CN110716053B - Novel method for detecting population reactive antibodies - Google Patents
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- CN110716053B CN110716053B CN201910802831.3A CN201910802831A CN110716053B CN 110716053 B CN110716053 B CN 110716053B CN 201910802831 A CN201910802831 A CN 201910802831A CN 110716053 B CN110716053 B CN 110716053B
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- G01N33/531—Production of immunochemical test materials
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- 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
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- 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
Abstract
The invention discloses a new method for detecting group reactive antibodies, which comprises the steps of selecting a certain number of representative donors from potential donor groups of transplanted recipients, collecting and separating cells of the representative donors, and purifying cell surface antigens; these purified antigens from different individual cells in the potential donor population are combined into an antigen combination which can represent the characteristics of the cell surface antigen population of the tissue of the potential donor population, and the antigen-antibody detection platform is used for detecting PRA in the biological sample to be detected. The method avoids the defects of the PRA detection method based on cells, such as limitation of the number and quality of cells and operation problems of technicians, and also overcomes the defect that the PRA detection method based on purified HLA protein antigen can only detect HLA antibody and miss detection of all non-HLA antibodies.
Description
Technical Field
The invention belongs to the technical field of group reactive antibody detection, and relates to a novel method for detecting group reactive antibodies.
Background
A Population Reactive Antibody (PRA) is a method for detecting the presence of antibodies specific to antigens associated with rejection of potential donor populations in biological samples such as peripheral blood of transplant recipients. The range of PRA detection results is 0-100%, with 0% indicating that no detectable antibodies to any potential donor are present in the graft recipient sample with minimal risk of rejection; 100% indicates that there are antibodies to all potential donors in the graft recipient sample and the highest risk of rejection.
Currently, the detection techniques for PRA commonly used in Transplantation clinical tests mainly include cell-based and purified HLA antigen-based PRA detection methods (Transplantation Reviews, vol 18, no 4 (October), 2004; surgToday (2005) 35).
1. Cytology PRA detection method using complement-dependent cell killing effect of antibody as mechanism
A certain number of representative potential donors are selected from a potential donor population of a transplant recipient, and their tissue cells (vascular endothelial cells, blood cells or fractions thereof, or tissue cells of the transplant, etc.) are collected and individually loaded into culture wells of a cell culture plate to form a cell combination (Panel) derived from the donor population and representing the characteristics of the transplantation-associated antigen population of the potential donor population to a certain extent. Subsequently, a biological sample of the graft recipient is added to each cell culture well of the cell combination of the potential donor population in the presence of Complement, and the antibodies in the biological sample of the graft recipient kill cells expressing the target antigens (including HLA and non-HLA antigens) to which the antibodies correspond according to the mechanism of Complement dependent cell killing (CDC) of the antibodies, resulting in cell death. The final results were interpreted as a percentage of all cell populations tested in which a proportion of the cells from the potential donor were killed by the antibody in the biological sample from the graft recipient, with the PRA test ranging from 0-100%,0% indicating that none of the cells from all selected potential donors were killed, and 100% indicating that all of the cells from selected potential donors were killed.
The disadvantage of this method is that only complement-activating, cell-killing antibodies can be detected, whereas antibodies that do not activate complement but can cause rejection by other routes are missed, and thus flow cytometry is introduced for the detection of PRA.
2. Flow cytometry PRA detection method with antibody combined with cell surface antigen as mechanism
The selection principle of potential donor group cell combination in the flow-type cell PRA detection technology is similar to the PRA detection method, but the interpretation of the result is not based on whether the cells are killed or not, but based on whether antibodies in the biological sample of the transplant recipient can perform binding reaction with the cells in the target cell group or not, which can detect the antibodies with cell killing function and non-complement activation function, therefore, the sensitivity of the flow-type cell PRA detection technology is higher than that of the PRA detection technology based on CDC. The range of results for flow cytometry PRA is also 0-100%, with 0% representing that the antibodies in the test sample from the graft recipient do not bind to the cells of any of the selected potential donors and 100% representing that the antibodies in the test sample from the graft recipient do bind to the cells of all of the selected potential donors (ASHI Laboratory Manual, edition 4.2.2000 V.I.B.1-8).
The two PRA detection methods are cell-based methods, but the two PRA detection methods are influenced by reasons of poor instant availability of donor group cells, complex cell separation and purification, high requirements on cell quantity and activity, high technical requirements on experimenters, more factors interfering with result interpretation and the like, are greatly limited in popularization and application, are often inconsistent in result comparability among laboratories, are poor in convenience in use and are not easy to popularize.
