WO2005029072A1 - Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet - Google Patents

Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet Download PDF

Info

Publication number
WO2005029072A1
WO2005029072A1 PCT/CN2003/000812 CN0300812W WO2005029072A1 WO 2005029072 A1 WO2005029072 A1 WO 2005029072A1 CN 0300812 W CN0300812 W CN 0300812W WO 2005029072 A1 WO2005029072 A1 WO 2005029072A1
Authority
WO
WIPO (PCT)
Prior art keywords
hla
complement
antibody
enzyme
antigen
Prior art date
Application number
PCT/CN2003/000812
Other languages
English (en)
Chinese (zh)
Other versions
WO2005029072A8 (fr
Inventor
Ge Chen
Original Assignee
Pel-Freez Biotechnology (Beijing) Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pel-Freez Biotechnology (Beijing) Ltd. filed Critical Pel-Freez Biotechnology (Beijing) Ltd.
Priority to PCT/CN2003/000812 priority Critical patent/WO2005029072A1/fr
Priority to AU2003272846A priority patent/AU2003272846A1/en
Publication of WO2005029072A1 publication Critical patent/WO2005029072A1/fr
Publication of WO2005029072A8 publication Critical patent/WO2005029072A8/fr

Links

Classifications

    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the invention relates to a method for measuring complement-fixing antibodies (CFAbs). More specifically, the present invention relates to a method for determining complement-fixing antibodies against an HLA antigen. The method of the present invention measures complement-fixed antibodies by measuring complement-fixed. The method of the invention is particularly useful for cross-matching tests in organ transplants. Background technique
  • Rejection in tissue and organ transplantation is the immune response of the biological body's immune system to foreign grafts (not already).
  • the protein component (non-antigen) on the surface of the transplant cell contacts the recipient's immune system, the non-antigen component is recognized through immunity and the recipient's humoral immune system and cellular immune system launch an immune attack on the graft to destroy and reject it.
  • Non-graft which causes tissue and organ transplant failure.
  • the recipient's immune system uses the MHC (tissue compatible complex) recognition mechanism to perform dual immune recognition of the transplant MHC phenotype and short peptide complex.
  • MHC tissue compatible complex
  • HLA human leukocyte antigen
  • the ultra-acute immune rejection is mainly mediated by the humoral immune component of the recipient, that is, the antibody molecule against the donor HLA that already exists in the recipient before transplantation.
  • These antibodies are produced by recipients who become pregnant, receive blood transfusions, organ transplants, etc., and expose their immune systems to non-existing HLA molecules.
  • This type of antibody is characterized by its ability to rapidly bind to donor HLA and cascade through complement fixation and activation of the complement system to induce complement-dependent cytotoxicity (CDC) effects.
  • CDC complement-dependent cytotoxicity
  • This type of ultra-acute immune rejection occurs abnormally quickly, usually within a few minutes to several hours; while acute and chronic immune rejection involve both the humoral and cellular immune systems of the recipient.
  • the donor lymphocytes and the serum of the recipient are used to cross-match before transplantation to identify the presence of antibodies against the HLA in the recipient.
  • the key to hyperacute rejection is of great importance in monitoring the occurrence of acute and chronic rejection reactions and in predicting the recipient's lifetime after transplantation.
  • HLA antibodies can be divided into two categories: One is to fix complement and activate complement system to induce CDC effect after binding with corresponding HLA. This type of HLA antibody is called Complement dependent cytotoxic antibody
  • CDC-Abs Complement-dependent Cytotoxic Antibodies
  • CFAbs Complement Fixing Antibodies
  • its role in inducing immune rejection has been very clear, which directly leads to immune rejection, especially The humoral immune component of the ultra-acute immune rejection reaction; while another type of HLA antibody, although it can specifically bind to HLA, does not fix complement and does not trigger the CDC effect, it is called a non-complement fixed antibody
