EBV (Epstein-Barr Virus) neutralizing antibody-specifically-immune plasma high-throughput screening method and application thereof

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to an EBV (Epstein-Barr Virus) neutralizing antibody-specifically-immune plasma high-throughput screening method and application thereof. EB virus envelope glycoprotein gp350 is selected as a screening target antigen, EBV specific plasma containing a high-titer EBV neutralizing antibody is rapidly screened in a high-throughput mode and used for preparing EBV specific human immune globulin, the titer of the EBV neutralizing antibody is obviously higher than that of listed intravenous gamma-C products, and the content of the EBV neutralizing antibody is about 4-8 times that of the EBV neutralizing antibody with the same concentration. The method provided by the invention can realize large-scale collection of EBV specific plasma through rapid high-throughput screening, reduces the workload of plasma screening, is suitable for large-scale production of EBV specific human immune globulin, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a high-throughput screening method for EBV neutralizing antibody-specific immune plasma and its application. Background Technology

[0002] Epstein-Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), belongs to the subfamily Gamma subfamily of the family Herpesviridae. It is a double-stranded DNA virus and one of the eight known human herpesviruses, as well as one of the most common pathogenic viruses in humans. It is estimated that more than 95% of the adult population worldwide is infected with this virus. EBV is associated with the development of various malignant tumors (such as nasopharyngeal carcinoma, various lymphomas, and gastric cancer), various acute and chronic infectious diseases (such as infectious mononucleosis), and autoimmune diseases (such as systemic lupus erythematosus). Globally, there are more than 200,000 new cases of EBV-related cancers and more than 140,000 deaths each year, causing a huge public health and financial burden.

[0003] Epstein-Barr virus (EBV) specifically targets lymphocytes, primarily infecting B lymphocytes and epithelial cells. Infection relies on the synergistic action of various viral surface glycoproteins, among which glycoproteins gp350, gp42, gH, and gL antigens play crucial roles. Antibodies binding to gp350 are a major component of antibodies neutralizing B cell infection in human serum. Research data indicates that gp350-positive tumor cells in mice can be effectively eliminated by CAR-T cell therapy using neutralizing antibodies. Immunoglobulin combined with acyclovir and ribavirin has shown significant clinical efficacy in treating infectious mononucleosis in children. Further research indicates that current antiviral and immunomodulatory treatments for EBV infection in conjunction with tumors or other diseases include the use of gamma globulin. Currently, research on biological products for the treatment of EBV infection in China is still in the exploratory stage, and there are no approved EBV vaccines or specific EBV immunoglobulin products on the market. The existing technology has the following problems: lack of rapid and accurate methods for detecting EBV neutralizing antibody titers in raw plasma, combined plasma, immunoglobulin stock solution and finished products; lack of high-titer standards for EBV neutralizing antibody screening and strategies for plasma donor return; and lack of process flow for preparing high-titer EBV neutralizing antibody immunoglobulin intermediates.

[0004] Therefore, the preparation and research of EBV-specific immune plasma / immunoglobulins are of great academic significance and broad clinical value, and there is an urgent need in this field to develop EBV-specific immunoglobulin products. Summary of the Invention

[0005] The technical problem to be solved by this invention is that, given the current lack of a rapid, large-scale method for detecting EBV neutralizing antibody titers in raw plasma and combined plasma, this invention provides a high-throughput rapid screening method for EBV neutralizing antibodies and its application in plasma screening.

[0006] This invention provides a high-throughput screening method for EB virus neutralizing antibodies, comprising: using an enzyme-linked immunosorbent assay (ELISA), pre-diluting the sample and mixing it with EB virus envelope glycoprotein gp350, and using a single-point standard titer calculation method to detect the titer of EB virus neutralizing antibodies.

[0007] Furthermore, the sample is raw plasma or combined plasma, and the dilution factor of the pre-diluted sample is 5-20 times.

[0008] Furthermore, the EB virus envelope glycoprotein gp350 is pre-coated on an enzyme-linked immunosorbent assay (ELISA) plate.

[0009] Furthermore, the enzyme-linked immunosorbent assay (ELISA) method sequentially includes the following steps: sample addition, incubation, plate washing, addition of enzyme-labeled reagent, incubation, plate washing, color development, and termination; the incubation conditions include: 37℃, 30±1 minutes; the volume ratio of the sample to the enzyme-labeled reagent is 50:50; and the detection wavelength of the ELISA method is 450nm.

