Method for determining titer of neutralizing antibody by using specific immune globulin

Abstract

The invention relates to the technical field of biological medicine, and discloses a titer determination method of a specific immune globulin neutralizing antibody, which comprises the following steps: step 1, preparing a cell maintenance fluid; 2, preparation of a virus solution for neutralization: diluting viruses (cytomegalovirus, syncytial virus and EV71 virus) to 100CCID50 by using a diluent according to the virus titer; step 3, diluting the test sample: carrying out double series dilution on the intravenous injection human immune globulin, the tetanus human immune globulin, the rabies human immune globulin and the human immune globulin until no cytotoxicity exists; 4, diluting a standard substance; 5, virus back dripping; step 6, neutralizing viruses; step 7, preparing cells for the test; step 8, infection; and 9, judging a result. According to the present invention, the accurate detection on the titer of the neutralizing antibody of the specific immune globulin can be achieved, and the cytotoxicity influence of the test sample and the fringe effect during the detection process can be removed so as to ensure the detection accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for determining the potency of a specific immunoglobulin neutralizing antibody. Background Art

[0002] Neutralizing antibodies (NAb) are a type of antibody that can specifically recognize and bind to specific antigenic epitopes on the surface of pathogens (such as viruses, bacteria, etc.), thereby preventing pathogens from binding to receptors on the surface of their host cells or entering the cells. Neutralizing antibodies play a vital role in immune defense and can effectively prevent the infection and spread of pathogens. Its mechanism of action is mainly to prevent pathogens from binding to receptors on the surface of their host cells by binding to key sites on the surface of pathogens; prevent pathogens from entering host cells, thereby preventing them from replicating and spreading in cells; and after neutralizing antibodies bind to pathogens, they can mark pathogens and promote their phagocytosis and clearance by immune cells (such as macrophages).

[0003] In the actual application stage, the determination of neutralizing antibody titer can help understand the immune status of individuals after natural infection and evaluate their resistance to reinfection; it is also helpful to study the immune mechanism of pathogens and understand the interaction between antibodies and pathogens; in the process of new drug development, the determination of neutralizing antibody titer can help evaluate the immune effect of candidate drugs and guide the design and optimization of drugs. At present, the methods for determining the titer of neutralizing antibodies usually include microneutralization test, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. However, in the actual detection process, the interference caused by the titer of cytomegalovirus neutralizing antibodies in the human immunoglobulin (pH4), tetanus human immunoglobulin, rabies human immunoglobulin, human immunoglobulin intermediates (stock solution, semi-finished products) and finished products developed by our company and their matrix, the linear range and whether the sample itself will cause CPE unknown for the cells (MRC-5, Vero, RD) used in the test method, and the blind transmission of cytomegalovirus titer unknown, which makes the accuracy of the test results subject to a certain amount of influence, and the test results of the test sample on the 96-well plate often have "edge effect", which is prone to false positives and may affect the result judgment. Summary of the invention

[0004] The present invention aims to provide a method for determining the titer of specific immunoglobulin neutralizing antibodies, so as to achieve accurate detection of the titer of specific immunoglobulin neutralizing antibodies.

[0005] To achieve the above object, the present invention adopts the following technical scheme: a method for determining the titer of specific immunoglobulin neutralizing antibodies, using the Karber method for detection, and diluting the test sample to be non-cytotoxic before detection;

[0006] Test product titer (U / ml) = B 标 ×N 标准品 / N 供试品 ; Where:

[0007] B 标 =National standard potency;

[0008] N 供试品 = neutralizing antibody titer of the test article;

[0009] N 标准品 = National standard neutralizing antibody titer.

[0010] Preferably, as an improvement, the calculation formula for the neutralization titer of the standard and the test sample is: N = 2 L+D(S-0.5) ; Where:

[0011] L = logarithm of the highest dilution;

[0012] D = difference between log dilutions;

[0013] S = composite of the percentage of wells showing no cytopathic effect (CPE).

[0014] Preferably, as an improvement, the test sample is intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin or human immunoglobulin.