  • the CDC serological method is a standard CDC measurement method that is generally considered classic. This method uses CDC-induced lymphocyte mortality as a criterion. Its main feature is to directly measure the final effect of CDC-cell death induced by CFAbs, which is a method for measuring the biological effect of CDC. This method has been widely used in cross-matching of clinical organ transplantation and PRA measurement; however, it has many disadvantages such as time-consuming, complicated technology, and difficult to control experimental conditions.
  • Another type of method can measure CFAbs and Non-CFAbs simultaneously; including flow cytometry to measure IgG antibody binding to the cell surface (Garovoy MR, et al, Transplant Proc 1983; 15: 1939-41), or using purified HLA-coated solid phase particle anti-HLA antibody assay; and purified HLA-coated 96-well microplate assay for HLA-binding IgG antibody ELISA method, etc.
  • HLA antibodies Non-CFAbs
  • these antibodies can block the HLA antigenic site after blocking the corresponding HLA, preventing HLA toxic antibodies- CFAbs bind to the corresponding HLA, thereby blocking the subsequent CDC effect and allowing the recipient to develop immune tolerance to the graft. Therefore, the above-mentioned methods have obvious defects in the principle of detection methodology, and cannot distinguish between the above two types of antibodies with completely different effects.
  • results of the above methods continue to be positive for organ transplant recipients who have suppressed treatment, while clinical patients are in a normal or better condition.
  • false positive results of this type of method often lead to misleading donor selection, causing some patients who are in urgent need of organ transplantation to miss the opportunity for transplantation and their condition worsens or even dies.
  • IVIG human IgG
  • Flow-type cross-matching can detect positive results, which is contradictory to the clinical outcome (via IVIG anti-rejection treatment) and can mislead clinicians to evaluate the patient's immune function status and cause incorrect treatment.
  • Another obvious shortcoming of all current methods for measuring HLA antibodies is the long time required, which is the leading cause of transplant failure in cadaver donor organ transplants.
  • the determination of CFAbs in recipients before organ transplantation is one of the key factors in determining the success of organ transplantation. After organ transplantation, it is important to monitor the recipients for immune rejection. Indicators; meanwhile, it has a predictive effect on the disease outcome of recipients after transplantation. Points meet the above requirements. Therefore, the establishment of a simple, fast, accurate, sensitive, specific, and stable standardized method for the determination of CFAbs has become an urgent need in organ transplantation.
  • the present invention is based on the basic principle of the CDC effect, A measurement method was established to determine the effect of CDC by measuring the amount of CFAbs fixed complement. Because CFAbs bind the corresponding cell HLA antigen and fixed complement is the initial stage of the CDC effect, and the amount of CFAbs fixed complement has a direct positive correlation with the intensity of CDC effect (cell death); therefore, the determination of the fixed amount of complement of CFAbs can be used directly Evaluation of the CDC effect. At the same time, this method introduces a solid-phase antibody against a characteristic antigen on the cell surface into the reaction system, which is used to identify and separate a specific cell population, and can selectively react to a specific cell population in a multi-cell mixed reaction system. The CDC effect was measured.
  • This CFAbs assay method abandons the shortcomings of various existing HLA antibody assay methods. It has high sensitivity, specificity, and unique features such as economy, simplicity, and speed. Summary of the invention
  • the present invention relates to a method for detecting complement-fixing antibodies against an HLA antigen, the method comprising the steps of contacting the HLA antigen with a sample that may contain complement-fixing antibodies against the HLA antigen and related complements, and labeling
  • the method further comprises adding a reaction substrate of the labeled enzyme to the reaction system to generate an enzymatic color or an enzymatic A step of luminescent reaction, and then measuring the optical density value or luminescent intensity of the reaction solution to determine the presence and / or amount of the complement-immobilized antibody in the sample.