[0010] Furthermore, in the enzyme-linked immunosorbent assay (ELISA) method, the theoretical efficacy range of the single-point working standard is 25-50 ng / L.

[0011] This invention provides the use of the screening method described in any of the preceding claims for preparing immunoglobulin intermediates, stock solutions, or immunoglobulin formulations that specifically target EB virus.

[0012] This invention provides a method for preparing an immunoglobulin intermediate, stock solution, or immunoglobulin formulation specifically targeting Epstein-Barr virus, comprising: Step 1: Screen the plasma of healthy individuals using the screening method described in any of the preceding steps; Step 2: Select high-titer plasma from the plasma of healthy individuals screened as raw plasma; Step 3: Mix the raw plasma.

[0013] Further, in step 3, the raw plasma is mixed and then purified; the purification method is plasma protein separation. And / or, in step 2, the criterion for selecting high-titer plasma is an EB virus neutralizing antibody titer ≥500 ng / L.

[0014] Furthermore, the intermediate products include: mixed raw plasma, collected liquid from the plasma protein separation process, and products after virus inactivation or removal.

[0015] The present invention provides a specific EB virus-targeting human immunoglobulin intermediate, stock solution or human immunoglobulin preparation prepared by any of the preparation methods described above.

[0016] In this invention, the "single-point standard potency calculation method" refers to a method that uses only one standard with a fixed dilution (instead of creating a 5-7 point standard curve) to convert the OD value of the sample to be tested into a potency. In other words, the standard is measured at only one dilution point, and the sample's dilution point is determined according to its type. The potency is calculated using a formula, without performing well-by-well serial dilution of the linear standard or four-parameter fitting.

[0017] The plasma protein separation methods include: low-temperature ethanol separation and chromatography purification.

[0018] In this invention, "specific immune plasma" refers to specific immune plasma, specifically raw plasma containing high titers of neutralizing antibodies against a specific pathogen, with antibody titers significantly higher than those of ordinary blood donors.

[0019] This invention selects EBV envelope glycoprotein gp350 as the screening target site for high-throughput rapid screening of EBV neutralizing antibody titers in raw plasma samples. The target titer is determined by sample dilution factor (5-20 times) and the theoretical value of single-point standard is determined to be 25-50 ng / L. The specific detection procedure is defined to meet the requirements of quantitative detection. This invention establishes a screening standard of ≥500 ng / L for high-titer EBV neutralizing antibody plasma. High-titer EBV neutralizing antibody plasma is mixed and a plasma protein separation method is used to prepare an intermediate product of high-titer EBV neutralizing antibody immunoglobulin. The EBV neutralizing antibody titer of this intermediate product is significantly higher than that of commercially available intravenous immunoglobulin (IVIG) products. The method of this invention can greatly improve the efficiency of EBV neutralizing antibody titer screening and reduce the workload of detection. The EBV-specific immunoglobulin products prepared based on this method have high antibody titers and promising application prospects.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0022] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0023] Materials and Methods (1) Combine plasma samples (for establishing high-titer screening criteria); (2) Plasma samples from healthy individuals (used for screening high-titer plasma containing EBV neutralizing antibodies); (3) EBV neutralizing antibody high-titer immunoglobulin stock solution (preparation process: collect EBV neutralizing antibody high-titer plasma (3 bags) → process preparation → stock solution → test).

[0024] The samples involved include: 22 batches of combined plasma samples, 3,560 retained plasma samples from healthy individuals, 3 bags of high-titer plasma, and several process samples.

[0025] Main reagents and instruments (1) Enzyme-linked immunosorbent assay kit for human EBV envelope glycoprotein gp350 antibody (EBV-gp350-Ab); (2) BioTek Elx508 fully automatic plate washing machine (USA); (3) BioTek 800TS microplate reader from the United States, with built-in GEN 5 analysis software, the equipment has been verified and calibrated; (4) Instruments used in the process: Cytiva AKTA AVANT chromatography system, constant temperature water bath, ultrafiltration device.

[0026] Example 1: Screening for EBV neutralizing antibodies in combined plasma samples and determination of high titer standards (1) Kit selection: Human EBV envelope glycoprotein gp350 antibody (EBV-gp350-Ab) enzyme-linked immunosorbent assay kit (commercial quantitative detection kit, manufacturer: Shanghai Fusheng, product number: 4037-96T, this kit comes with 160ng / L standard).

[0027] (2) Detection subjects: 22 batches of combined plasma samples put into production between 2022 and 2024. The combined plasma samples were pre-diluted 5 times using sample diluent. The ratio of combined plasma sample to sample diluent was 1:4.