[0015] Preferably, as an improvement, the minimum dilution multiples of intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin and human immunoglobulin diluted to no cytotoxicity are respectively 16 times, 16 times, 8 times and 32 times for MRC-5 cells; 16 times, 16 times, 32 times and 32 times for Vero cells; and 8 times, 16 times, 8 times and 16 times for RD cells.

[0016] Preferably, as an improvement, when the sample is added dropwise, PBS or purified water is added to the outer circle of the 96-well plate.

[0017] Preferably, as an improvement, 8 samples are tested in parallel for each concentration gradient of the test article.

[0018] Preferably, as an improvement, a method for determining the titer of a specific immunoglobulin neutralizing antibody comprises the following steps:

[0019] Step 1: Preparation of cell maintenance medium: Add inactivated fetal bovine serum to DMEM culture medium to obtain cell maintenance medium;

[0020] Step 2: Preparation of virus solution for neutralization: Dilute the cytomegalovirus to 100 CCID with diluent according to the virus titer. 50 , and the virus solution for neutralization is obtained;

[0021] Step 3. Dilution of test sample: dilute intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin, and human immunoglobulin until they are non-cytotoxic, and then perform two-fold serial dilutions in 6 dilution gradients;

[0022] Step 4: Standard dilution: dilute the standard to within the linear range, and then perform two-fold serial dilutions in a 96-well plate;

[0023] Step 5: Virus back titration: Take the neutralization virus solution and make a 10-fold serial gradient dilution, add the cell maintenance solution, and then transfer to a 96-well culture plate that has been pre-added with the cell maintenance solution. Make 8 wells in parallel for each dilution, for a total of 6 gradients;

[0024] Step 6: Virus neutralization: Add the neutralizing virus solution to each well of the test sample after the series of gradient dilutions, mix well, and then place each cell culture plate at 37°C and 5% carbon dioxide for neutralization;

[0025] Step 7. Preparation of test cells: Select well-growing monolayer cells and dilute the cells to the concentration of test cell suspension;

[0026] Step 8. Infection: After neutralization, add 50 μl of 1.5×10 5 cells / ml cell suspension and culture;

[0027] Step nine: Determine the results.

[0028] In the present technical solution, in step seven, the cytomegalovirus neutralizing antibody titer is determined using MRC-5 cells, the syncytial virus neutralizing antibody titer is determined using Vero cells, and the EV71 virus neutralizing antibody titer is determined using RD cells.

[0029] Preferably, as an improvement, in step six, the neutralization time is 1.5 h.

[0030] Preferably, as an improvement, in step 7, the concentration of the cell suspension is 1.5×10 5 cells / ml.

[0031] Preferably, as an improvement, in step eight, the culture conditions are 37° C. and 5% carbon dioxide.

[0032] The principle and advantages of this solution are: in actual application, in this technical solution, based on the immunoglobulin products produced by our company - intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin, human immunoglobulin, the test sample toxicity analysis was carried out, and the sample was diluted before the titer detection to make it non-cytotoxic, so as to avoid the test results being affected by cytotoxicity. In addition, in the process of titer detection (based on the Kabar method), the "edge effect" is usually a difficult problem to solve, which will lead to the generation of false positives, thereby affecting the result determination. Based on this, the inventor added PBS (purified water can also be used) to the outer circle of the 96-well plate to circumvent it. In the result characterization stage, the traditional method usually uses titer to represent the calculation result, but it is found through experiments that the repeatability and accuracy of the titer calculation result vary greatly. Based on this, the technical solution adds a standard sample to each experiment, and calculates the result of the test sample (U / ml, or IU / ml) by comparison. The coefficient of variation of the result obtained by adding a standard sample is significantly better than that of directly using the titer.

