WO2019211864A1 - Dosage multiplex fluorescent moléculaire pour identifier des allo-anticorps dirigés contre des antigènes du cmh - Google Patents

Dosage multiplex fluorescent moléculaire pour identifier des allo-anticorps dirigés contre des antigènes du cmh Download PDF

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WO2019211864A1
WO2019211864A1 PCT/IN2019/050327 IN2019050327W WO2019211864A1 WO 2019211864 A1 WO2019211864 A1 WO 2019211864A1 IN 2019050327 W IN2019050327 W IN 2019050327W WO 2019211864 A1 WO2019211864 A1 WO 2019211864A1
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mhc
antigen
antigens
allo
antibodies
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PCT/IN2019/050327
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Vimarsh Raina
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Chimera Translational Research Fraternity Private Limited
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease

Definitions

  • the present invention relates to a molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens. More specifically, the present invention relates to a cost-effective, specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention also relates to an economical kit for molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • Organ transplantation is one of the most complicated therapies. Many challenges are associated with each stage of organ transplant. Limited availability of organs, counseling the matched donor (in case of live donor transplant) or donor’s family (in cases of deceased donor transplant) challenges the organ donation process. These challenges are followed by the high possibility of organ rejection by the recipient’s body, post transplantation. This happens when the recipient’s immune system recognizes the transplanted organ as foreign and destroys it resulting in failure of the transplantation.
  • transplant rejection post transplantation There are two ways to minimize the risk of organ rejection post transplantation. Recipients undergoing organ transplantation are administered with immunosuppressive drugs. These drugs suppress the immune response in the recipient’s body to safeguard the transplanted organ from rejection. In addition to immunosuppressants transplant rejection risk can be minimized by determining donor-recipient compatibility before transplantation.
  • Cell-based and solid-phase antibody screening assays are employed for the detection of allo-antibodies in recipient’s serum against donor specific MHC antigens and hence they help in determining donor-recipient compatibility for transplant. If allo-antibodies against donor specific MHC antigens are detected in the recipient’s serum, it is recommended to not undergo transplant with the tested donor as organ rejection risk in such a case is very high.
  • CDC-XM complement-dependent cytotoxicity cross match test
  • FCXM flow cytometry crossmatch
  • Solid-phase antibody identification assays shows increased sensitivity as compared to cell-based assays.
  • solid phase assays There are two kinds of solid phase assays - ELISA (enzyme linked immunosorbent assay) and assay employing fluorescent beads coupled with MHC antigens. These assays involve adding recipient's serum on a fluorescently labelled surface coupled with MHC antigens. The antigen-antibody reaction between the allo-antibodies present in the recipient’s serum and MHC antigens coupled on the fluorescently labeled surface is analyzed, enabling the identification of allo-antibodies present in the recipient’s serum.
  • ELISA enzyme linked immunosorbent assay
  • Solid phase assay is reported to have high sensitivity, however limitation on the number of MHC antigens that can be coupled with the fluorescently labelled surface and high genetic diversity of MHC antigens poses the risk of missing out allo-antibodies present in recipient’s serum. This might further result in misinterpretation of potential donor- recipient incompatibility, increasing the chance of transplant failure by several folds.
  • MHC antigens are genetically very diverse. Extensive genetic diversity of MHC antigens is witnessed in Indian population. The Indian population, comprises of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts.
  • the Indian Genome Variation database (IGVdb) Consortium was established with the objective of building an SNP and repeat polymorphism database of the Indian population to identify susceptible biomarkers for any disease or understanding drug response in different subpopulations. Genetic diversity at similar scale exists in MHC antigens expressed in Indian population. MHC antigen based studies carried out in the Indian population by various groups have highlighted unique and extensive MHC diversity of the Indian population. For example, the HLA-A2 and A33 allele families in the Indian population show greater diversity with the existence of several‘unique’ alleles. The best example is the presence of A*02l l, an allele that occurs only in the Indian populations with almost complete absence in the Caucasoid groups.
  • HLA-A33-B44-DR7 HLA-A33-B58-DR3, and HLA-A2-B50- DR3.
  • HLA-A33-B44-DR7 is also observed in most other Asian countries like Malaysia, Thailand, South Korea, Bangladesh, Pakistan, China, but is not reported from‘rest of the world’.
  • HLA-A33-B58-DR3 is one of the commonest haplotypes in the Chinese population, which occurs with significantly high frequency in the Indian subcontinent suggesting racial admixture from the East.