[0028] (3) Setting up standard and blank well: Dilute the kit standard to 40 ng / L using standard diluent as working standard. The ratio of standard stock solution (160 ng / L) to standard diluent is 1:3.

[0029] (4) Detection process: In the enzyme-labeled reaction plate, each pre-diluted combined plasma is set as 2 wells, the working standard is set as 3 wells, and the blank well is set as 1 well. Except for the blank well, 50 μl of the corresponding sample is added to each well. After sealing the plate, it is incubated at 37℃ for 30 minutes. The plate is washed 5 times and then patted dry. 50 μl of enzyme-labeled reagent is added to each well, except for the blank well. After sealing the plate, it is incubated at 37℃ for 30 minutes. The plate is washed 5 times and then patted dry. 50 μl of chromogenic reagent A is added to each well first, followed by 50 μl of chromogenic reagent B. The plate is gently shaken and mixed. The plate is incubated at 37℃ in the dark for 10 minutes. 50 μl of stop solution is added to each well to stop the reaction. The blank well is used to zero the plate. The absorbance OD value of each well is measured at a wavelength of 450 nm. The colorimetric analysis is completed within 15 minutes after adding the stop solution.

[0030] (5) Calculation of efficacy value: The efficacy value of the EBV neutralizing antibody in the sample = OD value of the sample well / OD value of the working standard × 40 (efficacy value of the working standard) × 5 (pre-dilution factor of the sample).

[0031] (6) Test results: The EBV neutralizing antibody efficacy value range of the 22 batches of combined plasma samples was 55-164 ng / L, and the average titer was 108 ng / L.

[0032] (7) Screening criteria for high titer of EBV neutralizing antibody: Multiply the average titer of the combined plasma by 5 to get 540 ng / L, round down, and determine the screening criteria for high titer of EBV gp350 antibody as 500 ng / L. Samples with a titer ≥ 500 ng / L are high titer plasma, and those < 500 ng / L are low titer plasma.

[0033] Example 2: Application of a high-throughput screening method for EBV neutralizing antibodies (1) The reagent kit selection is the same as in Example 1.

[0034] (2) Subjects of testing: 3,560 plasma samples from healthy individuals from 4 single-donor plasma collection stations. The plasma samples were pre-diluted 5 times using sample diluent. The ratio of plasma sample to sample diluent was 1:4.

[0035] (3) Setting up standards and blank wells: The theoretical efficacy of the standards is greater than 5 ng / L and less than 80 ng / L. Based on the standard of high-titer raw plasma and combined plasma, the neutralizing antibody titer of EB virus is ≥500 ng / L. The theoretical efficacy range of the working standards is 25-50 ng / L. Specifically, the reagent kit standards are diluted to 40 ng / L using standard diluent as the working standards. The ratio of standard stock solution (160 ng / L) to standard diluent is 1:3.

[0036] (4) Detection process: Each sample of raw plasma is set to 1 well, and the other detection processes are the same as those in Example 1.

[0037] (5) Calculation of efficacy value: The OD value of the sample well is compared with the OD value of the control well. If the plasma value is higher than or equal to the plasma value of the control well, it is considered qualified plasma for screening, and the neutralizing antibody content is ≥500ng / L.

[0038] Example 3: Application of Specific Immunoglobulin in the Preparation of Specific Immunoglobulin (1) Three EBV neutralizing antibody high-titer raw plasma samples were melted and combined, and the original sample, namely 5% intravenous immunoglobulin stock solution (chromatographic method), was obtained by performing a low temperature ethanol + chromatography process.

[0039] The low-temperature ethanol + chromatography process specifically involves adding ethanol to the combined slurry to prepare F. I+II+III F is obtained after filtering. I+II+III Precipitation; F I+II+III After the precipitate dissolves, sodium caprylate is added for caprylic acid precipitation, and the filtrate is obtained by filtration. The pH and conductivity of the filtrate are adjusted, and then subjected to one-step anion exchange chromatography, two-step anion exchange chromatography, and three-step affinity chromatography to remove impurities. The flow-through is concentrated by ultrafiltration, sterilized, and filtered to obtain the immunoglobulin stock solution. The first-step anion exchange chromatography packing material is Capto Q, the second-step anion exchange chromatography packing material is Nuvia HP Q, and the chromatography process and buffer system are based on patent "CN102532307 B A method for preparing immunoglobulins". The third-step affinity chromatography packing material is Eshmuno P Anti A / Anti B, and the chromatography process and buffer system are based on patent "US 12441809 B2 Affinity chromatography media for removal of anti-A and / or anti-B antibodies".