[0033] During the technical research and development stage, during the neutralizing antibody titer determination process, the control of viral virulence is one of the research and development difficulties and key points of this technical solution. Due to the instability of the virus, there is a certain deviation in the test results. Before conducting the neutralizing antibody titer test, the virus titer was repeated 6 times, and the average value was used as the virus titer value for dilution to reduce the test error. In addition, the cytotoxicity of the test sample is also a key factor affecting the observation of the test results. Through continuous exploration, the dilution scheme of the test sample before the test was determined, and the minimum dilution multiple was determined to dilute it to no cytotoxicity, so as to avoid the influence of the cytotoxicity of the test sample on the accuracy of the test results. In addition, when testing the antibody titer based on the Kabar method, the test sample is usually diluted in a 10-fold gradient, and the present technical solution uses a 2-fold gradient dilution method, which can improve the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the linear regression equation of the logarithm of the theoretical value and the logarithm of the measured value of the cytomegalovirus neutralizing antibody titer in the embodiment of the present invention.

[0035] Figure 2 The linear regression equation (relative accuracy) of the logarithmic value of the titer of the cytomegalovirus neutralizing antibody in the embodiment of the present invention is:

[0036] Figure 3 It is the linear regression equation (linear) of the logarithm of the theoretical value and the logarithm of the measured value of the cytomegalovirus neutralizing antibody titer in the embodiment of the present invention.

[0037] Figure 4 It is the linear relationship between the logarithm of the theoretical value of the potency and the logarithm of the measured value of the potency of the standard substance in the embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0039] Example

[0040] A method for determining the potency of specific immunoglobulin neutralizing antibodies, which is applicable to intravenous human immunoglobulin (pH 4), tetanus human immunoglobulin, rabies human immunoglobulin, human immunoglobulin intermediates (stock solution, semi-finished products) and cytomegalovirus neutralizing antibodies in finished products. The specific determination method comprises the following steps:

[0041] Step 1. Preparation of cell maintenance solution (diluent): Take 2.5 ml of inactivated fetal bovine serum and add 47.5 ml of DMEM culture medium to obtain cell maintenance solution.

[0042] Step 2, preparation of virus solution for neutralization: Take cytomegalovirus seed (usually freeze-dried virus) and make appropriate dilution, inoculate well-growing MRC-5 cell suspension with an infection amount of 0.1-1 MOI, culture at 37°C and 5% carbon dioxide, shake once every 15 minutes in the first hour, and add appropriate amount of cell maintenance solution after 1 hour. Observe the cell pathological changes day by day, collect the supernatant 1-2 days after the cells are completely pathological, centrifuge at 4000 rpm for 10 minutes at 4°C to remove cell debris, take the supernatant and add 10% newborn fetal bovine serum, mix well and dispense into small tubes, freeze below -70°C for later use.

[0043] Virus titer determination: Take one frozen cytomegalovirus, thaw it quickly with running water, and make a series of gradient dilutions (such as 6 gradients of 10-fold dilution); take 100μl of virus solution and add it to 900μl of cell maintenance medium (DMEM culture medium containing 5% inactivated newborn fetal bovine serum), mix thoroughly, and transfer 50μl of each dilution to a 96-well culture plate that has been pre-added with 50μl of cell maintenance medium. Make 8 wells in parallel for each dilution, and add 100μl of the test cell suspension to each well. Set up 6 cell control wells, and add 100μl of the test cell suspension and 100μl of cell maintenance medium to each well. Culture at 37°C and 5% carbon dioxide for 2 to 7 days, observe the CPE situation daily and determine the endpoint, and calculate the CCID using the Karber method 50 Then, the cytomegalovirus was diluted to 100 CCID with diluent according to the virus titer. 50 , you will get the virus solution for neutralization, and put it in an ice bath for later use.

[0044] Step 3, test sample dilution: The test samples of different titer concentrations (22U / ml, 13U / ml, 8U / ml, 5U / ml and 3U / ml) were diluted 2 times in series. For example, 500μl of solution A was added to 500μl of diluent to obtain solution A1 with a dilution of 1:2. After sufficient mixing, 500μl of solution A1 was added to 500μl of diluent to obtain solution A2 with a dilution of 1:4. After sufficient mixing, dilute to the appropriate dilution according to this series. The specific operation steps are shown in Table 1. Take 50μl of each dilution and add it to a 96-well culture plate, and load the sample in the middle 8 wells in parallel. After one row, 6 parallel wells were used as negative cell control wells (100μl of cell maintenance solution was added first). In this process, in order to avoid false positives in the wells close to the edge of the 96-well plate, which affects the result determination, in this embodiment, PBS (purified water can also be used) was added to the outer circle of the 96-well plate to eliminate the edge effect.

