WO2005029072A1

WO2005029072A1 - A detection method of hla complement dependent cytotoxic antibodies based on enzyme-linked immunosorbent assay and the kit thereof

Info

Publication number: WO2005029072A1
Application number: PCT/CN2003/000812
Authority: WO
Inventors: Ge Chen
Original assignee: Pel-Freez Biotechnology (Beijing) Ltd.
Priority date: 2003-09-24
Filing date: 2003-09-24
Publication date: 2005-03-31
Also published as: AU2003272846A1; WO2005029072A8

Abstract

The present invention provides a method of detecting HLA complement-dependent cytotoxic antibodies by enzyme-linked immunosorbent assay in vitro and the kit thereof. The reaction system comprises fixed HLA antigens or target cells comprising HLA antigens and liquid complements or antibodies against the complements, which were labeled with enzymes. The CFAbs in the samples to be determined will combine with the fixed HLA antigens or the HLA antigens on target cells and at the same time fix the enzyme-labeled complements in the reaction system or combine as an enzyme-labeled antibodies against the complements with the complement in the HLA antigen-CFAbs complexes, and then adding the corresponding substrates of the enzymes to ensure the color development or luminous reaction; the presence or absence or the relative levels of the CFAbs in the samples will be determined by detecting the optical density, because the optical density is corresponding to the CFAbs contents in the samples. Adding antibodies against characteristic marker (e.g. CD et al.) of the cell surface to the reaction system based on cells to recognize and isolate specific cell panels, can detect specifically the CDC reactions of the cell panels.

Description

ELISA-based

HLA complement-dependent cytotoxic antibody detection method and kit

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). When 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.

In tissue and organ transplantation, 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. In humans, MHC is called human leukocyte antigen (HLA). If the transplant donor and recipient's HLA type do not match, the recipient's immune system recognizes the graft as non-existent and develops an immune response, leading to "immunological rejection" reaction".

In this type of immune rejection, 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. 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. Therefore, in order to prevent immune rejection during organ transplantation, In addition to selecting donors whose HLA type matches the HLA type 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. At the same time, the detection of HLA antibodies in recipients after organ transplantation, including the dynamic determination of PRA (population-reactive HLA antibodies), is of great importance in monitoring the occurrence of acute and chronic rejection reactions and in predicting the recipient's lifetime after transplantation.

According to whether antibodies can fix and activate complement and induce CDC effect, human 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

(Complement-dependent Cytotoxic Antibodies; CDC-Abs) or '1' HFA 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

(on- CFAbs; Non-Complement Fixing Antibodies). Such antibodies exist naturally in the human body even when the body's immune system does not come into contact with any non-existing HLA; they can bind to self (Autologous) or foreign (Allogeneic) non-existing HLA molecules, but do not activate complement , Inducing immune CDC attack effect. To date, the role of such antibodies has not been fully understood. But most scholars believe that these antibodies are a natural antibody

(Natural Antibody) component, has a protective effect on the organism. The United States, France, and others have done in-depth research on this. A large number of in vitro and in vivo experiments and clinical experimental results have obtained completely consistent conclusions: that is, natural antibodies exist in normal human bodies. Because of their immunoregulatory functions, they are important for maintaining normal biological body health It plays a beneficial protective role (Protective Effects) and can be effectively used to suppress immune rejection in organ transplantation. See Figure 1 for the mechanism of cytotoxic antibodies and non-cytotoxic antibodies in the occurrence of CDC effect.

At present, there are two methods for the determination of HLA antibodies in the world: One is Terasaki's cytotoxicity test (Patel R, Terasaki PL, N Engl J Med 1969; 280: 735-9), referred to as 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. The biggest disadvantage of these methods is that they ignore the existence of natural HLA antibodies (Non-CFAbs), they do not cause CDC effects; instead, 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. This is why many laboratories often produce results that are difficult to interpret or contradictory to the serology of CDC, or even completely opposite to the clinical outcomes of the recipient: such as receiving normal human immunoglobulin (IVIG) immunity The 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. In addition, in clinical organ transplantation, 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.