3. PRA detection technology based on purified HLA antigen
With the intensive research on rejection-associated antigens, MHC antigens encoded by Major Histocompatibility Complex (MHC) genes, i.e., human Leukocyte Antigens (HLA), have been found to be a major target Antigen with polymorphism that can cause rejection. Based on this finding, a significant portion of the scholars in the field of transplantation immunology believe that the positive response detected by cell-based PRA detection methods may be due to the critical role of HLA antibodies in them. Therefore, with the spread of protein biochemical and molecular cloning technologies in transplantation detection technologies, the purified HLA antigen-based PRA detection method has been widely used from the 90 s, and in recent years, there is a trend to replace the cell-based PRA detection method (Surg Today (2005) 35. The PRA detection method based on the purified HLA antigen mainly combines the HLA antigen protein purified from cells expressing the HLA antigen protein to a carrier medium of a stationary phase, and realizes the PRA detection by technical platforms such as enzyme linked immunosorbent assay (ELISA), a solid phase membrane, a protein chip, flow microspheres, luminex liquid phase chip microspheres and the like according to the principle of antigen-antibody combination.
Compared with the traditional PRA detection method based on cells, the PRA detection method based on the purified HLA antigen has the advantages of rapidness and convenience, definite target antigen, high sensitivity and the like. However, it has a fatal disadvantage that only PRA positive reactions caused by HLA antibodies can be detected, and antibodies against all graft rejection-associated antigens other than HLA antibodies are missed (Transplantation Reviews, vol 18, no. 4 (October), 2004.
Disclosure of Invention
Aiming at the problems, the invention provides a new method for detecting the group reactive antibodies, which solves the problem that the purified HLA antigen detection method can only detect PRA positive reaction caused by HLA antibodies.
In order to achieve the purpose, the invention adopts the following technical scheme:
a novel method for detecting population reactive antibodies comprising the steps of:
1) Selecting cells from a plurality of individuals that are characteristic of a population of cell surface antigens from a potential donor population;
2) Respectively purifying and separating all antigens on the cell surface of the selected cells;
3) Binding all antigens on the cell surface of the purified single individual to a carrier medium of the stationary phase respectively;
4) Population reactive antibodies are detected according to the antigen-antibody binding principle.
Further, in step 1), the cell comprises: vascular endothelial cells, blood cells or components thereof, or tissue cells of the graft, and the like, but are not limited thereto.
Further, the antigens in step 2) include HLA antigens and non-HLA antigens.
Still further, the non-HLA antigens include: the major histocompatibility complex class i molecular chain related genes, i.e., MIC genes encode protein-MIC antigen, minor histocompatibility antigens (minor histocompatibility antigens), angiotensin II receptor (AT 1R), endothelin-1A receptor (ETAR), etc., but are not limited thereto.
Further, step 2) comprises: directly purifying the cell surface antigen of the selected cell, or purifying the cell surface antigen after the selected cell is cultured and amplified in vitro.
Further, in step 3), the carrier medium of the stationary phase includes, but is not limited to, a plate for ELISA, a solid-phase membrane, a flow-type or Luminex fluorescent microsphere, or a protein chip.
Further, the detection method adopted in the step 4) comprises the following steps: enzyme-linked immunosorbent assay, immunofluorescence antibody assay, immuno-colloidal gold assay, chemiluminescence immunoassay, and protein chip assay, but are not limited thereto.
Selecting a certain number of representative donors from potential donor groups of transplanted recipients, collecting and separating cells of the donors, and purifying all antigens on the surfaces of the cells; the purified antigens (the cell surface contains HLA and non-HLA antigens) from different individual cells in the potential donor group are combined into an antigen combination which can represent the characteristics of the cell surface antigen group of the tissue of the potential donor group, and PRA in the tested biological sample is detected in vitro by utilizing the principle of solid-phase immunological antigen-antibody binding reaction.
The invention has the following beneficial effects:
compared with a cytological PRA detection method, the method can simultaneously detect the PRA positive reaction caused by HLA and non-HLA antibodies, but can avoid a plurality of defects of the cytological detection method, such as poor instant availability of cells, complicated cell separation and purification, high requirements on cell quantity and activity, high technical requirements on experimenters, more factors interfering with result interpretation and the like.
Compared with the conventional common method for detecting PRA by purifying HLA antigen, the method has the advantages of small influence of human factors, high sensitivity and stability and easy popularization, but solves the problem that the purified HLA antigen detection method can only detect PRA positive reaction caused by HLA antibody and can not detect PRA positive reaction caused by non-HLA antibody.
Drawings
FIG. 1 is a schematic diagram of the preparation of the detection reagent of the present invention.
FIG. 2 is a schematic view illustrating the interpretation of the detection results of the present invention.
FIG. 3 is a schematic diagram of the PRA calculation method of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Examples
In this embodiment, the novel method for detecting a population reactive antibody mainly comprises the following steps:
1) Based on the characteristics of the potential graft-derived population, a number of individual tissue cells, such as vascular endothelial cells, blood cells or fractions thereof, or tissue cells of the graft, are selected that are characteristic of the population of cell surface antigens of the potential donor population.
2) All antigens (including HLA antigens and other non-HLA antigens) on the cell surface were purified and isolated from the cell surface of the selected individuals or the tissue cells of the individuals after in vitro expansion culture, respectively, as shown in fig. 1A.
