A high-throughput detection method for serum neutralizing antibodies in respiratory syncytial virus vaccines

Abstract

The invention belongs to the technical field of high-throughput detection, and particularly relates to a high-throughput detection method for neutralizing antibodies in respiratory syncytial virus vaccine serum. The method comprises the following steps: using a diluted monoclonal D25 antibody to react with a respiratory syncytial virus diluent, adding the diluted monoclonal D25 antibody to cultured cells for adsorption, discarding the adsorption solution, washing, adding a virus culture solution, fixing the cultured cells, adding a diluted monoclonal 101 antibody to react, and then adding a diluted FITC fluorescent secondary antibody, taking photos, analyzing and counting, using software to process data and determine results, the whole process is read by an instrument, there is less human subjective judgment, the result calculation is more scientific and reliable, and has a good correlation with a gold standard method.

Description

Technical Field

[0001] The invention belongs to the technical field of high-throughput detection, and in particular relates to a high-throughput detection method for serum neutralizing antibodies of respiratory syncytial virus vaccines. Background Art

[0002] Respiratory Syncytial Virus (RSV) is a single-stranded RNA virus belonging to the genus Pneumovirus of the family Paramyxoviridae. It has only one serotype and is divided into two subtypes, A and B. It is one of the most important viral pathogens that cause acute lower respiratory tract infections in infants, immunocompromised people and the elderly. It can cause respiratory tract infections in people of all ages. Virus-infected patients and asymptomatic carriers are the main sources of transmission. Direct contact is the main route of transmission. It can also be transmitted through droplets and aerosols under certain conditions.

[0003] Currently, three vaccines have been launched globally, namely Arexvy from GlaxoSmithKline (GSK), Abrysvo from Pfizer, and mRNA vaccine from Moderna, which are used to prevent lower respiratory tract diseases caused by RSV infection in people aged 60 and above. In terms of anti-RSV monoclonal antibodies, the U.S. Food and Drug Administration (FDA) announced that the monoclonal antibody nevacor has been approved for the prevention of RSV-related lower respiratory tract diseases in all infants. Prior to this, only palivizumab was approved for the prevention of severe lower respiratory tract infections in children at high risk of severe diseases.

[0004] At present, the types of RSV vaccine research and development in the world are mainly divided into recombinant protein vaccines, virus-like particle vaccines, nucleic acid vaccines, recombinant vector vaccines, virus chimeric vaccines and live attenuated vaccines. Among the respiratory syncytial virus vaccine products, the challenge protection test is one of the main methods for judging the efficacy of the vaccine, but its experimental cost is high, it needs to be operated in a specific animal laboratory, it is time-consuming, and the animal model required is strict, so it is not suitable for the screening of large-scale vaccines in the early stage. The use of ELISA method to detect binding antibodies cannot reflect the protective effect of the vaccine on animals. Therefore, detecting the level of neutralizing antibodies produced by animals after vaccination has become one of the criteria for judging the efficacy of the vaccine. The traditional gold standard detection methods include microneutralization tests by observing cytopathic effect (CPE) and plaque reduction neutralization tests (PRNT) by counting the number of plaques. However, the test cycles of the above methods are all time-consuming. For example, the plaque reduction neutralization test is to culture the virus for about 7 days, and after producing plaques large enough to be visible to the naked eye, the neutralizing antibody titer is calculated. The micro-neutralization test requires manual judgment of cytopathic effect (CPE), which requires the experience of the experimenter, has large differences between personnel, and the reading is subjective, and is easily affected by the cell state, the edge hole effect of long-term culture of the cell culture plate, etc. In addition, the two traditional methods have low detection throughput and are labor-intensive projects.

[0005] Therefore, developing a rapid, standardized method to measure neutralizing antibodies produced by respiratory syncytial virus vaccines to increase testing throughput can greatly shorten the testing cycle of non-clinical and clinical samples during vaccine development, while providing more effective proof data. Summary of the invention

[0006] In order to solve the problems in the prior art of strong subjectivity in judging the results of neutralizing antibodies of respiratory syncytial virus vaccine, long detection time, low calculation and throughput, the present invention provides a high-throughput detection method for neutralizing antibodies in serum of respiratory syncytial virus vaccine. The experimental results are read by instruments throughout the process, with less subjective judgment, more scientific and reliable result calculation, and good correlation with the gold standard method.