  • the present invention relates to a method for cross-matching T lymphocytes and / or B lymphocytes in organ transplantation, which includes the following steps: incubating nucleated cells of an organ donor and a body fluid sample of a recipient,
  • the isolated cells are reacted with an enzyme-colored substrate or an enzymatic luminescent substrate in a buffer solution, 7) The optical density value or luminous intensity of the reaction solution is measured, and the T lymphocytes or B lymphocyte cross-matching results.
  • the present invention relates to a method for determining anti-HLA I and / or HLA II complement fixing antibodies, which comprises the following steps: '
  • the present invention also relates to a kit for the above method of the present invention, which contains containers each containing at least the following: an enzyme-labeled complement or an enzyme-labeled anti-complement antibody; a corresponding enzyme reaction substrate; Appropriate buffer; optionally coated with a solid-phase carrier against antibodies against target cell surface antigens or purified HLA antigen.
  • containers each containing at least the following: an enzyme-labeled complement or an enzyme-labeled anti-complement antibody; a corresponding enzyme reaction substrate; Appropriate buffer; optionally coated with a solid-phase carrier against antibodies against target cell surface antigens or purified HLA antigen.
  • the present invention relates to a method for detecting complement-fixing antibodies against HLA antigen, the method comprising the step of contacting the HLA antigen with a sample that may contain complement-fixing antibodies against the HLA antigen and a related complement, and
  • the step of contacting with the labeled complement or the labeled anti-complement antibody is characterized in that the label is an enzyme label
  • the method further comprises adding a reaction substrate of the labeled enzyme to the reaction system to generate an enzymatic color or Enzymatic luminescence reaction, and subsequently measuring the optical density value or luminous intensity of the reaction solution to determine the presence and / or amount of the complement-immobilized antibody in the sample.
  • the HLA antigen may be a target cell having a HLA antigen on its surface or a purified HLA antigen.
  • the target cell may be, for example, any nucleated cell of the donor of the organ to be transplanted, particularly T lymphocytes and / or B lymphocytes.
  • the HLA antigen may be an HLA type I antigen and / or an HLA type II antigen.
  • HLA antigens can also be distinguished as HLA -A, B, C, D (DR, DQ, DP) and their subtypes, etc., where any type or subtype or any mixture thereof can be used in the method of the present invention.
  • the HLA antigen is immobilized on a solid phase carrier.
  • the solid phase support may be a solid phase support commonly used in the art, such as a microplate or a magnetic bead.
  • the test sample may be a body fluid of a human or an animal.
  • the body fluid is, for example, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, saliva or urine.
  • the complement may be any complement component involved in complement fixation of antibodies and subsequent complement activation enzymatic chain reaction or protein molecules or other factors that regulate and control complement activation, including participation in the classical activation pathway or Replace any component of the activation pathway. That is, the term "complement" as used in the present invention is not limited to the conventional components of complement that are conventionally understood.
  • the complement in the present invention may be at least one selected from the group consisting of Cl (Clq, Clr, Cls), C2, C3, C4, C5, C6, C7, C8, C9; Clqrs, Clqrs, C2a, C2b, C3a, C3b, C4a, C4b, C4b2, iC3b, C4b2b, C4b2b3b, C3bBb, C3bnBb, C5a, C5b, C5b67, C5b- 8, C5b ⁇ 9; CI-inhibitory factor, C4-binding protein, D factor, B Factor, P factor (properdin), I-factor, H-factor, S-protein; Ba, Bb, MBP, MCP, DAF (CD55), CR1, CR2, CR3, CR4, CR5, C3aR, C2aR, C4aR , ClqR, CD59.
  • the complement is Clq or C3.
  • enzyme-labeled complement is used in one embodiment of the invention.
  • the labeled complement may be human, animal, biochemically prepared, chemically synthesized, or genetically engineered.
  • an enzyme-labeled anti-complement antibody is used.
  • the anti-complement antibody may be any source or any type of antibody capable of specifically binding to the complement to be tested, for example, it may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody or an antibody fragment.