Christiaans (Christiaans MH, et al., Transplantation 1996; 15: 1341 -1347) and others conducted a parallel study of flow and traditional serological cross-matching in 190 transplant patients in 1996. The results showed that the incidence of post-transplant rejection was not statistically different between the patients with positive and negative cross-matching flow cytometry. According to clinical observations, 28% of patients with positive cross-flow tying actually had good clinical results Outcome. Therefore, they concluded that: flow-type cross-matching is not superior to traditional serological cross-matching. Karuppan (Karuppan ss, et al, Transplantation 1992; 53: 666-673) has reached similar conclusions.

The results of Chia and Terasaki (Chia D, et al., Tissue Antigens 1991; 37: 49-55) in 1991 showed that: Normal human immunoglobulin IgG can bind through its non-hypervariable region HLA molecules, and this binding can be blocked by specific HLA antisera. Using different experimental designs and methodologies, we have further confirmed that normal human IgG can bind to human lymphocytes and block the complement cytotoxic effect of specific HLA antibodies. The above research shows that flow cross-matching can detect non-complement-fixed IgG and lead to false positive results, and this IgG is not closely related to, or even contradictory to, clinical outcome.

Koba (Koka P., Transplantation 1993; 56: 207-211) and Kerman (Kerman RH., Transplantation 1999; 68 (12): 1855-1858) published research results in 1993 and 1999, respectively: suggesting IgA and IgM classes The clinical significance of HLA antibody and its correlation with the occurrence of rejection. These two types of Ab cannot be detected by conventional flow cross-matching.

Dolly (Tyan et al. Transplantation 1994; 57: 553-562) et al. In a 1994 clinical study on the inhibition of transplant rejection by intravenous injection of human IgG (IVIG), proved that IVIG has good immunity for transplant patients treated for immune rejection Inhibitory effect; At the same time, in vitro experiments have also shown that IVIG can protect the graft by inhibiting the transplantation recipient's anti-transplant donor lymphocyte Ab effect. IVIG significantly inhibits cytotoxic effects induced by complement in vitro. Further research on its mechanism confirms (unpublished results): IVIG can block the cytotoxic effects of complement fixation Ab by binding to lymphocytes. 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.

In summary, 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

Accordingly, 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 step of contacting a complement or a labeled anti-complement antibody, characterized in that the label is an enzyme label, and 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.

Preferably, 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,

2) performing solid-liquid phase separation,

3) washing the solid phase carrier of the captured cells with an appropriate buffer,

4) incubating the T lymphocytes or / B lymphocytes adsorbed on the solid phase carrier with an enzyme-labeled anti-complement Clq or C3 antibody in a buffer,

5) Repeat steps 2) and 3),

6) 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.

Preferably, the present invention relates to a method for determining anti-HLA I and / or HLA II complement fixing antibodies, which comprises the following steps: '

1) The purified HLA I and / or HLA II antigen immobilized on a solid phase carrier and the test sample

2) performing solid-liquid phase separation,

3) washing the solid phase with a buffer solution,

4) incubating the washed solid phase with an enzyme-labeled anti-human Clq antibody in a buffer,

5) Repeat steps 2) and 3),

6) reacting the solid phase after washing with the chromogenic substrate or the enzymatic luminescent substrate of the labeled enzyme,

7) Measure the optical density value or luminous intensity of the reaction solution, and determine whether the sample is positive for HLA I and / or HLA II complement fixation antibodies.

In another aspect, 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. Detailed description of the invention

As described above, 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, and 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.

In the method of the present invention, 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.

In a preferred embodiment of the 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.

In the method of the present invention, 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.

In the method of the present invention, 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. Specifically, 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.

In a preferred embodiment of the invention, the complement is Clq or C3.

As mentioned above, 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.