[0007] Specifically, the high-throughput detection method of respiratory syncytial virus vaccine serum neutralizing antibodies of the present invention comprises:

[0008] Step 1: Cultivate cells in a 96-well cell culture plate, grade dilute monoclonal D25 antibody, dilute respiratory syncytial virus subtype A strain, and mix the diluted antibody with the virus solution for neutralization reaction;

[0009] Step 2: Add the neutralized sample in step 1 to a 96-well cell culture plate for cultured cells for adsorption reaction, then discard the adsorption solution, wash, and add virus culture medium for culture;

[0010] Step 3: Immunofluorescence experiment, discard the culture medium in the cell culture plate in step 2, add fixative to fix, then discard the fixative, wash 3 times, add diluted monoclonal 101 antibody to react, then add diluted FITC fluorescent secondary antibody, take pictures and analyze the counts;

[0011] Step 4: Use software to process data and determine results.

[0012] In some embodiments, the culturing cells in step 1 specifically comprises digesting and homogenizing the Hep-2 cells grown into a monolayer, and adjusting the cell concentration to: 2×10 5 100 μL per well of the cell culture plate was added and cultured in a 37°C, 5% CO2 incubator for 24-48 h.

[0013] In some embodiments, the neutralization reaction in step 1 specifically includes mixing equal volumes of diluted antibodies and diluted viruses, and neutralizing them in an incubator at 37°C, 5% CO2 for 1 hour; the virus control group is a mixture of equal volumes of diluted viruses and virus diluent, and the cell control directly uses the virus diluent.

[0014] In some embodiments, the adsorption reaction in step 2 specifically includes adding 100 μL of the neutralized sample to a 96-well cell culture plate and adsorbing it in a 37°C, 5% CO2 incubator for 1 hour; at the same time, a cell control group is set up, and 100 μL of virus dilution is added to each well of the cell control group.

[0015] In some embodiments, the fixative in step 3 is 80% acetone precooled at -20°C, added in an amount of 50 μL / well, and fixed at 4°C for 30 minutes.

[0016] In some embodiments, the photographing and analysis and counting in step 3 are performed using an ELISPOT analyzer.

[0017] In some embodiments, the data processing in step 4 includes: ① processing the fluorescence focus data using Excel, and calculating the mean of the same group of duplicate wells; ② calculating the difference between the detection value and the negative value, which is the true value; ③ using ELISACalc software to process the raw data, and drawing a four-parameter fitting curve with the logarithm of the sample dilution multiple as the horizontal axis and the corresponding number of fluorescence focus points as the vertical axis.

[0018] In some embodiments, the result determination in step 4 includes: using ELISA Calc software to calculate the neutralizing antibody titer, taking the number of fluorescent foci of 1 / 2 (i.e., 50% fluorescent foci) of the virus control group as a reference, substituting it into the calculation formula, and using the software to calculate the dilution multiple of the sample to be tested that inhibits 50% of the virus, which is the neutralizing antibody titer of the sample;

[0019] Calculation formula:

[0020] ;

[0021] Where, y: neutralizing antibody titer of the test sample

[0022] x: number of fluorescent spots

[0023] A: Asymptote valuation on the curve

[0024] D: Estimate of the asymptote under the curve

[0025] B: The slope of the curve

[0026] C: Dose corresponding to half-maximal binding.

[0027] In some embodiments, the logarithmic value of the sample dilution factor has a good linear relationship with the number of fluorescent foci, R 2 Greater than 0.950.

[0028] In some embodiments, after discarding the adsorption solution in step 2, the cells are washed with RPMI-1640, 100 μL / well of virus culture solution is added, and the cells are cultured in a 37° C., 5% CO 2 incubator for 24 to 48 hours.