[0040] (2) Detection objects: 1 mixed plasma sample of 3 EBV neutralizing antibody high titer raw material plasma, 1 5% intravenous immunoglobulin stock solution sample prepared from mixed plasma, 1 ordinary 5% intravenous immunoglobulin product and 1 plasma sample combined with it, 10% intravenous immunoglobulin product (chromatographic method) and 1 plasma sample combined with it.

[0041] (3) Setting up standard and blank wells: According to the instructions of the kit, use standard diluent to dilute the kit standard to 80ng / L, 40ng / L, 20ng / L, 10ng / L and 5ng / L for a total of 5 gradients. Set up 2 wells for each gradient standard and set up a single blank well per plate.

[0042] (4) Sample dilution: EBV neutralizing antibody high titer mixed plasma samples were diluted 40 times, 5% intravenous immunoglobulin stock solution samples were diluted 50 times; combined plasma samples were diluted 10 times, ordinary 5% intravenous immunoglobulin product was diluted 10 times, and 10% intravenous immunoglobulin product (chromatographic method) was diluted 25 times.

[0043] (5) Detection process: Follow the detection steps in the kit instructions (add sample - incubation - wash plate - add enzyme - incubation - wash plate - color development - termination), use the linear calculation method, and detect the absorbance value at 450nm wavelength after terminating the reaction.

[0044] (6) Calculation of efficacy value: Plot a standard curve with the concentration of the standard as the x-axis and the OD value as the y-axis to obtain a linear regression equation. Substitute the OD value of the sample into the linear regression equation to calculate the corresponding concentration, and then multiply by the dilution factor to calculate the efficacy value of the sample.

[0045] (7) Detection results: As shown in Table 1, the 5% intravenous immunoglobulin (IVIG) stock solution prepared from the mixed plasma with high EBV neutralizing antibody titer has a titer of 6754 ng / L and an IgG content of 44.1 g / L. The mixed plasma titer is 1145 ng / L, which increases the antibody titer of the mixed plasma by 5.9 times. Compared with ordinary 5% IVIG and 10% IVIG (chromatographic method), the ratio of EBV titer (ng / L) to IgG content (g / L) of the high-titer EBV neutralizing antibody stock solution is more than 10 times higher.

[0046] Table 1: Comparison of antibody titer levels in mixed plasma / stock solution prepared from ordinary combined plasma, ordinary 5% IVG and 10% IVG (chromatographic method) and high-titer EBV neutralizing antibody plasma. Note: 5% IVIG is a product prepared by low-temperature ethanol process, and 10% IVIG (chromatographic method) is a product prepared by chromatographic process. Both of these products are already on the market of Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0047] (8) Conclusion: Compared with ordinary combined plasma, ordinary 5% IVI and 10% IVI (chromatographic method), the mixed plasma and original solution prepared from high-titer EBV neutralizing antibody plasma have significantly higher efficacy values, proving that high-titer products can be prepared from high-titer raw plasma through chromatographic process.

[0048] The results of the above embodiments demonstrate that the method of the present invention has the following advantages: (1) gp350 is the most abundant glycoprotein on the surface of EBV envelope. It achieves viral adsorption by binding with CD21 and CD35 proteins on the surface of host cell membrane. Antibodies targeting gp350 are the main components of neutralizing B cell infection antibodies in human serum. Therefore, a double antigen sandwich enzyme-linked immunosorbent assay kit coated with EBV gp350 recombinant glycoprotein antigen was selected for screening EBV virus neutralizing antibodies in raw plasma.

[0049] (2) Enzyme-linked immunosorbent assay (ELISA) is a high-throughput detection method that can detect multiple samples at once and obtain test results quickly. It is suitable for the requirements of blood product companies for large-scale plasma screening.

[0050] (3) When using enzyme-linked immunosorbent assay (ELISA) for large-scale plasma screening, single-point standards are used as working standards. Compared with linear standards, this simplifies the standard dilution process and the calculation of efficacy value after result reading, thus meeting the requirements of high-throughput ELISA detection.

[0051] (4) Since the combined plasma is produced by mixing multiple raw plasma samples, the average value of EBV neutralizing antibody in multiple batches of ordinary combined plasma can accurately represent the average level of EBV neutralizing antibody titer in the general population. This method tested the EBV neutralizing antibody titer of 22 batches of combined plasma samples. The average titer was 108 ng / L. Five times the average titer was determined as the screening standard for high EBV neutralizing antibody titer. The average value was rounded up to 500 ng / L. Therefore, the screening standard for high EBV neutralizing antibody titer is 500 ng / L.