  • the anti-complement antibody used in the method of the present invention may be, for example, the C3a antibody C3a (H-300): sc-20137 C3 precursor and 5-chain antibody C3 (V-20): sc-14612, H factor from Santa Cruz Biotechnology Inc.
  • Antibody Factor H (Y-20): sc-17949, factor D antibody Adipsin (P-16): sc-12402, C5a N-terminal antibody C5a (H-196): sc-20136, etc .; Anti-Clq antibody C3900, anti-C3 antibody C7761, anti-C3c antibody C6025, and anti-C4 antibody C3402, etc .; #Cat: 68215 Sheep, Ami human Clq from ICN Biomedicals, Inc.
  • the labeling enzyme used in the method of the present invention may be any enzyme commonly used in ELISA, such as horseradish peroxidase (HRPO), alkaline phosphatase (AP), galactosidase, and glucose oxidase. Enzymatic labeling of complement or complement antibodies can be performed according to conventional methods in the art.
  • Enzyme substrates for color development reactions are known to those skilled in the art and vary depending on the labeling enzyme.
  • ABTS 2,2'-azino-bis (3-ethylbenzothiazolidine-6 sulfonic acid)
  • 73 ⁇ 4 3 , 3 ', 5,5'-Tetrafluorenylbenzidine
  • HPPA 3- (4-hydroxy) phenylpropionic acid
  • DAB 3,3, -diaminobenzidine
  • OAP anthraminophenol
  • alkaline phosphatase for example, p-NPP ( ⁇ -nitrophenyl phosphate) or MUP (4-fluorenylumbellylphosphonic acid) can be used.
  • p-NPP ⁇ -nitrophenyl phosphate
  • MUP 4-fluorenylumbellylphosphonic acid
  • galactosidase for example, ONPG (0-nitrophenyl- ⁇ -D-galactopyranoside) can be used.
  • ONPG (0-nitrophenyl- ⁇ -D-galactopyranoside
  • MPMS / NBT can be used for glucose oxidase.
  • the labeling enzyme uses horseradish peroxidase or alkaline enzyme.
  • enzyme-labeled complement is used. In this embodiment, it is sometimes necessary to inactivate complement components that may be present in the sample.
  • the cellular ELISA method of the present invention it is preferable to further include adding an antibody against a specific antigen on a surface of a target cell coated on a solid-phase carrier in the reaction system, so as to isolate a specific cell subset and determine complement on the surface of the cell subset Immobilized antibodies.
  • the antibody against a cell surface antigen is, for example, an antibody against a Tc / Bc CD antigen.
  • the present invention relates to a method for cross-matching T cells and / B cells, which method comprises the steps of: incubating nucleated cells of an official donor and a body fluid sample of a recipient,
  • the isolated cells are reacted in a buffer with an enzymatic chromogenic substrate or an enzymatic luminescent substrate,
  • the present invention provides the following method, which includes the following steps:
  • the solid-phase carrier used for immobilizing the antigen or antibody may be any solid-phase carrier commonly used in the art, such as magnetic beads or microplates.
  • the present invention also relates to a kit for carrying out the method of the present invention, which contains containers each containing at least the following: an enzyme-labeled complement or an enzyme-labeled anti-complement antibody; a corresponding enzyme reaction substrate; an appropriate Buffer.
  • the kit further comprises an antibody against a target cell surface antigen or a purified HLA antigen coated on a solid phase carrier, such as a coated magnetic bead or a microwell plate.
  • a solid phase carrier such as a coated magnetic bead or a microwell plate.
  • the CFAbs immunoassay method and kit established by the present invention include the following main components Solid phase components: Depending on the solid phase method and composition, there are the following choices: Solid phase antibodies: can directly target cell surface antigens (such as CD antigen series, etc.), receptors or other components (such as antibodies, complements) bound to the cell surface Etc.) attached to the surface of the microplate to capture and isolate specific cells. CFAbs on the cell surface were measured.
  • the above components can also be connected to the surface of magnetic beads, and the magnetic beads can be adsorbed by a magnet to achieve selective separation of specific cells.
  • Solid phase antigen mainly refers to purified HLA antigen; the known type (such as HLA
  • HLA type I or / and type II mixed HLA antigens attached to microplates or solid particles (such as plastics, polymer compounds , Magnetic particles, etc.) as the target substance component bound by CFAbs.