[0029] Compared with the prior art, the advantages and positive effects of the present invention are:

[0030] The indirect immunofluorescence method of the present invention detects serum after immunization with respiratory syncytial virus vaccine, successfully detects serum neutralizing antibody titer, and performs correlation analysis with the gold standard cytopathic effect method to find that the indirect immunofluorescence method of the present invention has high sensitivity and specificity, short detection time, the experimental results are read by the instrument throughout the process, less human subjective judgment, the result calculation is more scientific and reliable, and has a good correlation with the gold standard method. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0032] Figure 1 : Fluorescent foci produced by different dilution multiples of respiratory syncytial virus subtype A strains;

[0033] Figure 2: Fluorescent foci produced by different dilutions of binding antibodies 101;

[0034] Figure 3 : Repeatability verification sample four-parameter fitting curve results;

[0035] Figure 4 : Intermediate precision verification sample four-parameter fitting curve results;

[0036] Figure 5 : Linear verification sample four-parameter fitting curve results;

[0037] Figure 6 : Four-parameter fitting curve results of 101-2, D25 and AM22;

[0038] Figure 7 : Results of correlation analysis of neutralizing antibodies against respiratory syncytial virus vaccine detected by indirect immunofluorescence assay. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific embodiments:

[0040] To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0041] The embodiments of the present invention are as follows:

[0042] 1 Neutralization experiment

[0043] (1) After digesting and homogenizing the Hep-2 cells grown into a monolayer, adjust the cell concentration to: 2×10 5 100 μL per well of the cell culture plate was added and cultured in a 37°C, 5% CO2 incubator for 24-48 h.

[0044] (2) Dilute the monoclonal D25 antibody by a certain multiple and gradient and set aside.

[0045] (3) Respiratory syncytial virus subtype A strain, dilute it to a certain multiple using virus diluent and set aside.

[0046] (4) Mix the diluted antibody and diluted virus in equal volumes and neutralize in a 37°C, 5% CO2 incubator for 1 hour. For the virus control group, mix the diluted virus and virus diluent in equal volumes, and for the cell control, use the virus diluent directly.

[0047] (5) 100 μL of the neutralized sample was added to a 96-well cell culture plate and adsorbed in a 37°C, 5% CO2 incubator for 1 hour. A cell control group was set up at the same time, and 100 μL of virus dilution was added to each well of the cell control group.

[0048] (6) Discard the adsorption solution, wash with RPMI-1640, add 100 μL / well of virus culture medium, and culture in a 37°C, 5% CO2 incubator for 24 to 48 hours.

[0049] 2 Immunofluorescence experiments

[0050] (1) Take out the 96-well cell culture plate, discard the culture medium, and use 50 μL / well of -20℃ pre-cooled 80% acetone and fix it at 4℃ for 30 min.

[0051] (2) Discard the fixative solution and wash three times with 1× PBS solution, 200 μL / well.

[0052] (3) Add 50 μL / well of diluted monoclonal 101 antibody and place in a 37°C constant temperature incubator for 1 h.

[0053] (4) Discard the solution and wash three times with 1× PBS solution, 200 μL / well.

[0054] (5) Add 50 μL / well of diluted FITC fluorescent secondary antibody and place in a 37°C constant temperature incubator for 1 h.

[0055] (6) Discard the solution and wash three times with 1× PBS solution, 200 μL / well.

[0056] (7) Use an ELISPOT analyzer to take pictures of the fluorescent foci and analyze and count them.

[0057] 3. Calculation of results:

[0058] (1) Data processing

[0059] ① The fluorescence focus data were processed using Excel, and the mean values ​​of the duplicate wells in the same group were calculated;

[0060] ② Calculate the difference between the test value and the negative value, which is the true value;

[0061] ③ Use ELISA Calc software to process the raw data and draw a four-parameter fitting curve with the logarithm of the sample dilution factor as the abscissa and the corresponding number of fluorescent foci as the ordinate.

[0062] (2) Result determination

[0063] ① The logarithm of the sample dilution factor has a good linear relationship with the number of fluorescent foci, and R2 is greater than 0.950;

[0064] ② Calculation of neutralizing antibody titer: Use ELISA Calc software to calculate the neutralizing antibody titer. Take the number of fluorescent foci of 1 / 2 of the virus control group (i.e. 50% fluorescent foci) as a reference, substitute it into the calculation formula, and use the software to calculate the dilution multiple of the sample to be tested that inhibits 50% of the virus, which is the neutralizing antibody titer of the sample.