As described above, in another embodiment of the present invention, 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. For example, for horseradish peroxidase, ABTS (2,2'-azino-bis (3-ethylbenzothiazolidine-6 sulfonic acid)), (o-phenylenediamine), 7¾ (3 , 3 ', 5,5'-Tetrafluorenylbenzidine), HPPA (3- (4-hydroxy) phenylpropionic acid), DAB (3,3, -diaminobenzidine), OAP (anthraminophenol), etc. . As the alkaline phosphatase (AP), for example, p-NPP (^ -nitrophenyl phosphate) or MUP (4-fluorenylumbellylphosphonic acid) can be used. For galactosidase, for example, ONPG (0-nitrophenyl-β-D-galactopyranoside) can be used. For glucose oxidase, MPMS / NBT can be used.

In a preferred embodiment of the present invention, the labeling enzyme uses horseradish peroxidase or alkaline enzyme.

In another embodiment of the invention, enzyme-labeled complement is used. In this embodiment, it is sometimes necessary to inactivate complement components that may be present in the sample.

In 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.

Therefore, 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,

2) into solid phase-liquid phase separation,

3) Wash the isolated solid-phase carrier that has captured the target cells with an appropriate buffer, Anti-complement Clq or C3 antibodies are incubated in buffer,

5) Repeat steps 2) and 3),

6) The isolated cells are reacted in a buffer with an enzymatic chromogenic substrate or an enzymatic luminescent substrate,

7) Measure the optical density value or luminous intensity of the reaction solution, and determine the result of cross-matching of T lymphocytes or B lymphocytes. For the case of using a purified HLA antigen, for example, the present invention provides the following method, which includes the following steps:

Σ / JDL,

2) Perform solid-liquid phase separation,

3) Wash the solid phase with buffer,

4) incubating the washed solid phase with an enzyme-labeled anti-human C 1 q antibody in a buffer,

5) Repeat steps 2) and 3),

6) The washed solid phase reacts with the chromogenic substrate or the enzymatic luminescent substrate of the labeled enzyme,

7) Measure the optical density of the reaction solution and determine whether the sample is positive for HLA I and / or HLA II complement fixation antibodies. In the method of the present invention, 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. In another aspect, 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.

In a preferred embodiment of the present invention, 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. 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.

2.Solid phase antigen: mainly refers to purified HLA antigen; the known type (such as HLA

-A, B, C, D (DR, DQ, DP) and their subtypes, etc.) or 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.

(Two) signal amplification detection system

1. Tracer markers: Usually various labeled ligands, antibodies or complement components, especially enzymatic labels (oxidases such as HRP, AP).

2. Enzymatic substrate or chemiluminescent agent:

Usually 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.

(3) ^ "Photo samples

1. 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.

2. 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.

(4) 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.

(5) Other

Including wash buffer, sample diluent, reaction containers (such as test tubes, microplates, etc.), 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. Since 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.

Since the object measured by the present invention is the HLA-CFAbs-complement complex component at the initial stage of the occurrence of CDC, the reaction time required by this method is the shortest compared to the existing methods.

In the method of the present invention, a signal detection method using enzymatic coloration and / or enzymatic luminescence achieves high sensitivity sufficient for practical qualitative and quantitative determination.

Because 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.

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.

Because 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.

Since 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. According to the principle of the occurrence of the CDC effect, 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. 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. Because 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. Therefore, 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. The present invention is described in more detail below with reference to the accompanying drawings and embodiments, which are used to illustrate the present invention and are not intended to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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)

(1) Add a certain amount of anti-CD3 antibody-coated immunomagnetic beads, donor nucleated cells, recipient serum or plasma to the reaction system and mix well.

(2) Incubate for a certain period of time (30 minutes-3 hours) under a certain temperature condition. At this time: If CFAbs are present in the serum or plasma of the recipient, it can bind to HLA on the surface of the donor's nucleated cells. Then, the complement Clq or C3 molecule in the recipient sample is fixed; the HLA-CFAbs-Clq triple complex is formed, and at the same time, the anti-human CD3 antibody of the magnetic bead solid phase specifically binds to the CD3 molecule on the surface of T lymphocytes.

(3) 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.

(4) Wash the isolated T lymphocytes with a buffer containing a certain amount of protein, such as a phosphate buffer containing 2% BSA, to further remove the remaining other cellular components and non-specific protein components.

(5) Remove the magnet and suspend the isolated T lymphocytes in a buffer containing a certain amount of HRP-labeled anti-Clq or C3 antibody (HRP-ClqAb or HRP-C3Ab), and continue incubation (the conditions are the same as in step 2) At this time, HRP-ClqAb is combined with Clq or C3 of the "triple complex" in step (2) to form a "quadruplex".

(6) Repeat steps (3) and (4) to remove free HRP-ClqAb or HRP-C3Ab; and carefully suspend the cells in a small amount of buffer.

(7) A certain amount of HRP substrate is added to the above cell suspension, and the color reaction is catalyzed by HRP in the quadruplex.

(8) Measure the optical density absorption value of the above sample with an ELISA Reader to obtain an OD value (Optical Density).

(9) Compare the OD value of the sample with the OD value of the normal control; the OD value higher than the upper limit of the normal control sample is defined as positive TLA lymphocyte HLA cross-matching; otherwise it is defined as negative. Example 2: B lymphocyte HLA cross-matching CELISA method

(―) The anti-CD3 antibody-coated immunomagnetic beads in step (a) of Example 1 were changed to anti-human CD19 or CD20 antibody-coated immunomagnetic beads. Other ingredients are the same.

(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.

(3) Same steps (three to nine) as in Example 1, except that the T lymphocytes in the above steps are changed to B lymphocytes. In the above Examples 1 and 2, 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. Regarding the Tc / Bc HLA cross-matching by using a microplate or magnetic bead-fixed antibody ELISA assay, see Figures 2 and 3.

The 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. Example 3.ELISA method for HLA Class I / II CFAbs

(―) Coat the purified HLA Class I or / and Class II antigen with a certain concentration in a microtiter plate and incubate at 37 ° C for 1 to 4 hours or overnight at 4 ° C. Discard the coating solution.

(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.). The time and temperature are the same as in step (1), and then the remaining blocking is aspirated. liquid.

(3) Add 50 microliters each of the negative control serum, P control serum, background buffer and test serum to the corresponding microtiter plate (or diluted serum).

4. Incubate the microplates at 37 ° C or 4 ° C for 30 minutes, 3 hours or 4 ° C overnight.

(5) Aspirate and discard the reaction solution, and wash each well of the microplate with a washing solution containing a certain concentration of detergent (such as Tween-20; NP-40, etc.), usually three times, and then aspirate and discard the last wash liquid.

(6) Add HRP-labeled anti-human Clq antibody with a certain concentration in each well, and continue incubation under the same conditions as in step (4).

(G) Repeat step (f).

(8) Add a certain amount of HRP substrate (such as TMB, etc.) to each well.

(9) Measure the OD value of each well with a microplate reader, and collect and record the results.

(10) Judgment of the result: If the OD value is greater than the upper limit of the normal value, the result is expressed as HLA-

Class I or / and Class II CFAbs test results are positive; otherwise, the test results are negative. Example 4: HLA-I or / and HLA-II immunomagnetic bead ELISA method

(1) Add magnetic beads coated (or connected) with HLA-I or / and HLA-II antigen to each well of the microplate.

(2) Same steps (3) and (4) as in Example 3.

(3) Place the microwell plate on a magnetic plate for several minutes; make the immunomagnetic beads adhere to the bottom or one side of the microwell. Then aspirate the liquid part; add washing solution to each well, then repeat the above magnetic plate adsorption and suction steps 2-3 times, and aspirate the last washing solution.

(4) Same steps (8), (9), and (ten) as in Example 3. Example 5: Population Responsive HLA CFAbs Measurement Method (CFAbs-PRA)

(1) Preparation of 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.

(2) Add a certain amount of the same test sample serum to each well of the above PRA plate, and incubate with the corresponding target cells or solid-phase target antigen; the incubation conditions are the same as step (4) of Example 3.

(3) Wash the above-mentioned cell wells or solid-phase HLA antigen wells; for example, cells can be centrifuged; magnetic beads can be separated by magnetic fields; micro-well plates can be washed directly, etc.