  • haplotype HLA-A2-B50-DR3 is observed exclusively in India particularly in patients with type 1 diabetes and is therefore‘unique’ to this population.
  • the most commonly encountered haplotype in western population showing a strong association with autoimmune diseases is HLA-A1-B8-DR3, often referred to as the ancestral haplotype 8.1 (AH 8.1).
  • the population sharing Indian gene pool does not follow the western trend, and AH 8.1 occurs either with a very low frequency or is absent in the Indian subcontinent. Instead, the haplotype HLA-A26-B8-DR3 designated as AH8.2 is the most frequently occurring haplotype in the population sharing Indian gene pool, and this shows strong association with several autoimmune diseases just as AH 8.1 does in the western population.
  • the limitation of the assays described above and genetic diversity of MHC antigens demands identification of commonly expressed MHC antigens to be coupled on microsphere beads to increase the accuracy, specificity and sensitivity of allo-antibodies detection and minimize the possibility of graft rejection.
  • the present invention identifies common MHC antigens to be coupled with fluorescently labelled microsphere beads with the aim to increase the specificity of allo-antibodies detection in the population sharing Indian gene pool globally.
  • the present invention provides a cost-effective, specific and sensitive molecular fluorescence based multiplex assay to identify acquired allo-antibodies in patients’ serum against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the main object of the present invention is to provide a molecular fluorescent multiplex assay to identify allo- antibodies against MHC antigens.
  • Yet another object of the present invention is to provide a cost-effective, specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • Yet another object of the present invention is to provide a kit for molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • Yet another object of the present invention is to provide an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the present invention provides molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens. More specifically, the present invention provides a specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally. The present invention also provides an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the MHC antigens in the design panel have been coupled with fluorescently labeled microsphere beads and then these beads have been tested with patients’ sera for detection of allo- antibodies against identified MHC antigens using a standard flow cytometer.
  • the present invention comprises the steps of identification of antigens in the design panel specifically conserved in population sharing Indian gene pool, coupling of the antigens with a bead under predetermined conditions with predetermined reagents, determination of coupling efficiency in singleplex assay and multiplex assay employing Fluorescent Microsphere -based Immunoassay (FMIA) and testing cross -reactivity of allo-antibodies with the coupled MHC antigens.
  • FMIA Fluorescent Microsphere -based Immunoassay
  • diagnostic kit prototypes After determining the efficiency, diagnostic kit prototypes have been developed and its specificity and sensitivity has been validated with stored patient’s sera. The prototype was then scaled up as a molecular fluorescent multiplex diagnostic assay to identify allo- antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the present invention provides a molecular fluorescent multiplex assay for identifying specific allo-antibody which binds to a purified MHC antigen coated over microsphere bead.
  • the process to obtain said purified MHC antigen comprises identification of MHC alleles conserved in population sharing Indian gene pool globally, manufacturing of MHC antigens specific to said MHC alleles and synthesis of monoclonal anti-MHC antibodies specific to said manufactured MHC antigens.
  • the invention utilizes fluorescently labeled microsphere beads coated with a coupling agent such as avidin, coupled with manufactured MHC antigen for detection of allo-antibodies against MHC antigens in human serum by a standard flow cytometer.
  • a coupling agent such as avidin
  • manufactured MHC antigen for detection of allo-antibodies against MHC antigens in human serum by a standard flow cytometer.
  • mixture of different microsphere beads, each coated with manufactured MHC antigens is used to detect specific allo-antibody.
  • the invention also provides an array of microsphere beads coated with different purified MHC antigens which can be distinguished on the basis of intensity of fluorescent labels.
  • Such use of microsphere beads labeled with fluorophores allows the identification and/or separation of different beads by a standard flow cytometer.
  • microsphere beads also referred to as microbeads coated with MHC antigen have been incubated with serum to be tested for 30 minutes at 20°-25° C on a rotating platform at a predetermined dilution, preferably in the range of 1:8.
  • the microbeads were then washed with a buffer, preferably PBS and incubated with anti-Human IgG antibodies conjugated with phycoerythrin (PE) for 30 minutes at 20°-25° C on a rotating platform.
  • PE phycoerythrin
  • the microbeads are then re-suspended with wash buffer and analyzed on a standard flow cytometer. Sera which contain allo-antibodies against the tested microsphere (MHC antigens) will show a high median fluorescent intensity in comparison to the negative controls.