[0065] According to the four-parameter fitting principle combined with the software operation, the calculation formula is:

[0066] ;

[0067] Where, y: neutralizing antibody titer of the test sample

[0068] x: number of fluorescent spots

[0069] A: Asymptote valuation on the curve

[0070] D: Estimate of the asymptote under the curve

[0071] B: The slope of the curve

[0072] C: Dose corresponding to half-maximal binding.

[0073] ③ In precision verification, compared with physical and chemical determination methods, the coefficient of variation of various biological determination methods is larger, and the variation of biological determination methods for different types of products is also different. With reference to the "General Principles for Technical Review of Validation of Analytical Methods for Quality Control of Biological Products" issued by the Drug Review Center of the State Food and Drug Administration, and based on this test method, it is stipulated that the relative standard deviation (RSD) of this test method is ≤40%.

[0074] The technical solution of the present invention is described in detail below in conjunction with the establishment, verification and preliminary application of a specific immunofluorescence method for detecting anti-RSV neutralizing antibodies.

[0075] Reagent Information:

[0076]

[0077] Instrument information:

[0078]

[0079] Preparation of monoclonal 101 antibody:

[0080] The expression plasmids (pcDNA3.4-h101H / pcDNA3.4-h101L, pcDNA3.4-m101H / pcDNA3.4-m101L) carrying the h101 heavy chain nucleotide sequence shown in SEQ ID NO:1, the h101 light chain nucleotide sequence shown in SEQ ID NO:3, the m101 heavy chain nucleotide sequence shown in SEQ ID NO:5, and the m101 light chain nucleotide sequence shown in SEQ ID NO:7 were transfected into Expi293 cells using PEI. The day before transfection, Expi293 cells were cultured at 3×10 6 cells / mL, and the cell density was maintained at 4.5-5.5×10 cells / mL on the day of transfection. 6 cells / mL. Dilute the cells to 3.0×10 cells / mL using pre-warmed fresh culture medium. 6 cells / mL. Prepare a polypropylene centrifuge tube, first add MEM medium with a final culture volume of 8% as a diluent, then add DNA below the liquid surface (heavy chain to light chain ratio: 1:1), and mix well by inverting 4-5 times. Add the transfection reagent to the DNA diluent and mix immediately, then let the Reagent-DNA mixture stand at room temperature for 15-20 minutes. Slowly drop the incubated Reagent-DNA mixture into the shake flask. When adding, gently shake the shake flask to help mix the cell suspension and transfection mixture. 18-22 hours after transfection, add 293-ProFeed at 5% of the starting culture volume, and then continue to culture. Add 293-ProFeed at 5% of the starting culture volume again on the second day after transfection. On the 4th to 5th day after transfection, the cell supernatant was collected by centrifugation and purified to obtain humanized monoclonal 101 antibody (h101) and murine monoclonal 101 antibody (m101). The amino acid sequences of the monoclonal 101 antibody are shown in SEQ ID NO: 2 (heavy chain of h101) / SEQ ID NO: 4 (light chain of h101) and SEQ ID NO: 6 (heavy chain of m101) / SEQ ID NO: 8 (light chain of m101).

[0081] The reagent information used in the above preparation process is as follows:

[0082]

[0083] >heavy chain nucleotide sequence of h101 (SEQ ID NO: 1)

[0084]

[0085] >Heavy chain amino acid sequence of h101 (SEQ ID NO: 2)

[0086] MEFGLSWLFLVAILKGVQCQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0087] >Light chain nucleotide sequence of h101 (SEQ ID NO: 3)

[0088] ATGGATATGCGAGTGCCAGCACAACTACTAGGACTGCTGCTGCTGTGGTTTCCCGGATCTCGGTGCGACATCGTGCTGACCCAGAGCCCAGCTTCTCTGGCCGTGTCCCTGGGCCAACGGGCTACCATCTTCTGCAGAGCCTCTCAGTCCGTGGATTATAATGGAATCTCCTACATGCACTGGTTCCAGCAGAAGCCTGGCCAGCCTCCTAAGCTGCTGATCTACGCTGCTTCCAACCCCGAGTCCGGCATCCCTGCCAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGCCGCCACCTACTACTGCCAGCAGATCATTGAGGATCCTTGGACCTTCGGCGGCGGCACCAAACTGGAAATCAAGCGGACCGTCGCCGCTCCTAGCGTGTTCATCTTTCCTCCATCCGACGAACAGCTGAAGTCTGGCACAGCTAGCGTCGTGTGTCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCCCTGCAGTCTGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCCACCTACTCCCTCAGCTCTACCCTGACACTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACATCAGGGCCTGTCCTCTCCTGTGACCAAGTCCTTCAACAGAGGCGAGTGTTGA