[0045] Table 1

[0046] Solution A1 A2 …… A5 A6 Dilution 1:2 1:4 …… 1:32 1:64 Dilution (μl) 500 500 …… 500 500 Diluent (μl) Take A 500 Take A1 500 …… Take A8 500 Take A9 500

[0047] Step 4. Dilution of standard: Take 1 standard of 10U / ml and perform 2-fold series gradient dilution. For example, add 500μl of solution A to 500μl of diluent to obtain solution A1 with a dilution of 1:2. After fully mixing, take 500μl of solution A1 and add 500μl of diluent to obtain solution A2 with a dilution of 1:4. After fully mixing, dilute to the appropriate dilution according to this series (as shown in the table below). Take 50μl of each dilution and add it to a 96-well culture plate, and load the middle 8 wells in parallel. After every other column, add 6 parallel wells as negative cell control wells (first add 100μl of cell maintenance solution).

[0048] Step 5. Virus back titration: Take the neutralization virus solution and make a series of gradient dilutions (such as 6 gradients of 10-fold dilution), take 100μl of virus solution and add it to 900μl of cell maintenance medium (DMEM culture medium containing 5% inactivated newborn calf serum), mix thoroughly, and transfer 50μl of each dilution to a 96-well culture plate that has been pre-added with 50μl of cell maintenance medium. Make 8 wells in parallel for each dilution, and add 100μl of the test cell suspension to each well. Set up 6 cell control wells, add 100μl of the test cell suspension and 100μl of cell maintenance medium to each well. Culture at 37°C and 5% carbon dioxide for 2 to 7 days and determine the endpoint, and use the Karber method to calculate the CCID 50 .

[0049] Step 6. Virus neutralization: Add 50 μl of the neutralizing virus solution to each well of the test sample after the series of gradient dilutions, mix well, and place each cell culture plate at 37°C and 5% carbon dioxide for neutralization for 1.5 hours.

[0050] Step 7. Preparation of test cells: Within 1.5 hours after the test sample and virus are neutralized, select a well-growing monolayer of MRC-5 cells. Collect and count the well-growing MRC-5 cells before use. Dilute the MRC-5 cells to the test cell suspension (1.5×10 5 cells / ml).

[0051] Step 8. Infection: After neutralization, add 1.5×10 5 100 μl of MRC-5 cell suspension with a concentration of 1 cell / ml was cultured and observed at 37°C and 5% carbon dioxide.

[0052] Step 9. Result determination: Place the 96-well plate that has completed the above operation in a 37° C., 5% carbon dioxide incubator for culture, observe the cell pathological changes day by day (generally for 6 to 8 days), record the CPE results and determine the endpoint.

[0053] Test establishment conditions:

[0054] 1. The test is established if the sample is non-cytotoxic and the cell control is non-pathological.

[0055] 2. There is no lesion in the smallest dilution wells of the standard and test samples, and all lesions in the largest dilution wells.

[0056] 3. Attack virus titer is 32-320CCID 50 If it is within the range, the test is established.

[0057] The formula for calculating the potency is:

[0058] The formula for calculating the neutralization titer of standard and test products is: N = 2 L+D(S-0.5)

[0059] Where L = logarithm of the highest dilution;

[0060] D = difference between log dilutions;

[0061] S = composite of the percentage of wells showing no cytopathic effect (CPE).

[0062] Test product titer (U / ml) = B 标 ×N 标准品 / N 供试品

[0063] Where B 标 =National standard potency;

[0064] N 供试品 = neutralizing antibody titer of the test article;

[0065] N 标准品 = National standard neutralizing antibody titer.