  • Tracer markers Usually various labeled ligands, antibodies or complement components, especially enzymatic labels (oxidases such as HRP, AP).
  • corresponding substrates of oxidases such as HRP, AP, such as OPD, 4-CN, ABTS, PNPP, TMB, etc .; or the above enzymes can be used as chemiluminescence (lumino and its derivatives), bioluminescence catalysts and Other luminous enhancers co-catalyze and enhance the chemiluminescence signal to obtain high sensitivity for CFAbs detection.
  • Positive control Set according to the specific testing requirements, usually a single serum or mixed serum containing a known HLA type CFAbs; this serum can also be appropriately diluted as needed.
  • the positive control is usually set at three dose points, which are located in the three dose areas of the standard curve, high, middle and low.
  • Negative control Normally a normal male, AB blood type, individual serum that has never received blood transfusion or organ transplantation; the serum source can be the same individual or multiple individuals' mixed serum.
  • Standard products Made from a series (usually 5-6) of known levels of CFAbs serum After the test is completed, a standard curve is prepared for the dose value (horizontal axis) of CFAbs with the detection signal value (such as OD value; luminescence unit) of this series as the ordinate; as a basis for quantitative calculation of the CFAbs content of the sample, a more accurate result.
  • the detection signal value such as OD value; luminescence unit
  • CDC immunological CDC
  • HLA antibodies HLA antibodies
  • test kit instructions and other materials and items related to the test.
  • the present invention is based on the principle of immunological CDC, that is, CDC must be bound to the corresponding target cells by HLA antibodies, and then the first complement component can be fixed to activate the classical or / and alternative pathway of complement. With the participation of factors, a cascade of complement activation occurs until the formation of a membrane attacker (MAC), which damages the biological cell membrane and forms small pores leading to lower intracellular osmotic pressure and cell lysis and death.
  • MAC membrane attacker
  • the relative number of CFAbs fixed complements has a direct positive correlation with the concentration of CFAbs and the degree of CDC effect in the sample, therefore, to determine the level of HLA-CFAbs fixed complement or to measure any factor in the complement activation pathway or the complement itself and its complex, On the one hand, it can directly reflect the presence or relative concentration of HLA-CFAbs; on the other hand, it can indirectly reflect the relative degree and number of CDC effects or cell damage and death.
  • the reaction time required by this method is the shortest compared to the existing methods.
  • a signal detection method using enzymatic coloration and / or enzymatic luminescence achieves high sensitivity sufficient for practical qualitative and quantitative determination.
  • the present invention uses complements present in the recipient's own serum as the response measurement object, and the main components of the reaction system are the recipient's serum components and donor cells; therefore, the physiological internal environment in the recipient can be most realistically simulated.
  • the method Since the activity of CFAbs and complement components in serum can be stored for a long time (at least several years) at low temperature, the method has methodological stability and good reproducibility.
  • the method uses a "dual recognition system"; specific antibodies against two different antigen components on the same target cell can be used; meanwhile, identification and selection of specific target cells are achieved Use: For example, using solid-phase antibodies against target cell antigen 1 (cell-specific CD antigen) to identify and isolate target cells; using labeled antibodies against target cell antigen 2 (HLA antigen or complement) as the tracer signal for CFAbs
  • the detection means realizes the simultaneous identification, isolation and detection of specific target cells and detection of CFAbs signals, thereby simplifying the experimental steps and shortening the experimental cycle, and improving the methodological specificity.