  • Fig. 1 represents mean MFI values of the monoclonal antibodies detected against the identified MHC antigens
  • Fig. 2 represents mean MFI values of the pooled monoclonal antibodies, diluted in the ratios of 1:16, 1:32 and 1:64, detected against identified MHC antigens using the developed prototype.
  • Fig. 3 represents mean MFI values of allo-antibodies detected against identified MHC antigens in pooled sera, prepared from the serum collected from ten MHC sensitized individuals.
  • Fig. 4 depicts a heatmap representing the mean MFI values of allo-antibodies identified in ten MHC sensitized individuals against common MHC antigens, represented in the developed kit.
  • the assay of the present invention is capable of detecting allo-antibody against single MHC antigen, thus identifying donor specific antibodies in patient’s serum.
  • This assay has promising potential to determine allo-antibodies against donor specific MHC antigens in recipient’s serum with high specificity and sensitivity.
  • the assay involves incubation of recipient’s serum with fluorescently labelled microsphere beads coupled with MHC antigen, allowing allo-antibodies present in the added serum to react with the MHC antigen coupled to the fluorescently labelled microsphere beads.
  • the antigen-antibody reaction can be determined via fluorescence (MFI) generated with the help of labelled secondary antibodies added post the antigen-antibody reaction.
  • the present invention also allows detection of multiple allo-antibodies in a unified multiplexed alloantibody detection assay where each microsphere bead is coupled to a unique MHC antigen and has a unique fluorescence, this helps in identification of all the different MHC antigens coupled on the beads in a single assay. Addition of fluorescently labeled secondary antibodies against the anti human IgG chain generates a fluorescence spectrum which helps in analyzing the antigen-antibody reactions.
  • the experiments described herein suggests that the identification of MHC antigens conserved in population sharing Indian gene pool globally is clinically important for optimizing the method of allo-antibodies detection in said population, and hence improving the transplantation outcome.
  • the present invention provides molecular fluorescent multiplex assay to identify allo- antibodies against MHC antigens. More specifically, the present invention provides a specific and sensitive molecular fluorescent multiplex assay to identify allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the present invention also provides an economical kit for molecular fluorescent multiplex assay to identify said allo-antibodies against MHC antigens comprising of common MHC antigens and antigens conserved in population sharing Indian gene pool globally.
  • the present invention provides multiplex bead based assay to detect acquired allo- antibodies in individuals who have been sensitized due to exposure to corresponding antigens.
  • the assay is carried out with MHC antigens conserved in population sharing Indian gene pool globally.
  • sera of a particular individual previously reported with the history of organ transplant, pregnancy or blood transfusions, is allowed to react with multiplex bead based assay of the present invention, the corresponding beads would hybridize with the allo- antibodies.
  • fluorescence is generated which is detected by using a standard flow cytometer
  • the present invention involves the identification of conserved MHC alleles specific to the population sharing Indian gene pool globally.
  • Genetic database of Genebandhu (Adult stem cell registry) is used through a formal Memorandum of Understanding (MOU).
  • MHC alleles conserved in the population sharing Indian gene pool globally has been identified.
  • the nucleotide sequences of said MHC alleles have been searched/identified using a freely available online tool (IMGT database). Said nucleotide sequences have been converted to amino acid sequences. Proteins have been manufactured from said amino acid sequences.
  • Table 1 represents the list of conserved MHC alleles specific to the population sharing Indian gene pool globally.
  • Said manufactured protein has been coupled with avidin or an alternate coated fluorescently labeled bead to form activated protein coupled beads.
  • the manufactured protein coupled beads are incubated under predetermined conditions to obtain pellets. Said pellets have been re-suspended in a buffer and stored at 2-8 °C in the dark.
  • Samples of highly MHC sensitized subjects are taken for analysis. Samples of highly MHC sensitized subjects are collected from bio-bank of Chimera Transplant Research Foundation (CTRF) and obtained under a formal Memorandum of Understanding.
  • CRF Chimera Transplant Research Foundation
  • Sera taken from highly MHC sensitized subjects are pooled and mixed in a tube.
  • the pooled sera is diluted in the ratio of 1:8 in buffer and then incubated with manufactured protein coupled with fluorescent beads under predetermined reaction conditions followed by washing with PBS buffer.
  • goat anti-Human immunoglobulin conjugated with fluorescent dye labeled secondary antibodies are added to the tube, mixed well and allowed to react for a specific time.
  • buffer is added to the tube and analyzed using a standard flow cytometer.
  • the coupling method is selected from biotinylation, carbodiimide coupling.
  • the present invention is carried out using biotinylation to yield experimental data.