[0066] Experimental Example 1 The lowest dilution factor without cytotoxicity of different test products

[0067] The toxicity analysis of the test samples was conducted on intravenous human immunoglobulin (pH 4), tetanus human immunoglobulin, rabies human immunoglobulin, and human immunoglobulin, and the lowest dilution multiple without cytotoxicity was obtained. The lowest dilution multiples of each immunoglobulin are shown in Table 2:

[0068] Table 2

[0069]

[0070] The theoretical titers of cytomegalovirus neutralizing antibodies in the test samples are shown in Table 3:

[0071] Table 3

[0072]

[0073]

[0074] The linearity test results of the titer determination method for cytomegalovirus neutralizing antibody titer are shown in Table 4:

[0075] Table 4

[0076] Group Theoretical titer (U / ml) Logarithmic value of theoretical potency Measured value (U / ml) Logarithm of potency measurement value 1 32 3.5 30.8 3.4 2 20 3.0 18.3 2.9 3 16 2.8 15.4 2.7 4 8 2.1 9.2 2.2 5 4 1.4 5.9 1.8 6 2 0.7 2.7 1.0

[0077] In summary, the linear regression equation of the logarithm of the theoretical value and the logarithm of the measured value of the cytomegalovirus neutralizing antibody titer is obtained as follows: Figure 1 As shown, within the range of 2U / ml-32U / ml, the logarithm of the theoretical potency value (abscissa) is plotted against the logarithm of the corresponding measured potency value (ordinate), and the least squares method is used for linear regression. The results are as follows Figure 1 The fitted linear regression equation is y=0.8248x+0.5048, and the correlation coefficient (R 2 )=0.9906; The minimum dilution times required for the cytomegalovirus neutralizing antibody titer in various samples are as follows:

[0078] Table 5

[0079] Sample name Minimum dilution factor <![CDATA[Human Immune Globulin for Intravenous Injection (pH4)]]> 16 Tetanus immune globulin 16 Rabies immunoglobulin 8 Human immunoglobulin 32

[0080] Experimental Example 2 Verification of relative accuracy, intermediate precision, linearity and range:

[0081] The acceptable standards for relative accuracy, precision, linearity and range of potency detection in this protocol are shown in Table 6:

[0082] Table 6

[0083]

[0084]

[0085] (1) Relative accuracy

[0086] The relative bias and confidence interval of the relative potency determination value of each potency level were calculated according to the calculation formula under "(II) Validation strategy for each validation indicator 2. Relative accuracy (2) Evaluation method" in Part III of the Chinese Pharmacopoeia 9401 Validation guidelines for biological activity / potency determination methods for biological products. The results are shown in Table 7 and Figure 2 .

[0087] Table 7

[0088]

[0089] The results showed that the relative bias of each potency level was within 15%. The logarithm of the theoretical potency value (abscissa) was used to make a linear regression with the logarithm of the corresponding measured potency value (ordinate), and the regression equation was y = 1.0235x + 0.0222. The slope of 1.0235 was between 0.8 and 1.25, which met the acceptance criteria.

[0090] (2) Intermediate precision

[0091] The geometric standard deviation, geometric coefficient of variation and upper confidence limit of the relative potency measurement value of each potency level were calculated according to the calculation formula under "(II) Validation strategy for each validation indicator 3. Precision (2) Evaluation method" in the Validation Guidelines for Biological Activity / Potency Determination Methods of Biological Products in Part III of the Chinese Pharmacopoeia. The results are shown in Table 8.

[0092] Table 8

[0093]

[0094]

[0095] Conclusion: The geometric coefficient of variation of the relative potency measurement values ​​of each potency level was less than 20%, which met the acceptance criteria.