[0089] >Light chain amino acid sequence of h101 (SEQ ID NO: 4)

[0090] MDMRVPAQLLGLLLLWFPGSRCDIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

[0091] >Heavy chain nucleotide sequence of m101 (SEQ ID NO: 5)

[0092]

[0093] >Heavy chain amino acid sequence of m101 (SEQ ID NO: 6)

[0094] MEFGLSWLFLVAILKGVQCQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLMITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK*

[0095] >Light chain nucleotide sequence of m101 (SEQ ID NO: 7)

[0096] ATGGATATGCGAGTGCCAGCACAACTACTAGGCCTGCTGCTGCTGTGGTTCCCCGGCTCCAGATGCGACATCGTGCTGACCCAGTCTCCTGCCTCTCTGGCCGTGTCTCTGGGCCAACGGGCTACCATCTTCTGCAGAGCCTCTCAGTCCGTGGACTACAACGGCATCTCCTACATGCACTGGTTCCAGCAGAAGCCTGGCCAGCCTCCTAAGCTGCTGATCTACGCCGCCTCCAACCCCGAGTCTGGCATCCCTGCTAGATTCACCGGATCCGGCAGCGGAACAGATTTTACCCTGAACATCCATCCTGTGGAAGAAGAGGACGCTGCTACCTACTACTGTCAACAGATCATCGAGGATCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGGCCGACGCCGCTCCTACCGTGTCCATCTTTCCTCCAAGCTCTGAGCAGCTGACCTCTGGCGGCGCTTCTGTGGTGTGCTTCCTGAACAACTTCTACCCCAAAGATATCAACGTGAAGTGGAAGATCGACGGCTCTGAAAGACAGAACGGCGTGCTGAATTCCTGGACCGACCAGGACTCCAAGGACAGCACCTATTCCATGAGCTCCACACTCACACTGACAAAAGACGAGTACGAGCGGCACAATTCCTACACCTGCGAGGCCACCCACAAGACCTCCACCAGCCCAATTGTCAAGTCCTTCAACCGGAACGAGTGTTGA

[0097] >Amino acid sequence of the light chain of m101 (SEQ ID NO:8)

[0098] MDMRVPAQLLGLLLLWFPGSRCDIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIED PWTTFGGGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* ;

[0099] Example 1 Method establishment:

[0100] In the indirect immunofluorescence method, the main factors affecting the detection results include: effector cell culture time, virus titer of neutralization sample, and working concentration of binding antibody.

[0101] 1.1 Determination of effector cell culture time:

[0102] After digesting and blowing Hep-2 cells grown into a monolayer, adjust the cell concentration to 2×105 cells / mL, add 100 μL per well of a 96-well cell culture plate, and the cell seeding density is 2×104 cells / well. Culture in a 37°C, 5% CO2 incubator for 24 to 48 hours, and observe the cell morphology and density under a microscope.

[0103] 1.2 Determination of the dilution multiple of RSV subtype A strain for verification:

[0104] Respiratory syncytial virus subtype A strain (4.6×105 PFU / mL) was diluted with virus diluent at 300 / 500 / 1000 / 1500 / 2000 PFU / well to determine the optimal dilution multiple of the virus.

[0105] 1.3 Determination of the dilution multiple of monoclonal 101 antibody:

[0106] The monoclonal 101 antibody (h101) was diluted with PBS at 1:100, 1:200, 1:400, and 1:800 to determine the optimal dilution multiple of the monoclonal 101 antibody.

[0107] Example 2 Method Verification:

[0108] 2.1 Specificity: refers to the ability of the analytical method to correctly determine the analyte when the product may contain other components (such as impurities, degradation products, excipients, etc.).