  • the CFAbs assay of the present invention can use the most common ELIS A method as a "technical platform" for detection, it does not require other complicated and expensive instruments and equipment (such as flow cytometers), the methodology is simple and easy to master, so it has low cost It has the characteristics of strong practicability and can complete the simultaneous measurement of large samples in a short time.
  • the present invention is based on the method of immune response, and combines the characteristics of high sensitivity detection of enzymatic coloration and / or enzymatic luminescence signals, and establishes the fixation of complements by measuring the surface of target cells or the surface of solid phase target substances CFAbs assay.
  • CDC During the occurrence of CDC, it takes a long time from CFAbs to bind to cell surface HLA antigens, complement fixation, and activation to the end effector of CDC that can be measured in vitro-cell death appears, and the present invention measures the earliest stage of CDC (initial induction effect)
  • the HLA-CFAbs-complement complex formed at this stage is used as an index to evaluate the level of CFAbs and the degree of CDC effect in the sample; therefore, the detection cycle is shortened, and the methodological stability and sensitivity are significantly improved.
  • this method is based on the most commonly used enzyme-linked immunoassay method, it has the advantages of simple operation, no special equipment and training of technicians; another significant feature of the present invention is that it can use the complement component contained in the sample itself, Simulate the physiological microenvironment in vivo, measure the early immunological binding effect of HLA CFAbs fixed complement to reflect the end effect of CDC immunobiology, and be able to synchronize selective separation of a specific cell population in multiple cell mixed samples. Achieving a measurement of CDC effects that occur in that particular cell population. At the same time, the method can simultaneously measure large samples of CFAbs in a short time.
  • the CFAbs measurement method or kit established in the invention has unique advantages not available in all other methods existing in the world.
  • the above method and its kit can be widely used to replace the traditional serological cytotoxicity measurement of HLA, such as Serology HLA Typing (HLA Typing); HLA Cross Match (HLA Cross Match), and group reactive HLA antibody measurement (Panel Reactive Antibodies, PRA) And ABO blood type matching and other serological methods; at the same time, it can also be directly applied to the determination of CFAbs in samples, providing an economical method for the evaluation of CDC immunological effects in biomedicine and the study of complement biological activity and complement activation mechanism , Fast, simple, and sensitive specific method.
  • HLA typing Serology HLA Typing
  • HLA Cross Match HLA Cross Match
  • PRA group reactive HLA antibody measurement
  • ABO blood type matching and other serological methods at the same time, it can also be directly applied to the determination of CFAbs in samples, providing an economical method for the evaluation of CDC immunological effects in biomedicine and the study of complement biological activity and complement activation mechanism , Fast, simple, and sensitive specific method.
  • Figure 1 Mechanisms of cytotoxic antibodies (CFAbs) and non-cytotoxic antibodies (Non-CFAbs) in the occurrence of CDC effects.
  • Figure 2 Cellular ELISA method for detection of complement-fixed antibodies, Tc / Bc HLA cross-matching method.
  • Figure 3 ELISA method for detection of complement-fixed antibodies, Tc / Bc HLA cross-matching method.
  • Figure 4 ELISA for detection of complement-fixed antibodies, population-reactive antibody ELISA.
  • Figure 5 Immunomagnetic beads ELISA method for detection of complement-fixed antibodies, population-reactive antibody ELISA method.
  • Figure 6 Cell ELISA method for detection of complement-fixed antibodies, T lymphocyte cross-matching results. detailed description
  • Example 1 T-lymphocyte HLA cross-matching cell ELISA method (Cellular ELISA; CELISA)
  • the magnet is brought into contact with the outside of the reaction container to form an effective magnetic field, and the T lymphocytes are adsorbed and separated by adsorbing immunomagnetic beads, and other cell components and liquid components that are not adsorbed are sucked away.
  • step (B) Same as step (b) in Example 1, but the difference is that the anti-CD19 or CD20 antibody of the magnetic bead solid phase binds to the CD19 or CD20 molecules on the surface of B lymphocytes.