[0096] (3) Linear

[0097] According to the Guiding Principles for Validation of Biological Activity / Potency Assay Methods for Biological Products in Part III of the Chinese Pharmacopoeia, “(II) Validation Strategy for Each Validation Parameter 4. Linearity (2) Evaluation Method”, the logarithms of the theoretical potency values ​​of the five potency levels and their corresponding logarithms of the measured potency values ​​were calculated. The logarithms of the theoretical potency values ​​(abscissa) and their corresponding logarithms of the measured potency values ​​(ordinate) were plotted, and linear regression was performed using the least squares method. The results are shown in the figure. Figure 3 .

[0098] Verification results: Plot the logarithm of the theoretical potency value (abscissa) against the logarithm of the corresponding measured potency value (ordinate), and perform linear regression using the least squares method. The results are as follows: Figure 3 As shown: the fitted linear regression equation is y=1.0258x+0.0173, and the correlation coefficient is 0.991.

[0099] (4) Scope

[0100] Acceptability criteria: The range of potency levels for which relative accuracy, intermediate precision, and linearity are reported as meeting requirements, which at least covers the specification range for the relative potency.

[0101] Verification results: The relative accuracy, intermediate precision and linearity of this method all met the required potency level range of 38-275%.

[0102] Experimental Example 3: Specificity Verification

[0103] ① Acceptable standard: The sample matrix should have no effect on the potency determination results, that is, the value of (measured value of spiked sample - measured value of sample) / theoretical potency value of standard * 100% is within the range of ±20%; at the same time, the slope of the linear regression equation of the dose-response curve of the standard, sample and spiked sample is within the range of 0.8-1.25.

[0104] Table 9 Sample spike recovery

[0105]

[0106]

[0107] Table 10 Linear relationship between the logarithm of the theoretical value of the standard and the logarithm of the measured value of the standard

[0108]

[0109] Verification results: The recovery rates of the spiked samples were all within the range of 85%-115%, and the logarithm of the theoretical potency values ​​of the samples and spiked samples was used as the abscissa, and the logarithm of the potency measured value was used as the ordinate to plot the linear regression equation. Figure 4 As shown: the slopes are all in the range of 0.8-1.25, meeting the acceptance criteria.

[0110] Experimental Example 4 Durability Test

[0111] Table 11 Verification of cytomegalovirus neutralization time

[0112]

[0113]

[0114] Table 12 Cell concentration verification

[0115]

[0116] Conclusion: ① It has been verified that the virus neutralization time of the cytomegalovirus neutralizing antibody titer determination method is within the range of 1-2 hours, and the relative bias of the titer determination value meets the acceptance criteria. When the virus neutralization time is 1.5 hours, the relative bias of the determination result is the smallest, that is, the optimal virus neutralization time of this method is 1.5 hours;

[0117] ② When the cell concentration is 0.5-2×10 5 cells / ml, the relative bias of the titer determination value meets the acceptance criteria. 5 cells / ml, the relative bias of the determination result is the smallest, that is, the optimal concentration of this method is 1.5×10 5 cells / ml.

[0118] In addition, this technical solution also verifies the linearity, range, relative accuracy, precision, specificity and durability of the cytomegalovirus neutralizing antibody titer. The minimum dilution multiple of the sample was explored before the titer test. The results are shown in Table 13:

[0119] Table 13

[0120] Sample name Minimum dilution factor <![CDATA[Human Immunoglobulin for Intravenous Injection (pH4)]]> 16 Tetanus immune globulin 16 Rabies immunoglobulin 8 Human immunoglobulin 32

[0121] The relative accuracy, precision, linearity, range, specificity and robustness test results are as follows:

[0122] (1) Relative accuracy

[0123] ① Acceptable standard: The relative bias of each potency level relative to the potency measurement value should be within the range of ±20%; the logarithm of the theoretical potency value (abscissa) is used to make a linear regression on the logarithm of the corresponding potency measurement value (ordinate), and the slope of the regression equation should be within the range of 0.80 to 1.25.

[0124] ②Verification results: A linear regression was made between the logarithm of the theoretical titer value (abscissa) and the logarithm of the corresponding titer measurement value (ordinate), and the linear regression equation of the logarithm of the theoretical titer value and the measured value of the cytomegalovirus neutralizing antibody titer was y=1.0235x+0.0222. The slope of 1.0235 was between 0.8 and 1.25, which met the acceptance criteria.