[0109] (1) Purpose: To evaluate the cross-reactivity of the established method, 1× PBS solution, m101 antibody, negative serum and monoclonal D25 antibody were used as test samples to verify the specificity of the method.

[0110] (2) Experimental procedures

[0111] ① Take 1× PBS solution, m101 antibody, negative serum and monoclonal D25 antibody as the samples to be tested and perform the test according to the established steps;

[0112] ② Fluorescence focus counting: Use ELISPOT analyzer to count the fluorescent foci of the specificity verification sample.

[0113] (3) Result analysis: Data processing and analysis are performed according to the calculation method.

[0114] (4) Analytical method validation evaluation indicators

[0115] Specificity: The number of fluorescent foci in PBS solution and negative serum was comparable to that in the cell control group, and much smaller than that in the monoclonal D25 antibody.

[0116] 2.2 Precision: It refers to the closeness of the test results of a series of tests conducted on the sample under specified conditions. Precision can be considered from three levels, namely repeatability, intermediate precision and reproducibility.

[0117] 2.2.1 Repeatability: refers to the precision of the results obtained by the same experimenter under the same conditions.

[0118] (1) Purpose: The same operator tested the monoclonal D25 antibody on the same day, repeated three times, with duplicate wells, and the results were statistically analyzed.

[0119] (2) Experimental procedures

[0120] ① The monoclonal D25 antibody was tested three times according to the established procedures;

[0121] ② Fluorescent focus counting: Use ELISPOT analyzer to count the fluorescent foci.

[0122] (3) Result analysis: Data processing and analysis are performed according to the calculation method.

[0123] (4) Analytical method validation evaluation indicators

[0124] Repeatability: The RSD of the test results of the same sample, the same date, and the same experimenter is ≤40%.

[0125] 2.2.2 Intermediate precision: refers to the precision between the results obtained by different experimenters under the same conditions.

[0126] (1) Purpose: Different operators tested the monoclonal D25 antibody three times on the same day, with duplicate wells, and calculated the intermediate precision.

[0127] (2) Experimental procedures

[0128] ① Operators A and B tested the monoclonal D25 antibody three times on the same day, in duplicate, according to the established procedures;

[0129] ② Fluorescent focus counting: Use ELISPOT analyzer to count the fluorescent foci.

[0130] (3) Result analysis: Data processing and analysis are performed according to the calculation method.

[0131] (4) Analytical method validation evaluation indicators

[0132] Intermediate precision: For the same sample, the RSD of the test results of operator A and operator B is ≤40%.

[0133] 2.3 Linearity: refers to the degree to which the test result and the sample concentration are linearly related within the set range.

[0134] (1) Purpose

[0135] The monoclonal D25 antibody was assayed at least three times, and the R2 of each standard curve was greater than 0.950.

[0136] (2) Experimental procedures

[0137] ① Take the monoclonal D25 antibody and repeat the test 6 times according to the established steps;

[0138] ② Fluorescent focus counting: Use ELISPOT analyzer to count the fluorescent foci.

[0139] (3) Result analysis: Data processing and analysis are performed according to the calculation method.

[0140] (4) Analytical method validation evaluation indicators

[0141] Linearity: 6 standard curves R2≥0.950.

[0142] Example 3 Experimental results:

[0143] 3.1 Determination of effector cell culture time

[0144] The effector cells were seeded at a density of 2×10 4 / well, cultured in a 37°C, 5% CO2 incubator for 24 to 48 hours. According to the observation of cell morphology and density under a microscope, 24 hours after cell inoculation, the cell confluence was good and the cell growth morphology was good. 48 hours after cell inoculation, the cells were too dense and grew to a single layer without shedding, and some cells were rounded. According to the growth characteristics of effector cells, the inoculation density of effector cells was determined to be 2×10 4 / well, and can be used for experiments after 24 hours of growth.