  • said MHC antigens are coupled with fluorescently labelled microsphere beads.
  • the specificity of the coupled beads is validated by adding monoclonal antibodies specific to the coupled MHC antigen.
  • Antigen - antibody reaction between the coupled MHC antigen and monoclonal antibodies nonspecific to the said MHC antigen is also carried out to rule out the possibility of nonspecific binding.
  • the process of the present invention comprises of (a) coupling of said identified MHC antigen with the avidin or an alternate coupled- fluorescent labeled beads by incubating 1.0x105 avidin or an alternate labelled beads with 250 pi of 4000 nM diluted biotinylated protein (example B *40:06) and incubated at predetermined conditions on a rotating platform, (b) followed by washing with phosphate buffer saline (PBS) three times at >8000 g for 1-2 minutes to obtain the formed pellet, (c) resuspending the separated pellet in 500 pl PBS-Tris-NaCl Blocking buffer and storing it at 2-8 °C in the dark, (d) procuring monoclonal antibodies against said conserved MHC antigens labeled with fluorescent dye preferably, phycoerythrin (PE), (e) diluting the procured monoclonal antibodies in the ratios of 1:8 using PBS buffer (f) Incubating 50m1 biotinylated coupled proteins to react with
  • the said procedure is then repeated with monoclonal antibodies nonspecific to the coupled antigen to rule out the possibility of nonspecific binding.
  • the said procedure is carried out for all the identified MHC antigens represented in the prototype.
  • the sensitivity of the developed prototype has been tested by following the above said procedure with pooled monoclonal antibodies (pooling the procured monoclonal antibodies specific to each identified, conserved MHC antigens together) diluted in the ratios of 1:16, 1:32 and 1:64.
  • Fig. 1 represents mean MFI values of the monoclonal antibodies detected against the said MHC antigens.
  • X-axis represents conserved MHC antigens and Y-axis represents mean MFI values of the detected monoclonal antibodies.
  • the figure represents MFI values of specific and nonspecific monoclonal antibodies added against the coupled MHC antigens. The MFI values are detected using flow based technique - standard flow cytometer. High mean MFI values corresponding to the detection of monoclonal antibodies, specific to the coupled MHC antigen and low mean MFI values corresponding to the detection of monoclonal antibodies nonspecific to the coupled MHC antigen suggests reliable specificity of the developed prototype.
  • Fig. 1 represents mean MFI values of the monoclonal antibodies detected against the said MHC antigens.
  • X-axis represents conserved MHC antigens and Y-axis represents mean MFI values of the detected monoclonal antibodies.
  • the figure represents MFI values of specific and nonspecific monoclonal antibodies added against the coupled M
  • MFI values of the pooled monoclonal antibodies diluted in the ratios of 1:16, 1:32 and 1:64, detected against said MHC antigens using the developed prototype.
  • X-axis represents conserved MHC antigens and Y-axis represents mean MFI values of the detected monoclonal antibodies.
  • Mean MFI values of the detected monoclonal antibodies is significantly higher than the negative control even at 1:64 dilution suggesting high sensitivity of the developed prototype.
  • serum of 10 highly MHC sensitized subjects is used. 300pl of the sera is taken from all of the samples, pooled and mixed in a tube to obtain pooled sera. Said pooled sera is diluted up to 1:8 dilution using PBS buffer. Then 10 m ⁇ of the diluted pooled sera is incubated with 50 m ⁇ of the stored pellets for 30 minutes on a rotating platform at room temperature, followed by washing with PBS three times. Now 50 m ⁇ of Goat anti-Human IgG conjugated to phycoerythrin (PE) secondary antibodies diluted 1:10 in PBS are added and mixed. The solution is allowed to react for 30 minutes, followed by the addition of 100 m ⁇ PBS. The analysis is carried out by using a standard flow cytometer 200 analyzer.
  • PE phycoerythrin
  • Fig. 3 represents mean MFI values of allo-antibodies detected against said MHC antigens in pooled sera, prepared from the serum collected from ten MHC sensitized individuals.
  • X-axis represents said MHC antigens and Y-axis represents mean MFI values of the detected allo-antibodies in individual’s serum.
  • High MFI values of the allo-antibodies detected against identified, conserved MHC antigens - B*35:02 and B*40:06 suggests that the frequency of occurrence of the identified MHC antigens in the population sharing Indian gene pool globally is significant and hence it is essential to detect allo-antibodies against the said MHC antigens to reduce the chances of fallacious results.