[0125] (2) Intermediate precision

[0126] ① Acceptable standard: The geometric coefficient of variation (GCV, %) of the relative potency measurement value of each potency level should not be greater than 20%.

[0127] ②Verification results: The geometric coefficient of variation of the relative potency measurement values ​​of each potency level was less than 20%, which met the acceptance criteria.

[0128] (3) Linear

[0129] ①Acceptable standard: The correlation coefficient of the linear regression equation should not be less than 0.98.

[0130] ②Verification results: The logarithm of the theoretical potency value (abscissa) was plotted against the logarithm of the corresponding measured potency value (ordinate), and linear regression was performed using the least squares method. The fitted linear regression equation was y=1.0258x+0.0173, and the correlation coefficient was 0.991, which met the acceptance criteria.

[0131] (4) Scope

[0132] ① Acceptable standard: The range of potency levels when the reported relative accuracy, intermediate precision and linearity meet the requirements. This range should at least cover the quality standard range of relative potency (80% to 150%).

[0133] ②Verification results: The relative accuracy, intermediate precision and linearity of this method all met the required potency level range of 38% to 275%, covering its quality standard range.

[0134] (5) Exclusivity

[0135] ① Acceptable standard: The sample matrix should have no effect on the potency determination results, that is, the value of (measured value of spiked sample - measured value of sample) / theoretical potency value of standard * 100% is within the range of ±20%; at the same time, the slope of the linear regression equation of the dose-response curve of the standard, sample and spiked sample is within the range of 0.8-1.25.

[0136] ②Verification results: Meets acceptance criteria.

[0137] (6) Durability

[0138] ① It has been verified that the virus neutralization time of the cytomegalovirus neutralizing antibody titer determination method is within the range of 1-2 hours, and the relative bias of its titer determination value meets the acceptance criteria. When the virus neutralization time is 1.5 hours, the relative bias of the determination result is the smallest, that is, the optimal virus neutralization time of this method is 1.5 hours;

[0139] ② When the cell concentration is 0.5-2×10 5 cells / ml, the relative bias of the titer determination value meets the acceptance criteria. 5 cells / ml, the relative bias of the determination result is the smallest, that is, the optimal concentration of this method is 1.5×10 5 cells / ml.

[0140] The above are the results of cytomegalovirus neutralizing antibody titer determination. In addition, this technical solution also verifies the determination effect of this method in syncytial virus neutralizing antibody titer determination (using Vero cells) and EV71 virus neutralizing antibody titer determination (using RD cells). The results show that:

[0141] 1. Durability test:

[0142] Acceptance criteria: The relative bias (RB, %) of the titer determination value was within the range of ±20%; the results showed that this method met the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0143] 2. Relative accuracy test:

[0144] Acceptable standard: The relative bias of each titer level relative to the titer measurement value should be within the range of ±20%; the logarithm of the theoretical titer value (abscissa) is used to make a linear regression with the logarithm of the corresponding titer measurement value (ordinate), and the slope of the regression equation should be within the range of 0.80 to 1.25. The results show that this method meets the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0145] 3. Intermediate precision test:

[0146] Acceptable standard: The geometric coefficient of variation (GCV, %) of each titer level relative to the titer determination value should be no greater than 20%. The results show that this method meets the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0147] 4. Linearity:

[0148] Acceptable standard: The correlation coefficient of the linear regression equation should be no less than 0.98. The results show that this method meets the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0149] 5. Scope:

[0150] Acceptable standard: Acceptable standard: Report the range of titer levels when relative accuracy, intermediate precision and linearity meet the requirements, which should at least cover the quality standard range of relative titer (80% to 150%). The results show that this method meets the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0151] 6. Exclusivity Verification

[0152] Acceptable standard: The sample matrix should have no effect on the titer determination results, that is, the value of (spiked sample determination value - sample determination value) / theoretical value of the standard's titer × 100% is within ±20%; at the same time, the slope of the linear regression equation of the dose-response curve of the standard, sample, and spiked sample is within the range of 0.8-1.25. The results show that this method meets the acceptance criteria in the determination of syncytial virus and EV71 virus neutralizing antibody titers.