[0052] (5) The established high-throughput rapid screening method for EBV tested a total of 3,560 raw plasma samples, which is suitable for large-scale detection of the EBV neutralizing antibody efficacy value of raw plasma samples.

[0053] (6) This product adopts a low-temperature ethanol + chromatography purification process, that is, firstly, FI+II+III / II+III precipitate is prepared by low-temperature ethanol method, and then purified by octanoic acid precipitation, two-step anion exchange chromatography, and one-step affinity chromatography. The virus safety is ensured by a two-step virus inactivation / removal step of low pH incubation + nanofiltration, to obtain a 5%-10% protein concentration intravenous immunoglobulin stock solution. When the 5% concentration intravenous immunoglobulin stock solution is prepared, the EBV antibody titer in the stock solution is also about 5 times higher than that in human plasma. Compared with ordinary 5% IVIG and 10% IVIG (chromatographic method), the ratio of EBV titer (ng / L) to IgG content (g / L) in the high-titer EBV neutralizing antibody stock solution is more than 10 times higher.

[0054] In summary, this invention selects EBV viral envelope glycoprotein gp350 as the screening target site for high-throughput rapid screening of EBV gp350 neutralizing antibody titers in raw plasma samples. The samples are screened by dilution of 5-20 times to meet the requirements for quantitative detection. This invention establishes a screening standard of ≥500 ng / L for high-titer EBV gp350 neutralizing antibody plasma. By mixing high-titer EBV gp350 neutralizing antibody plasma, a high-titer EBV gp350 antibody immunoglobulin product is prepared, exhibiting a significantly higher EBV gp350 antibody titer than existing intravenous immunoglobulin products. The method of this invention can greatly reduce the workload of repeated testing and reagent costs. The EBV-specific immunoglobulin product prepared based on this method has high antibody titers and shows promising application prospects.

Claims

1. A high-throughput screening method for EB virus neutralizing antibodies, characterized in that, It includes: The EB virus neutralizing antibody titer was detected by pre-diluting the sample with EB virus envelope glycoprotein gp350 using an enzyme-linked immunosorbent assay (ELISA). The titer was calculated using a single-point standard titer method.

2. The screening method according to claim 1, characterized in that: The sample is raw plasma or combined plasma, and the pre-diluted sample is diluted by a factor of 5-20.

3. The screening method according to claim 1, characterized in that: The EB virus envelope glycoprotein gp350 was pre-coated on an enzyme-linked immunosorbent assay (ELISA) plate.

4. The screening method according to claim 1, characterized in that: The enzyme-linked immunosorbent assay (ELISA) method comprises the following steps in sequence: sample addition, incubation, plate washing, addition of enzyme-labeled reagent, incubation, plate washing, color development, and termination; the incubation conditions include: 37℃, 30±1 minutes; the volume ratio of sample to enzyme-labeled reagent is 50:50; and the detection wavelength of the ELISA method is 450nm.

5. The screening method according to claim 1, characterized in that: In the enzyme-linked immunosorbent assay (ELISA) method, the theoretical efficacy range of the single-point working standard is 25-50 ng / L.

6. The use of the screening method according to any one of claims 1-5 for preparing immunoglobulin intermediates, stock solutions or immunoglobulin preparations specifically targeting EB virus.

7. A method for preparing an immunoglobulin intermediate, stock solution, or immunoglobulin preparation specifically targeting Epstein-Barr virus, characterized in that, It includes: Step 1: Screen healthy human plasma using the screening method described in any one of claims 1-5; Step 2: Select high-titer plasma from the plasma of healthy individuals screened as raw plasma; Step 3: Mix the raw plasma.

8. The preparation method according to claim 7, characterized in that: In step 3, the raw plasma is mixed and then purified; the purification method is plasma protein separation. And / or, in step 2, the criterion for selecting high-titer plasma is an EB virus neutralizing antibody titer ≥500 ng / L.

9. The preparation method according to claim 8, characterized in that: The intermediate products include: mixed raw plasma, collection liquid from the plasma protein separation process, and products after virus inactivation or removal.

10. A specific EB virus-targeting human immunoglobulin intermediate, stock solution, or human immunoglobulin preparation prepared by the preparation method according to any one of claims 7-9.

Images