Depending on the cell source, non-HLA antigens on the cell surface include, but are not limited to, the following classes: major histocompatibility complex class I molecular chain related genes, namely MIC gene coding protein-MIC antigen, minor histocompatibility antigens (minor histocompatibility antigens), angiotensin II receptor (AT 1R), endothelin-1A receptor (ETAR) and the like.
3) Respectively binding all purified antigens on the cell surface of a single individual to a carrier medium of a stationary phase in a covalent or non-covalent binding mode, taking a flow type or Luminex fluorescent microsphere as an example, coating the cell surface antigens from a No. 1 donor with corresponding No. 1 fluorescent microsphere, and so on; the carrier medium such as the same-number homogeneous microspheres can also be divided into different groups in equal proportion, and the groups are formed after the antigens from different individual sources are respectively coated, as shown in figure 1B.
The carrier medium for the stationary phase may include a plate for ELISA, a solid phase membrane, a flow-type or Luminex fluorescent microsphere, or a chip carrier for detection such as a titer plate, a filter, a slide glass, or the like.
4) Interpretation of the detection results: the carrier medium combined with the purified cell surface antigen reacts with the sample to be detected, taking the flow-type or Luminex fluorescent microspheres coated with the purified cell surface antigen as an example, under the indication effect of the labeled target antibody specificity 2 antibody, the microspheres without the antigen specificity antibody combined on the surface are not combined with the secondary labeled 2 antibody, and the negative result of the labeled antibody is shown (fig. 2A); the fluorescent microspheres bound with the antigen-specific antibody bound with the secondary label 2 antibody, indicating a positive reaction of the labeled antibody (fig. 2B).
5) PRA calculation method: the results of the antigen-antibody binding reaction between the antibody in a test sample and the population of 50 antigen-coated microspheres are shown in FIG. 3, which contains 5 rows and 10 columns, and a total of 50 different sets of fluorescent microspheres as a population, wherein each set of microspheres (represented by a single microsphere) is coated with purified antigen from the surface of a single potential donor cell, and 50 sets of antigen-coated microspheres represent the combination of cell surface antigens of a population of 50 individuals.
It is shown in the figure that all 10 groups of microspheres in row 1 exhibited positive reactions with antibody binding to the microsphere surface antigen, and thus the PRA calculation =10/50=20%.
Claims (5)
1. A novel method for detecting population reactive antibodies, comprising the steps of:
1) Selecting cells from a plurality of individuals that are characteristic of a population of cell surface antigens from a potential donor population;
2) Separating and purifying all antigens on the cell membrane surface of the selected cell, including HLA antigens and non-HLA antigens;
3) Binding all antigens on the surface of the cell membrane of the purified single individual to a carrier medium of a stationary phase respectively;
4) According to the antigen-antibody binding principle, PRA positive reactions caused by HLA and non-HLA antibodies are simultaneously measured, and population reactive antibodies are detected.
2. The novel method of claim 1, wherein in step 1), the cells comprise: vascular endothelial cells, blood cells or components thereof, or tissue cells of the graft.
3. The novel method of claim 1, wherein step 2) comprises: directly purifying the cell surface antigen of the selected cell, or purifying the cell surface antigen after the selected cell is cultured and amplified in vitro.
4. The novel method for detecting antibodies reactive to a population according to claim 1, wherein in step 3), the carrier medium of the stationary phase comprises a plate of ELISA, a solid-phase membrane, a flow-type or Luminex fluorescent microsphere, or a protein chip.
5. The novel method for detecting population-reactive antibodies of claim 1, wherein the detection method used in step 4) comprises: enzyme-linked immunosorbent assay, immunofluorescence antibody assay, immune colloidal gold assay, chemiluminescence immunoassay, and protein chip assay.
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| CN111257566B (en) * | 2020-01-22 | 2023-03-24 | 苏州才博医学科技有限公司 | Methods for detecting allogeneic, or donor-specific, non-HLA antibodies |
| CN111474368B (en) * | 2020-04-16 | 2023-03-24 | 苏州才博医学科技有限公司 | Antigen purification method for comprehensively detecting non-HLA class donor specific antibody |
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| US5482841A (en) * | 1994-05-24 | 1996-01-09 | Sangstat Medical Corporation | Evaluation of transplant acceptance |
| CN1423130A (en) * | 2002-04-26 | 2003-06-11 | 帕弗瑞生物技术(北京)有限公司 | HLA cross matching method for stream measuring fixed complement antibody and kit thereof |
| CN101696974B (en) * | 2009-09-29 | 2013-03-06 | 才新 | HLA antibody specificity detecting method, cell dish and reagent kit |
| US20170082619A1 (en) * | 2015-09-23 | 2017-03-23 | One Lambda, Inc. | Methods of Detecting Alloantibodies Using HLA and Non-HLA Antigens |
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