(4) Add HRP-ClqAb or HRP-C3Ab to the above reaction wells to continue incubation (conditions are the same as above), and then further wash (conditions are the same as above).

(5) Add a certain amount of HRP substrate to each of the above reactions, and then measure the OD value of each well.

(6) Judgment of results: Those with OD values higher than the upper limit of normal values are judged to be CFAbs positive; then calculate the percentage of positive reaction wells to the total number of PRA reaction wells; the calculation formula is as follows:

CFAbs positive wells ÷ total PRA wells X 100 = (P A%)

Example: The number of CFAbs positive wells is 25; and the total number of PRA wells is 50; then? 1 ^% is 50

%. Example 6: CFAbs determination of total HLA-I and HLA-II

(―) The 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. .

(B) HFA-I CFAbs determination can also use mixed human platelets as a solid-phase HLA-I source.

(3) The above 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

(1) Coat the microtiter plate or magnetic beads with purified B27 antigen, and then perform the CFAbs measurement of B27 according to the corresponding conditions in the above example.

(2) It is known that cells of type B27 can also be used as the target cell source of B27 antigen, which can be directly used for the determination of CFAbs. Example 8: Validation of the method of the invention by CDC serology

Six organ transplant recipient serum samples were cross-matched in parallel by the ELISA complement fixation antibody assay and CDC serology assay of the present invention. The ELISA method for measuring complement-fixing antibodies of the present invention was carried out according to the method of Example 1. The specific steps are as follows:

1. Add 50 microliters (μΐ) of fresh anticoagulated whole blood from an organ transplant donor to an Eppdorf test tube containing 1 ml (ml) of red blood cell lysate, and mix at room temperature for 5 minutes.

2. Centrifuge at a speed of> 400g, centrifuge for 1-10 minutes, aspirate and discard the supernatant; then wash the pelleted cells 2-3 times with a buffer containing a certain concentration of protein, PBS, and aspirate and discard the supernatant.

3. Suspend the cells in 20-50 microliters of recipient serum, and continue to incubate at room temperature (about 25 ° C) for 10-30 minutes. 4. Add HRP-labeled anti-human Clq antibody (ICN Biomedicals, Inc. #Cat: 68215 Sheep, Anti human Cl q) to the above reaction tube and let stand for 30 minutes at room temperature. Repeat step 2.

5. Add 10 microliters of magnetic beads (Dynabeads M450 CD3 (pan T) #Cat: 111.13) coated with anti-human CD3 antibody. After mixing, continue to stand at room temperature for 30 minutes.

6. 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.

7. Add 100 microliters of enzyme substrate TMB to the above bead-cell-containing test tube, and place at room temperature for 15 minutes.

8. Then, add 100 ml of reaction stop solution to stop the color reaction.

9. Measure the absorbance (OD) in a microplate reader. The above-mentioned anti-human Clq antibody is labeled as follows:

(1) Weigh 5mg of HRP (Sigma P8375) and dissolve it in 1ml of distilled water.

(2) Add 0.2 ml of a freshly prepared 0.1M NaIC (Sigma SI 147) solution to the supernatant and stir at room temperature for 20 minutes in the dark.

(3) Put the above solution into a dialysis bag, and dialyze against 1 mM sodium acetate buffer, pH 4.4, overnight at 4 ° C.

(4) Add 20μ1 0.2M pH 9.5 carbonate buffer to increase pH to 9.0 ~ 9.5, and then immediately add 10mg IgG (ICN Biomedicals, Inc. #Cat: 68215 Sheep, Anti human Clq) in 1ml 0.01M Stir gently in carbonate buffer for 2 hours at room temperature, protected from light.

(5) Add 0.1ml of newly prepared 4mg / ml NaBH ₄ (Sigma S 9125) solution, mix well, and set it at 4 ° C for 2 hours.

(6) Put the above solution into a dialysis bag and dialyze against 0.15M pH7.4 PBS, overnight at 4 ° C.

(7) Add an equal volume of saturated ammonium sulfate dropwise with stirring, and set at 4 ° C for 1 hour.

