriate (HLA containing) bands to be excised from identically-electrophoresed gels and these bands to be analysed together or individually via tandem mass spectrometry.
  • the limited coverage of the HLA C sequence by the amino acid sequences identified by the mass spectrometry analysis meant that it was not possible with the data produced to determine which one or two of the three HLA C alleles identified was expressed by the individual. Repeated analysis of the same sample is expected to produce additional sequence coverage and should assist in resolving the identification of which of these HLA is actually expressed.
  • the purified HLA polypeptides are digested with trypsin and separated by strong cation exchange and reversed-phase HPLC. Tryptic peptides bound to the Cl 8 column are sequentially eluted using a gradient concentration of solvent in water (typically acetonitrile is used as the solvent). A voltage is applied to the flow and positive ions are generated via electrospray and analysed in the first stage of mass spectrometry. The most abundant ions are sequentially isolated and fragmented in a linear ion trap via collisionally induced dissociation. The mass-to-charge ratios of the tryptic peptides and the mass-to-charge ratios of the fragment ions produced by CID are used to determine the peptide sequence. These peptide sequences are collectively compared and aligned against a database of known protein sequences (most commonly Swiss-Prot or NCBI) to identify the proteins most likely to be within the sample.
  • a database of known protein sequences most commonly Swiss-Prot or NCBI

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Abstract

La présente invention concerne un procédé de caractérisation de l’expression de l’antigène des leucocytes humains (HLA) d’un sujet, comprenant a) l’enrichissement d’un échantillon biologique provenant du sujet pour un polypeptide HLA provenant du sujet, b) l’obtention d’au moins une séquence partielle d’acide aminé du polypeptide HLA provenant du sujet et c) la comparaison de la ou des séquences partielles d’acide aminé du polypeptide HLA provenant du sujet avec une banque de séquences de polypeptides HLA connus et l’attribution de la ou des séquences partielles d’acide aminé du polypeptide HLA provenant du sujet à un ou plusieurs polypeptides HLA connus.
PCT/AU2009/000382 2008-03-28 2009-03-30 Procédé de typage tissulaire hla WO2009117785A1 (fr)

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AU2008901498A AU2008901498A0 (en) 2008-03-28 Method of HLA tissue typing
AU2008901498 2008-03-28

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US20130096024A1 (en) * 2010-04-27 2013-04-18 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Method for detecting the susceptibility to develop adverse side effects related to bioimplants

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WO2005036180A1 (fr) * 2003-10-08 2005-04-21 The Government Of The United States Of America As Represented By The Secretary Of Department Of Health And Human Services Methodes d'analyse utilisant des biomarqueurs concentres avec des molecules d'attraction de biomarqueurs
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130096024A1 (en) * 2010-04-27 2013-04-18 Fundació Hospital Universitari Vall D'hebron - Institut De Recerca Method for detecting the susceptibility to develop adverse side effects related to bioimplants

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