[0145] 3.2 Determination of dilution multiples for RSV subtype A strains

[0146] During the establishment of the detection method, the monoclonal D25 antibody was used, and the dilution multiple was set as: 800 times, 4-fold serial dilution, 8 gradients, the dilution multiples of the respiratory syncytial virus subtype A strain were 300 / 500 / 1000 / 1500 / 2000 PFU / well, and the dilution multiple of the monoclonal 101 antibody (h101) was 1: 100. The dilution multiples of the respiratory syncytial virus subtype A strain were determined according to the established detection method with different virus dilution multiples.

[0147] When the virus was 2000 PFU, the results showed that the counting background was dark, the fluorescence intensity was high, and the number of fluorescent foci was better than the other dilution multiples. Therefore, this multiple was selected as the dilution multiple of the respiratory syncytial virus subtype A strain. The experimental results are shown in Table 1, and the fluorescent foci results are shown in Figure 1 , Figure 1 Medium dilution multiples: A: (2000); B: (1500); C: (1000); D: (500); E: (300).

[0148] Table 1: Number of fluorescent foci at different dilutions of respiratory syncytial virus subtype A strains

[0149]

[0150] 3.3 Determination of the dilution multiple of monoclonal 101 antibody

[0151] During the establishment of the detection method, the monoclonal D25 antibody was used, and the dilution factor was set at 800 times, 4-fold serial dilution, 8 gradients, 2000 PFU of respiratory syncytial virus subtype A virus, and the monoclonal 101 dilution factors were 1:100, 1:200, 1:400, and 1:800. Different dilution factors of the binding antibody were used to determine the dilution factor of the binding antibody according to the established detection method.

[0152] When the dilution factor of monoclonal 101 was 1:100, the antibody binding was stable, the outline of the fluorescent focus was clear, and the counting was accurate. Therefore, 1:100 was selected as the dilution factor of the binding antibody 101. The experimental results are shown in Table 2, and the fluorescent focus results are shown in Figure 2 , Figure 2 Medium dilution multiples: A: (1:100); B: (1:200); C: (1:400); D: (1:800).

[0153] Table 2: Number of fluorescent foci at different dilutions of binding antibodies

[0154]

[0155] 3.4 Specificity results

[0156] Acceptable criteria: The number of fluorescent foci in 1×PBS solution and negative serum was equivalent to that in the cell control group and significantly less than that in the antibody control group.

[0157] The test results show that the number of fluorescent foci of negative serum and 1×PBS solution is comparable to that of the cell control group, and significantly less than that of the antibody sample and virus control. This indicates that there is no anti-RSV neutralizing antibody. Therefore, it is determined that negative serum and 1×PBS solution have no response in this detection method, and the specificity is passed. The specificity verification sample measurement results are detailed in Tables 3 and 4.

[0158] Table 3: Specificity verification sample measurement results (inhibition rate)

[0159]

[0160] Table 4: Specificity verification sample test results

[0161]

[0162] 3.5 Reproducibility of results

[0163] Acceptable standard: RSD of the test results of the same sample and the same person on the same date is ≤40%.

[0164] The D25 monoclonal antibody was tested three times by the same researcher on the same day to verify the repeatability of the method.

[0165] The test results show that: the same person, on the same date, measured the same sample, RSD is less than 40%, and the repeatability is passed. The repeatability result data is shown in Table 5, and the four-parameter fitting curve of the repeatability verification sample is shown in Figure 3 (From left to right, they correspond to No.1, No.2, and No.3).

[0166] Table 5: Repeatability verification sample measurement results

[0167]

[0168] 3.6 Intermediate precision results

[0169] Acceptable standard for intermediate precision: For the same sample, the RSD of the test results of personnel A and personnel B is ≤40%;

[0170] Different experimenters used the established method to perform three replicate tests on the validated monoclonal D25 antibody to verify the intermediate precision of the method.

[0171] The test results show that after the determination by personnel A and personnel B, the RSD value of the same sample is less than 40%, and the intermediate precision passes. The intermediate precision result data is detailed in Table 6, and the four-parameter fitting curve of the intermediate precision verification sample is detailed in Figure 4 A in (from left to right, corresponding to No.1, No.2, No.3 of person A), Figure 4 B in (from left to right corresponding to No.1, No.2, No.3 of person B).

[0172] Table 6: Intermediate precision verification sample measurement results

[0173]

[0174] 3.7 Linear Results

[0175] Acceptable standard: at least 3 standard curves with R2 ≥ 0.950

[0176] The experimenters used the established method to test the monoclonal D25 antibody for verification, thereby verifying the linearity results of the method.