  • Example 3 Same steps (three to nine) as in Example 1, except that the T lymphocytes in the above steps are changed to B lymphocytes.
  • the corresponding T or B lymphocytes were separated by using immunomagnetic beads with different CD antibodies, and the T or B lymphocytes could also be identified and separated by directly connecting the CD antibodies to the microtiter plate.
  • the CFAbs on the cell membrane surface were measured.
  • Tc / Bc HLA cross-matching by using a microplate or magnetic bead-fixed antibody ELISA assay, see Figures 2 and 3.
  • HRP-ClqAb or HRP-C3Ab in the above Examples 1 and 2 can be replaced with HRP-hlgGAb, and the total lgG bound on the surface of T or B lymphocytes is measured to achieve the simultaneous determination of CFAbs and Non-CFAbs.
  • step (2) Block the microplate with a buffer (blocking solution) containing a certain amount of non-specific protein (such as BSA, skimmed milk powder, calf serum, etc.).
  • a buffer blocking solution
  • non-specific protein such as BSA, skimmed milk powder, calf serum, etc.
  • HRP substrate such as TMB, etc.
  • Example 4 HLA-I or / and HLA-II immunomagnetic bead ELISA method
  • Example 5 Population Responsive HLA CFAbs Measurement Method (CFAbs-PRA)
  • PRA plate Select multiple known HLA type cells and add them to each well of the microtiter plate at a certain number (0.01-3 X 10 6 / well); as the target cells for the CELISA reaction; or by purification Each type of HLA antigen is coated with a microplate or immunomagnetic beads as a solid phase reaction material.
  • microtiter plate or magnetic beads are coated with purified and mixed HLA-1 or HLA-II antigen, and then the CFAbs for total HLA-1 or I and / or II can be measured according to the corresponding conditions in the above examples. .
  • HFA-I CFAbs determination can also use mixed human platelets as a solid-phase HLA-I source.
  • HLA-I or II antigens should include statistically> 95% of HLA types. See Figures 4 and 5 for the method of performing population-reactive antibody measurement by complement-fixed antibody ELISA assay using 4-well plates and magnetic beads, respectively.
  • Example 7 CFAbs determination of HLA-B27
  • test tube Place the test tube in a magnetic field (on a magnetic stand) for 1 minute, aspirate the liquid from the reaction, and then wash the magnetic bead cells 3 times. Aspirate the last wash.
  • reaction stop solution 100 ml of reaction stop solution to stop the color reaction.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de détection d'anticorps cytotoxiques dépendant du complément HLA au moyen d'un essai in vitro d'immunoabsorption à liaison enzymatique. L'invention concerne également un nécessaire à cet effet. Le système de réaction comprend des antigènes HLA fixés, ou des cellules cibles comprenant des antigènes, et des compléments liquides ou des anticorps dirigés contre ces compléments, lesquels ont été marqués par des enzymes. Les CFAbs des échantillons à traiter viennent se combiner aux antigènes HLA fixés ou aux antigènes HLA sur les cellules cibles, et en même temps, ils fixent les compléments marqués par des enzymes dans le système de réaction, ou se combinent au complément dans les complexes antigènes HLA CFAbs, sous forme d'anticorps dirigés contre les anticorps marqués par des enzymes. On ajoute ensuite les substrats correspondants des enzymes pour garantir le développement de la couleur ou la réaction lumineuse, la présence ou l'absence, voire les niveaux relatifs des CFAbs dans les échantillons étant alors évalués par détection de la densité optique, étant donné que la densité optique est en correspondance avec la teneur en CFAbs des échantillons. Pour détecter de façon spécifique les réactions CDC du groupe de cellules, on prend des anticorps dirigés contre les marqueurs caractéristiques (CD et autres) de la surface des cellules et on les ajoute au système de réaction en fonction des cellules.
PCT/CN2003/000812 2003-09-24 2003-09-24 Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet WO2005029072A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2003/000812 WO2005029072A1 (fr) 2003-09-24 2003-09-24 Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet
AU2003272846A AU2003272846A1 (en) 2003-09-24 2003-09-24 A detection method of hla complement dependent cytotoxic antibodies based on enzyme-linked immunosorbent assay and the kit thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2003/000812 WO2005029072A1 (fr) 2003-09-24 2003-09-24 Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet

Publications (2)

Publication Number Publication Date
WO2005029072A1 true WO2005029072A1 (fr) 2005-03-31
WO2005029072A8 WO2005029072A8 (fr) 2005-06-09

Family

ID=34318859

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2003/000812 WO2005029072A1 (fr) 2003-09-24 2003-09-24 Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet

Country Status (2)

Country Link
AU (1) AU2003272846A1 (fr)
WO (1) WO2005029072A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113791206A (zh) * 2021-09-03 2021-12-14 珠海高瑞特医疗科技有限公司 一种封闭抗体的抗原包被酶标板的制备方法及其试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05172812A (ja) * 1991-07-31 1993-07-13 Denka Seiken Co Ltd 補体結合性抗体量の測定方法及び試薬
CN1423130A (zh) * 2002-04-26 2003-06-11 帕弗瑞生物技术(北京)有限公司 流式测定固定补体抗体的hla交叉配型方法和试剂盒

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05172812A (ja) * 1991-07-31 1993-07-13 Denka Seiken Co Ltd 補体結合性抗体量の測定方法及び試薬
CN1423130A (zh) * 2002-04-26 2003-06-11 帕弗瑞生物技术(北京)有限公司 流式测定固定补体抗体的hla交叉配型方法和试剂盒

Also Published As

Publication number Publication date
AU2003272846A1 (en) 2005-04-11
WO2005029072A8 (fr) 2005-06-09

Similar Documents

Publication Publication Date Title
EP0619884B1 (fr) Tests alloantigeniques par etude des liaisons
US5270169A (en) Detection of HLA antigen-containing immune complexes
US5223397A (en) Soluble hla cross-match
US5482841A (en) Evaluation of transplant acceptance
Minucci et al. Methodologies for anti-HLA antibody screening in patients awaiting kidney transplant: a comparative study
JPH03502241A (ja) ヒト血清におけるｈｉｖ抗原を検出する酵素免疫検定法
US20050277158A1 (en) Method and kit for donor specific complement-fixing antibodies crossmatch
WO1993009434A1 (fr) Detection de complexes immuns aux allo-antigenes solubles
JP2022500677A (ja) ジカウイルスイムノアッセイの方法および試薬
Chou et al. The detection of the HLA-B27 antigen by immunomagnetic separation and enzyme-linked immunosorbent assay—comparison with a flow cytometric procedure
CN1444044A (zh) 以酶联免疫测定为基础的hla补体依赖性细胞毒性抗体检测方法和试剂盒
JPWO2009084369A1 (ja) Ｈｉｖ−１抗原の検出用試薬及び検出方法
Yang et al. Detection of donor-specific anti-HLA class I and II antibodies using antibody monitoring system
WO2005029072A1 (fr) Procede de detection d'anticorps cytotoxiques dependant du complement hla, a base d'essai d'immunoabsorption a liaison enzymatique, et necessaire a cet effet
Malheiro et al. Clinical implications of anti-HLA antibodies testing in kidney transplantation
Jaramillo et al. The crossmatch assay in solid organ transplantation
CA3010587C (fr) Test de sous-typage d'igg pour determiner la transplantabilite de prelevements tissulaires
Wienecka et al. Detection of Giardia antigen in stool samples by a semi-quantitative enzyme immunoassay (EIA) test
EP0664886B1 (fr) Essai ameliore a alloantigenes
Gebel et al. Chee L. Saw, Denise L. Heaney
CN111474368A (zh) 全面检测非hla类供者特异性抗体的纯化抗原法
Lown et al. Application of the proteolytic enzyme papain in routine platelet serology
JPH01292254A (ja) Ｈｔｌｖ−ｉ抗体の確認試験方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NI NO NZ OM PG PH PT RO RU SC SD SE SG SK SL SY TJ TN TR TT TZ UA UG US UZ VC VN ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR HU IE IT LU NL PT RO SE SI SK TR BF BJ CF CI CM GA GN GQ GW ML MR NE SN TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
CFP Corrected version of a pamphlet front page

Free format text: UNDER (54) PUBLISHED TITLE IN CHINESE REPLACED BY CORRECT TITLE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP