Method for preparing human HLA specific antibody detection standard substance by using protein A immunoadsorption waste liquid

Abstract

The invention belongs to the technical field of biological medicine detection, and particularly relates to a method for preparing a human HLA specific antibody detection standard substance by using protein A immunoadsorption waste liquid. The preparation method comprises the following steps: neutralizing the protein A immunoadsorption waste liquid with a Tris-HCl buffer solution, adjusting the pH value to 6.9-7.2, and adding a protein protective agent to maintain the IgG activity; the method comprises the following steps: firstly, preparing an HLA-specific adsorption column, removing non-IgG miscellaneous protein by adopting protein G affinity chromatography, removing viruses from the obtained enriched liquid through nano filtration, and finally, carrying out epitope specific enrichment by adopting the HLA-specific adsorption column, so as to obtain the HLA specific antibody detection standard substance. The eluent generated after the protein A immunoadsorption column treatment is innovatively used as a basic raw material, the uniform and stable HLA specific antibody detection standard substance is prepared through a specific process, and the standard substance has the advantage of low cost and can truly reflect the HLA antibody specificity of a patient.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a method for preparing a human HLA-specific antibody detection standard product by utilizing protein A immunoadsorption waste liquid. Background Art

[0002] Human leukocyte antigen (HLA) is a component of the major histocompatibility complex (MHC) and is an important immune molecule. HLA molecules are located on the cell membrane and participate in the body's immune response, including important physiological functions such as antigen presentation, immune surveillance and immune tolerance. HLA antibodies are specific immune response products produced by the immune system to allogeneic HLA molecules. They can trigger rejection reactions, cause damage to transplanted organs, and affect the long-term survival rate of transplants. Before kidney transplantation, the detection of HLA antibodies is particularly important. Since the presence of HLA antibodies may cause acute rejection of the graft or long-term chronic rejection, testing HLA antibodies in the recipient before and after transplantation can help assess the risk of transplant failure.

[0003] At present, the detection methods of HLA antibodies mainly include cytological methods, enzyme-linked immunosorbent assay (ELISA), flow cytometry and single-molecule immunoassay. Although these methods can detect the presence of HLA antibodies, the problem of standardization has always plagued clinical diagnosis due to the differences between different detection methods. Especially in terms of standards, the current common HLA antibody standards rely on human serum or allogeneic tissue extraction, which has poor stability and limited sources. Therefore, the development of high-quality, stable and standardized HLA antibody detection standards has become an important task to improve the accuracy of HLA antibody detection and improve the success rate of transplantation. The shortcomings of HLA antibody detection standards are mainly reflected in the following aspects: 1. Limited sources: Most of the existing HLA antibody standards are derived from human serum or prepared through animal models. The acquisition process of these standards is complicated and the quantity is limited, which cannot meet the needs of large-scale clinical testing. 2. Poor stability: The stability of HLA antibody standards is poor, especially when stored for a long time, antibody degradation or reduced activity may occur, which directly affects the accuracy of the test results. 3. Lack of specific epitope standards: Due to differences in the sources of standards between different laboratories and detection methods, such as polyclonal serum or animal-derived polyclonal antibodies, there are differences in test results, which affects the widespread application of HLA antibody testing.

[0004] Protein A adsorption is one of the technologies currently widely used to separate and purify antibodies. Protein A is a surface protein derived from bacteria that can specifically bind to IgG antibodies and is widely used in the separation, purification and immunotherapy of antibodies. Due to its efficient binding ability, protein A adsorption is widely used in the production of monoclonal antibodies, immunotherapy and plasma therapy. In kidney transplantation treatment, protein A adsorption is used to remove adverse immune response antibodies in the body, thereby reducing rejection reactions and improving the success rate of transplantation. In practical applications, the waste liquid generated during protein A adsorption often contains a large amount of immunoglobulins, including HLA antibodies. These waste liquids are usually discarded or simply treated. However, waste liquids often contain potential resources-HLA antibodies. If these antibodies can be effectively recovered, their resource utilization will be greatly improved, and even raw materials will be provided for the production of HLA antibody detection standards. Developing an efficient waste liquid recovery process can not only improve economic benefits, but also effectively reduce environmental pollution and provide new sources of materials for research in related fields. Therefore, the adsorption of HLA-specific antibodies requires HLA proteins for adsorption.

[0005] HLA protein-specific adsorption technology selectively extracts antibodies associated with HLA molecules from biological samples using specific adsorbents, providing higher efficiency and accuracy for the purification and detection of HLA antibodies. This technology can not only improve the detection sensitivity of HLA antibodies, but also ensure the stability of antibodies, provide a reliable source for the production of HLA antibody standards, and further promote the standardization and precision of HLA antibody detection technology. For example, in organ transplantation surgeries such as kidney transplantation and heart transplantation, accurate detection of HLA antibodies in transplant recipients can help assess the risk of rejection and provide a reference for immunotherapy before and after transplantation. In "Antibodies targeting complexes containing atypical HLA-I and neoantigens and methods of use thereof" (application number: CN202180057675.8), antibodies that selectively bind to atypical HLA-I and neoantigens such as HLA-E are provided. This type of antibody has high specificity and can help screen antigens associated with immune escape, providing a more accurate treatment tool for immunotherapy, especially in cancer immunotherapy and immune monitoring. It has important application value. Summary of the invention

[0006] Based on the above prior art, the primary purpose of the present invention is to provide a method for preparing human HLA-specific antibody detection standards using protein A immunoadsorption waste liquid.

[0007] Another object of the present invention is to provide a human HLA-specific antibody detection standard prepared by the above method.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid comprises the following steps:

[0010] (1) Neutralize the protein A immunoadsorption waste liquid with Tris-HCl buffer, adjust the pH to 6.9-7.2, and add a protein protectant to maintain IgG activity;

[0011] (2) removing non-IgG proteins from the solution obtained in step (1) by protein G affinity chromatography to obtain an enriched solution;

[0012] (3) The enriched solution obtained in step (2) is filtered to remove viruses, and then epitope-specific enrichment is performed using an HLA-specific adsorption column to obtain an HLA-specific antibody detection standard.

[0013] Furthermore, the protein A immunoadsorption waste liquid in step (1) comes from the eluate of blood from patients who have received HLA-related treatment or transplantation and have been treated with protein A immunoadsorption columns.

[0014] Furthermore, the Tris-HCl buffer in step (1) uses a Tris-HCl buffer with a concentration of 1 M and a pH value of 9.0-9.3.

[0015] Furthermore, the protein protective agent and the addition amount in step (1) are 5 wt% D-trehalose + 2 wt% mannitol.

[0016] Furthermore, the nanofiltration to remove viruses in step (3) uses a membrane with a pore size of 35-45 nm for nanofiltration, and the filtration pressure is 0.2 MPa.

[0017] Furthermore, the HLA-specific adsorption column in step (3) is prepared by the following method:

[0018] The HLA protein is mixed with a dispersion of cross-linked agarose microbeads (such as Thermo Amino Link Plus coupling resin) for reaction and incubation, and then the excess activation sites are blocked with an amino reagent to obtain microbeads coupled with HLA proteins, which are then loaded into an adsorption column to obtain an HLA-specific adsorption column.

[0019] Further preferably, the mass concentration of the cross-linked agarose microbead dispersion is 4 wt %; and the mass volume ratio of the HLA protein and the cross-linked agarose microbead dispersion is 2:3 (mg / mL).

[0020] More preferably, the mixed reaction is incubated at a temperature of 37° C., a pH of 7.2-7.4, and a time of 1 h.

[0021] More preferably, the amino reagent is a Tris-HCl solution with a pH value of 7.2 to 7.4.

[0022] Furthermore, the epitope-specific enrichment step in step (3) is as follows:

[0023] The virus-free enriched solution is injected into an HLA-specific adsorption column for adsorption. After the adsorption is completed, the adsorption column is rinsed with a Tris-NaCl equilibrium buffer, and the adsorbed specific antibodies are eluted with a Tris-glycine buffer. The eluate is neutralized to neutrality with a Tris-NaCl solution, and the resulting product is subjected to protein quantification and antibody binding epitope identification to obtain an HLA-specific antibody detection standard.

[0024] Furthermore, the HLA-specific antibody detection standard obtained in step (3) is further prepared into a lyophilized preparation by the following method:

[0025] The HLA-specific antibody detection standard product is dissolved in a lyophilizing protective agent, filtered and sterilized, and then freeze-dried. The product is filled with an inert gas and sealed for storage to obtain a lyophilized preparation of the HLA-specific antibody detection standard product.

[0026] Further preferably, the lyoprotectant composition comprises: 0.1 M citrate buffer (pH 6.5), 0.01 wt% Tween-80, 0.1 wt% BSA, and 5 wt% trehalose.

[0027] Further preferably, the freeze-drying procedure is: first pre-freeze at -40 to -80°C to completely solidify the solution, then evacuate to 10-50 mTorr, heat to -40 to -10°C to sublimate and remove most of the water, and then heat to 20 to 30°C to remove residual water.

[0028] An HLA-specific antibody detection standard or freeze-dried preparation is prepared by the above method.

[0029] The principle of the present invention is that the eluate of the protein A immunoadsorption column is derived from the blood of patients who have received HLA-related treatment or transplantation, and may contain HLA multigene, polyclonal, multi-epitope antibodies and other complex components. The main components in the waste liquid include:

[0030] (1) IgG antibodies: IgG1 and IgG3 are the core components of HLA antibody standards. IgG2, IgG4, and autoantibodies (such as ANA, RF, etc.) may cause false positives and affect the accuracy of HLA antibody testing.

[0031] (2) Contaminants: Fc binding proteins, complement proteins, plasma proteins, etc. may interfere with antibody purification and need to be removed.

[0032] (3) Viral contaminants: such as HBV, HCV, HIV, HAV, etc., need to be effectively removed to ensure the biosafety of the product.

[0033] (4) Endotoxin: Residual endotoxin may affect the safety of the product and needs to be controlled below 0.5EU / mL.

[0034] Through the sequential neutralization and protection treatment (since protein A is generally eluted with a low pH (2.5-3.5) buffer (such as 0.1M acetic acid), direct recovery may cause partial denaturation of IgG and decreased antigen binding ability. The present invention uses Tris-HCl buffer for rapid neutralization, adjusts the pH of the eluate to 6.9-7.2, and adds D-trehalose and mannitol as protein protectants to maintain IgG activity), protein G affinity chromatography to remove foreign proteins (using protein G to have a strong affinity for HLA-specific antibodies, it is used to remove non-IgG foreign proteins and increase the enrichment of HLA antibodies), and nanofiltration to remove viruses pretreatment steps, the waste liquid meets the quality requirements of HLA antibody standards. Finally, epitope-specific enrichment is performed through an HLA-specific adsorption column to obtain an antibody detection standard that can target the characteristic epitopes of HLA antigens.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) Raw material innovation: Using the eluate produced after protein A immunoadsorption column treatment as the basic raw material can reduce costs and truly reflect the patient's HLA antibody specificity.

[0037] (2) Process innovation: Multiple steps are used, including neutralization protection treatment, protein G affinity chromatography to remove impurities, nanofiltration to remove viruses, epitope-specific enrichment, and freeze-drying stability modification, to ensure product uniformity and stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the preparation process of a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid in Example 1.

[0039] Figure 2 This is a heat map of epitope coverage of the HLA-A2 antibody obtained in Example 1.

[0040] Figure 3 This is a graph showing the results of multiple adsorption and elution stability tests on the HLA-A2 adsorption column obtained in Example 1.

[0041] Figure 4 This is a graph showing the results of multiple adsorption efficiency tests of the HLA-A2 adsorption column obtained in Example 1.

[0042] Figure 5This is a diagram showing the specific identification results of the HLA-A2 antibody obtained in Example 1 for the 43R epitope.

[0043] Figure 6 This is a diagram showing the specific identification results of the HLA-A2 antibody obtained in Example 1 for the 62GK epitope.

[0044] Figure 7 This is a diagram showing the specific identification result of the HLA-A2 antibody obtained in Example 1 for the 107W epitope.

[0045] Figure 8 This is a graph showing the fluorescence intensity analysis results of the total HLA-I type protein presented by three batches of positive quality control serum of the existing kit in Comparative Example 1. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0047] Example 1

[0048] A method for preparing a human HLA-specific antibody detection standard using protein A immunoadsorption waste liquid, the preparation process diagram is as follows Figure 1 As shown, the following steps are included:

[0049] (1) The protein A immunoadsorption waste liquid (the eluate from the blood of patients after transplantation and adsorption with a protein A immunoadsorption column) was quickly neutralized with 1M Tris-HCl buffer (pH = 9), the pH of the eluate was adjusted to 6.9-7.2, and 5wt% D-trehalose + 2wt% mannitol were added as protein protectants to maintain IgG activity.

[0050] (2) The solution obtained in step (1) was subjected to protein G affinity chromatography (catalog number: SA020005, Tiandirenhe Co., Ltd.) to remove non-IgG impurities to obtain an enriched solution.

[0051] (3) The enriched liquid obtained in step (2) was nanofiltered using a membrane with a pore size of 35 nm. The positive pressure filtration condition was 0.2 MPa, which effectively removed non-enveloped viruses. After inactivation verification, the residual virus was ≤ 1×10 -6 IU / mL. Then, an HLA-specific adsorption column is used for epitope-specific enrichment to obtain HLA-specific antibody detection standards. The specific steps of epitope-specific enrichment are as follows:

[0052] 1. Take 0.8 mg of HLA-A2 protein and 1.2 mL of 4% cross-linked agarose beads (Thermo Amino Link Plus coupling resin) for mixed reaction incubation, control the pH to 7.4, the temperature to 37°C, and the time to 1 hour. Then use amino reagent (Tris-HCl solution, pH=7.4) to block the excess activation sites to obtain beads coupled with HLA-A2 protein.

[0053] 2. Load 1 mL of microbeads coupled with HLA-A2 protein into the adsorption column to form the HLA-A2 adsorption column.

[0054] 3. The enriched solution after filtration and virus removal was adsorbed onto the HLA-A2 adsorption column, with a loading volume of 5 mL and a loading speed of 0.5 mL / min. After adsorption, the adsorption column was rinsed with equilibrium buffer (Tris-NaCl, pH=7.0) at 0.5 mL / min for 5 min.

[0055] 4. Elute the HLA-A2 antibody. Elute the adsorbed antibody with 1.5 mol / L Tris-glycine buffer (pH=3.5) with an elution volume of 1 mL, and then add 1M Tris-NaCl (pH=9.0) to neutralize the eluate to neutrality (pH 7.0-7.2).

[0056] 5. Perform protein quantification on the eluted and collected antibodies, and the obtained antibodies are HLA-A2 antibodies.

[0057] 6. Perform antibody binding epitope identification (LIFECODES LSA Single Antigen, Immucor) on the obtained antibodies to specify the characteristic epitope type bound by the obtained antibodies. The epitope coverage heat map is as follows: Figure 2 As shown, the coverage of the epitope database verified by next-generation sequencing was >95%.

[0058] The results of the multiple adsorption and elution stability and multiple adsorption efficiency tests of the HLA-A2 adsorption column are as follows: Figure 3 and Figure 4 From the above results, it can be concluded that the HLA-specific adsorption column of the present invention has good adsorption stability for HLA-specific antibody serum derived from protein A immunoadsorption waste liquid.

[0059] The specific identification results of the above HLA-A2 antibodies for different epitopes are as follows: Figures 5 to 7As shown, epitope-specific antibodies for HLA-A2 protein epitopes 43R, 62GK and 107W were obtained. For example, epitopes A*02:02, A*02:05, A*32:01, A*33:0 were positive, while genes A*01:01, A*43:01, A*80:01 were negative. The specificity of other epitopes was similar.

[0060] (4) Preparation of freeze-dried preparation of HLA-A2 antibody standard:

[0061] 1. The obtained HLA-A2 antibody was dissolved in a lyophilization protectant (0.1 M citrate buffer (pH = 6.5), 0.01wt% Tween-80, 0.1wt% BSA, 5wt% trehalose) to prepare a solution with a concentration of 1 mg / mL.

[0062] 2. Filtration and sterilization: Filter through a 0.22 μm filter membrane to remove impurities and microorganisms.

[0063] 3. Divide the protein solution into freeze-drying bottles and pre-freeze them at -80°C for 24 hours to completely solidify the solution.

[0064] 4. Transfer the pre-frozen samples to a freeze dryer and evacuate to 10-50 mTorr.

[0065] 5. Then slowly raise the temperature to -40°C and keep it warm for 24 hours. The ice crystals will sublime directly and remove most of the water.

[0066] 6. Raise the temperature to 25°C and continue vacuuming for 4 hours to remove residual moisture.

[0067] 7. After reaching the drying target, fill the freeze-drying bottle with inert gas such as nitrogen to prevent protein oxidation.

[0068] 8. Seal and store: Seal the freeze-dried bottle and store at -20°C.

[0069] The key characteristic parameters and stability test results of the lyophilized preparation obtained in this example are shown in Tables 1 and 2 below.

