阅读说明：本技术 检测群体反应性抗体的新方法 (Novel method for detecting population reactive antibodies ) 是由 蔡俊超 于 2019-08-28 设计创作，主要内容包括：本发明公开了检测群体反应性抗体的新方法,在移植受者潜在的供者群体中选取一定数量的有代表性的供者,采集并分离其细胞,纯化细胞表面抗原；把这些来源于潜在供者群体中不同个体细胞的纯化抗原组成一个可以代表潜在供者群体组织细胞表面抗原群体特征的抗原组合,通过抗原-抗体检测平台,用于被检生物学样本中PRA的检测。该方法即避免了以细胞为基础的PRA检测方法中受限于细胞数量和质量,以及技术人员操作问题相关的诸多缺点,也排除了以纯化的HLA蛋白抗原为基础的PRA检测法只能检测HLA抗体而漏检所有非HLA抗体的缺陷。(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.)

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) 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.

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 the antigens in step 2) comprise HLA antigens and non-HLA antigens.

4. The novel method of claim 3, wherein the non-HLA antigens comprise: MIC gene coding protein-MIC antigen, minor histocompatibility antigen, angiotensin II receptor and endothelin-1A receptor.

5. 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.

6. The novel method of claim 1, wherein in step 3), the carrier medium of the stationary phase comprises ELISA plate, solid phase membrane, flow or Luminex fluorescent microsphere, or protein chip.

7. 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.

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 transplant recipient sample and the highest risk of rejection.

At present, the detection techniques for PRA commonly used in Transplantation clinical examinations mainly include cell-based and purified HLA antigen-based PRA detection methods (Transplantation Reviews, Vol 18, No 4 (October),2004: pp 192-); SurgToday (2005)35: 605-.

1. Cytological 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 also ranged from 0 to 100%, with 0% indicating that the antibodies in the test sample from the graft recipient did not bind to the cells of any of the selected potential donors and 100% indicating that the antibodies in the test sample from the graft recipient did 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), were found to be a class of polymorphic major target antigens 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, PRA detection methods based on purified HLA antigens have been widely used since the 90 s, and in recent years, there has been a trend to replace cell-based PRA detection methods (Surg Today (2005)35: 605-. 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: pp 192-203).

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 purification 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 genes related to class I molecular chains of the major histocompatibility complex, namely MIC genes encode protein-MIC antigen, minor histocompatibility antigens (minor histocompatibility antigens), angiotensin II receptor (AT1R), endothelin-1A receptor (ETAR), and the like, 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, immunocolloidal 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 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 PRA in the biological sample to be detected 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.