  • all the common biotinylated proteins are coupled with avidin coated fluorescently labeled beads. All the common biotinylated proteins coupled with avidin coated fluorescent-labeled beads are then pooled into one single tube to obtain multiplexed beads. Then the differentiation between the substrates is done according to the fluorescent coating on beads. The level of fluorescence is analyzed by solid state laser of 488 nm in a standard flow cytometer.
  • multiplexed beads When said multiplexed beads are allowed to react with serum containing allo- antibodies against the coupled proteins, an antigen- antibody reaction takes place. The unbound antibodies are removed by washing with buffer. Then fluorescently labeled Goat anti-Human IgG is added which binds on the antigen-antibody complex and upon analysis through two lasers at the same time, (one for differentiation of substrate and other for detection of analytes) using a standard flow cytometer, a unique fluorescence is generated which is detected by using a specific filter and detectors.
  • a mixture of fluorescently labeled microsphere beads coated with MHC antigens are incubated with a serum of ten highly MHC sensitized individuals, for 30 minutes at 20°-25° C on a rotary shaker.
  • the microbeads are then washed with a wash buffer (PBS) three times and incubated with anti-Human IgG antibodies conjugated with phycoerythrin, for 30 minutes at 20°-25° C on a rotary shaker.
  • the microsphere beads are then re suspended with wash buffer and analyzed on a standard flow cytometer.
  • the power of multiplexing allow us to differentiate between the microbeads according to their fluorescence and further the fluorescence of secondary antibodies helps in determining the presence of allo-antibodies against MHC antigens in the tested serum.
  • Serum which contains allo-antibodies against the tested microsphere beads (MHC antigens) show a high median fluorescent intensity in comparison to the negative controls.
  • the present invention also provides an economical kit for molecular fluorescent multiplex diagnostic assay to identify allo-antibodies against common MHC antigens.
  • the kit comprises of fluorescently labelled beads coupled with said MHC antigens and containers for the following reagents - wash buffer (Phosphate Buffer Saline), secondary antibodies (anti-human IgG), negative control (sera with known absence of allo-antibodies, representing negligible MFI values for secondary antibodies) and positive control (sera with known presence of allo- antibodies against coupled MHC antigens, representing higher MFI values for secondary antibodies.
  • Fig. 4 depicts a heatmap representing mean MFI values of allo-antibodies identified in ten MHC sensitized individuals against common MHC antigens, represented in the developed kit.
  • X-axis represents the common MHC antigens coupled with fluorescently labelled microsphere beads
  • Y-axis represents the anonymous id of MHC sensitized individuals tested for the presence of allo- antibodies
  • Z-axis represents mean MFI values of the detected allo-antibodies.
  • Red colour represents the presence of allo-antibodies and blue colour represents the absence of allo-antibodies.
  • the first column of the heat map is positive control and the second column of the heat map represents the negative control.
  • the solid phase multiplex assay of the present invention is capable of being conducted on multiplex solution comprising of different fluorescently labeled microsphere beads coated with different corresponding MHC antigens.

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Abstract

La présente invention concerne un dosage multiplex fluorescent spécifique, sensible et économique pour identifier des allo-anticorps dirigés contre des antigènes du CMH conservés dans une population partageant globalement un groupe de gènes indiens. La présente invention concerne également une trousse économique pour le dosage par multiplexage pour identifier des allo-anticorps dirigés contre des antigènes du CMH comprenant des antigènes du CMH communs et des antigènes conservés dans une population partageant globalement un groupe de gènes indiens.
PCT/IN2019/050327 2018-04-29 2019-04-23 Dosage multiplex fluorescent moléculaire pour identifier des allo-anticorps dirigés contre des antigènes du cmh WO2019211864A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
US20060263836A1 (en) * 2001-12-10 2006-11-23 Zeus Scientific, Inc. Composition for homogeneous multiplexed microparticle-based assay
US20100261203A1 (en) * 2009-04-07 2010-10-14 National Institute Of Transplantation Foundation Methods and kits for screening transplant recipients and candidates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060263836A1 (en) * 2001-12-10 2006-11-23 Zeus Scientific, Inc. Composition for homogeneous multiplexed microparticle-based assay
US20040072262A1 (en) * 2002-10-11 2004-04-15 Montero-Julian Felix A. Methods and systems for detecting MHC class I binding peptides
US20100261203A1 (en) * 2009-04-07 2010-10-14 National Institute Of Transplantation Foundation Methods and kits for screening transplant recipients and candidates

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