[0153] 7. The minimum dilution multiples required for determining the titer of syncytial virus neutralizing antibodies in various samples are as follows: 16 times for intravenous human immunoglobulin (pH 4); 16 times for tetanus human immunoglobulin; 32 times for rabies human immunoglobulin; and 32 times for human immunoglobulin.

[0154] 8. The minimum dilution multiples required for the determination of EV71 neutralizing antibody titers in various samples are as follows: 8 times for intravenous human immunoglobulin (pH 4); 8 times for tetanus human immunoglobulin; 8 / 16 times for rabies human immunoglobulin; and 16 times for human immunoglobulin.

[0155] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for determining the titer of a specific immunoglobulin neutralizing antibody, characterized in that: The Karber method was used for detection, and the test samples were diluted to be non-cytotoxic before detection; Test sample titer (U / ml) = B 标 ×N 标准品 / N 供试品 ; Mode middle: B 标 =National standard potency; N 供试品 = neutralizing antibody titer of the test article; N 标准品 = National standard neutralizing antibody titer.

2. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 1, characterized in that: The formula for calculating the neutralization titer of standard and test products is: N = 2 L+D(S-0.5) ; Where: L = logarithm of the highest dilution; D = difference between log dilutions; S = composite of the percentage of wells showing no cytopathic effect (CPE).

3. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 2, characterized in that: The test sample is intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin or human immunoglobulin.

4. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 3, characterized in that: The minimum dilution multiples of intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin and human immunoglobulin without cytotoxicity were 16 times, 16 times, 8 times and 32 times for MRC-5 cells; 16 times, 16 times, 32 times and 32 times for Vero cells; and 8 times, 16 times, 8 times and 16 times for RD cells.

5. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 4, characterized in that: When adding samples, add PBS or purified water to the outer circle of the 96-well plate.

6. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 5, characterized in that: Each concentration gradient of the test sample was tested in parallel with 8 samples.

7. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 6, characterized in that: The steps include: Step 1: Preparation of cell maintenance medium: Add inactivated fetal bovine serum to DMEM culture medium to obtain cell maintenance medium; Step 2: Preparation of virus solution for neutralization: Dilute the cytomegalovirus to 100 CCID with diluent according to the virus titer. 50 , and the virus solution for neutralization is obtained; Step 3. Dilution of test sample: dilute intravenous human immunoglobulin, tetanus human immunoglobulin, rabies human immunoglobulin, and human immunoglobulin until they are non-cytotoxic, and then perform two-fold serial dilutions in 6 dilution gradients; Step 4: Standard dilution: dilute the standard to within the linear range, and then perform two-fold serial dilutions in a 96-well plate; Step 5: Virus back titration: Take the neutralization virus solution and make a 10-fold serial gradient dilution, add the cell maintenance solution, and then transfer to a 96-well culture plate that has been pre-added with the cell maintenance solution. Make 8 wells in parallel for each dilution, for a total of 6 gradients; Step 6: Virus neutralization: Add the neutralizing virus solution to each well of the test sample after the series of gradient dilutions, mix well, and then place each cell culture plate at 37°C and 5% carbon dioxide for neutralization; Step 7. Preparation of test cells: Select well-growing monolayer cells and dilute the cells to the concentration of test cell suspension; Step 8. Infection: After neutralization, add 50 μl of 1.5×10 5 cells / ml cell suspension and culture; Step nine: Determine the results.

8. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 7, characterized in that: In step six, the neutralization time is 1.5 h.

9. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 8, characterized in that: In step 7, the concentration of the cell suspension was 1.5×10 5 cells / ml.

10. The method for determining the titer of a specific immunoglobulin neutralizing antibody according to claim 9, characterized in that: In step eight, the culture conditions are 37° C. and 5% carbon dioxide.

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