[0070] Table 1. Key characteristic parameters and test methods

[0071] parameter scope Detection Methods Titer (MFI) 8000–12000 Luminex200 System Linear range 1:8–1:512 Serial dilution analysis Lyophilization reconstitution rate 98%–102% UV spectrophotometry / KarlFischer method

[0072] Table 2. Stability test results

[0073] Test conditions result Freeze-thaw cycles (3 times) MFI decreases ≤5% Vibration test (ISTA3A) The appearance is not damaged and the re-dissolution rate meets the requirements High temperature shock (45℃ / 48h) MFI change ≤ 8%

[0074] Comparative Example 1

[0075] At present, there are still no antibodies targeting characteristic epitopes of HLA antigens available on the market. For example, Imumuco's LSA I and LSA II detection kits do not contain separate epitope antibodies. Only polyclonal serum of HLA protein can be used for experimental control and as a positive quality control product. Figure 8 The following is a graph showing the fluorescence intensity analysis results of the total HLA-I protein of the three batches of positive quality control serum of the above kit. Figure 8 and Figures 5 to 7 The comparison results show that the positive quality control serum is essentially different from the characteristic epitope human IgG antibody of this patent. The antibody presented in this patent has a very specific recognition site, and the fluorescence intensity is stable, and it has good stability as a standard.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a human HLA-specific antibody detection standard using protein A immunoadsorption waste liquid, characterized in that: The steps include: (1) Neutralize the protein A immunoadsorption waste liquid with Tris-HCl buffer, adjust the pH to 6.9-7.2, and add a protein protectant to maintain IgG activity; (2) removing non-IgG proteins from the solution obtained in step (1) by protein G affinity chromatography to obtain an enriched solution; (3) The enriched solution obtained in step (2) is filtered to remove viruses, and then epitope-specific enrichment is performed using an HLA-specific adsorption column to obtain an HLA-specific antibody detection standard.

2. A method for preparing a human HLA-specific antibody detection standard using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The protein A immunoadsorption waste liquid in step (1) comes from the eluate of blood from patients who have received HLA-related treatment or transplantation and have been treated with protein A immunoadsorption columns.

3. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The Tris-HCl buffer in step (1) uses a Tris-HCl buffer with a concentration of 1 M and a pH value of 9.0-9.3; the protein protective agent and the added amount are 5 wt% D-trehalose + 2 wt% mannitol.

4. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The nanofiltration virus removal in step (3) uses a membrane with a pore size of 35-45 nm for nanofiltration, and the filtration pressure is 0.2 MPa.

5. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The HLA-specific adsorption column in step (3) is prepared by the following method: The HLA protein is mixed with a cross-linked agarose microbead dispersion for reaction and incubation, and then an amino reagent is used to block excess activation sites to obtain microbeads coupled to the HLA protein, which are then loaded into an adsorption column to obtain an HLA-specific adsorption column.

6. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 5, characterized in that: The mass concentration of the cross-linked agarose microbead dispersion is 4 wt %; the mass volume ratio of the HLA protein and the cross-linked agarose microbead dispersion is 2:3; the temperature of the mixed reaction incubation is 37° C., the pH is 7.2-7.4, and the time is 1 hour; the amino reagent uses a Tris-HCl solution with a pH value of 7.2-7.

4.

7. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The steps of epitope-specific enrichment in step (3) are as follows: The virus-free enriched solution is injected into an HLA-specific adsorption column for adsorption. After the adsorption is completed, the adsorption column is rinsed with a Tris-NaCl equilibrium buffer, and the adsorbed specific antibodies are eluted with a Tris-glycine buffer. The eluate is neutralized to neutrality with a Tris-NaCl solution, and the resulting product is subjected to protein quantification and antibody binding epitope identification to obtain an HLA-specific antibody detection standard.

8. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 1, characterized in that: The HLA-specific antibody detection standard obtained in step (3) is further prepared into a lyophilized preparation by the following method: The HLA-specific antibody detection standard product is dissolved in a lyophilizing protective agent, filtered and sterilized, and then freeze-dried. The product is filled with an inert gas and sealed for storage to obtain a lyophilized preparation of the HLA-specific antibody detection standard product.

9. The method for preparing a human HLA-specific antibody detection standard product using protein A immunoadsorption waste liquid according to claim 8, characterized in that: The freeze-drying protective agent comprises: 0.1 M citrate buffer, 0.01 wt% Tween-80, 0.1 wt% BSA, and 5 wt% trehalose; the freeze-drying procedure comprises: first pre-freezing at -40 to -80°C to completely solidify the solution, then vacuuming to 10 to 50 mTorr, heating to -40 to -10°C to sublimate and remove most of the water, and then heating to 20 to 30°C to remove the residual water.

10. An HLA-specific antibody detection standard or lyophilized preparation, characterized in that: It is prepared by the method according to any one of claims 1 to 9.

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