(8) Centrifuge at 3000 rpm for half an hour and discard the supernatant. The precipitate was washed twice with ammonium half-saturated stone, and finally the precipitate was dissolved in a small amount of 0.15M PBS, pH 7.4.

! 8 (9) Put the above solution into a dialysis bag, dialyze the 0.15M ρΗ7.4 PB buffered saline, and centrifuge at 10,000 rpm for 30 minutes to remove the precipitate. The supernatant is the enzyme label. Shown in Figure 6. According to the CELISA method of the present invention, the cross-matching results were 4 cases positive (samples 1-4) and 2 cases negative (samples 5 and 6). This is completely consistent with the results of traditional CDC serological methods (not shown). As can be seen from the above description, various changes or modifications can be made to the invention disclosed in this specification to suit various conditions and applications. These modified embodiments are also within the scope of the invention.

Claims

Rights request

What is claimed is: 1. 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 corpse Or a step of contacting a labeled anti-complement antibody, characterized in that the label is an enzyme label, and the method further comprises adding a reaction substrate of the labeled enzyme to the reaction system to generate an enzymatic color reaction, and then measuring A step of determining the presence and / or amount of the complement-fixing antibody in the sample by the optical density value of the reaction solution.

2. The method according to claim 1, wherein the HLA antigen is a target cell having a HLA antigen on its surface or a purified HLA antigen.

3. The method according to claim 1 or 2, wherein the HLA antigen is immobilized on a solid support.

4. The method according to claim 1, wherein the sample is a body fluid of a human or an animal.

5. The method according to claim 4, wherein the body fluid is serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, saliva or urine.

6. The method according to claim 1, wherein the HLA antigen is an HLA type I antigen and / or an HLA type II antigen.

7. The method according to claim 2 or 6, wherein the target cells are T lymphocytes and / or B lymphocytes.

8. The method according to claim 1, wherein the complement is 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.

9. The method according to claim 8, wherein the complement is Clq or C3.

10. The method according to claim 1, 8 or 9, wherein the labeled complement is a human Derived, derived from animals, obtained by chemical synthesis or obtained by genetic engineering methods.

11. The method according to claim 1, wherein the anti-complement antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody or an antibody fragment.

12. The method according to claim 1, wherein the labeling enzyme is selected from the group consisting of horseradish peroxidase, alkaline enzyme.

13. The method according to claim 2, further comprising adding an antibody against a specific antigen on a surface of a target cell coated on a solid-phase carrier to the reaction system in order to isolate a specific cell subgroup and determine complement fixation on the surface of the cell subgroup Sexual antibodies.

14. The method according to claim 13, wherein the antibody is an antibody against a Tc / Bc CD antigen.

15. The method according to claim 1, comprising the steps of: incubating the nucleated cells of the organ donor and the body fluid sample of the recipient together,

2) performing solid-liquid phase separation,

3) washing the separated solid-phase carrier that has captured the target cells with an appropriate buffer,

5) Repeat steps 2) and 3),

6) reacting the isolated cells with an enzyme chromogenic substrate in a buffer,

7) Measure the optical density of the reaction solution, and determine the cross-matching results of T lymphocytes or B lymphocytes.

16. The method according to claim 1, comprising the steps of:

1) incubating the purified HLA I and / or HLA II antigen immobilized on a solid phase carrier with the test sample,

2) performing solid-liquid phase separation,

3) washing the solid phase with a buffer solution,

5) Repeat steps 2) and 3),

6) the washed solid phase reacts with the chromogenic substrate of the labeled enzyme, 7) Measure the optical density of the reaction solution, and determine whether the sample is positive for HLA I and / or HLA II complement fixation antibodies.

17. The method according to claim 15 or 16, wherein the solid support is a magnetic bead or a microplate.

18. A kit for carrying out the method of claim 1, comprising containers each containing at least the following: an enzyme-labeled complement or an enzyme-labeled anti-complement antibody; a corresponding enzyme reaction substrate; a suitable buffer.

19. The kit according to claim 18, further comprising a solid-phase antibody against a target cell surface antigen.

20. The kit according to claim 18, further comprising a purified solid-phase HLA antigen.