[0177] The test results show that: after using the established detection method to measure the same sample, the R2 of each standard curve is greater than 0.950, and the linearity is passed. The linear result data is detailed in Table 7. The four-parameter fitting curve of the linear verification sample is detailed in Table 7 and Figure 5 A in (from left to right corresponds to the first curve, the second curve, and the third curve), Figure 5 B in (corresponding to the fourth curve, the fifth curve, and the sixth curve from left to right).

[0178] Table 7: Four-parameter fitting curve results of linear verification samples

[0179]

[0180] 3.8 Preliminary test sample results

[0181] After the detection method was initially established, anti-RSV antibody 101-2, monoclonal D25 antibody and AM22 were tested to determine the stability of the detection method.

[0182] The results of this experiment showed that the established immunofluorescence method can detect anti-RSV antibody 101-2 (m101), monoclonal D25 antibody and AM22. By drawing the four-parameter fitting curve, the R2 of each standard curve was greater than 0.950, indicating that the established detection method is stable and the fluorescent foci are clearly identifiable. The four-parameter fitting curves are detailed in Table 8 and Figure 6 .

[0183] Table 8 Four-parameter fitting curves of 101-2, D25 and AM22

[0184]

[0185] 3.9 Positive Control Result Range

[0186] The monoclonal D25 antibody detection data were analyzed during the validation process, and the mean ± 2SD of the neutralizing antibody titer of 18 repeated tests was used as the range of the positive control. After the positive control result Lg, the result was: 5.26 ± 0.46, which can be used as a criterion for determining the effectiveness of the detection method.

[0187] 3.10 Correlation analysis with micro-neutralization test

[0188] The immune serum after immunization with the respiratory syncytial virus vaccine was used to compare the neutralizing antibody results of the respiratory syncytial virus vaccine detected by the microneutralization experiment using the established indirect immunofluorescence method, and a correlation analysis was performed, such as Figure 7 The results showed that the correlation coefficient R=0.8735 (correlation coefficient 0.8-1.0 is extremely strong correlation; 0.6-0.8 is strong correlation; 0.4-0.6 is moderate correlation; 0.2-0.4 is weak correlation), which has good correlation.

[0189] In summary, the present invention has established a high-throughput detection method for neutralizing antibodies in serum of respiratory syncytial virus vaccine based on indirect immunofluorescence technology. The optimal conditions of the method are: using Hep-2 cells for 24 hours, adding neutralized immune serum for 48 hours, using acetone for fixation, adding diluted monoclonal 101 antibody (1 mg / ml) (1:100), and FITC fluorescent secondary antibody (1:800). The method has the advantages of rapid and efficient detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine, and the result determination is less affected by human factors, and has a good correlation with the traditional gold standard, and can be used for large-scale preclinical vaccine screening of respiratory syncytial virus vaccine.

[0190] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0191] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A high-throughput detection method for serum neutralizing antibodies of respiratory syncytial virus vaccine, characterized in that: The method comprises: Step 1: Cultivate cells in a 96-well cell culture plate, grade dilute monoclonal D25 antibody, dilute respiratory syncytial virus subtype A strain, and mix the diluted antibody with the virus solution for neutralization reaction; Step 2: Add the neutralized sample in step 1 to a 96-well cell culture plate for cultured cells for adsorption reaction, then discard the adsorption solution, wash, and add virus culture medium for culture; Step 3: Immunofluorescence experiment, discard the culture medium in the cell culture plate in step 2, add a fixative to fix, then discard the fixative, wash 3 times, add the diluted monoclonal 101 antibody for reaction, then add the diluted FITC fluorescent secondary antibody, take pictures and analyze and count, the heavy chain and light chain amino acid sequences of the monoclonal 101 antibody are shown in SEQ ID NO: 2 and 4; the method adopts indirect immunofluorescence method; Step 4: Use software to process data and determine results.

2. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The cultured cells in step 1 specifically include digesting and homogenizing the Hep-2 cells grown into a monolayer, and adjusting the cell concentration to: 2×10 5 100 μL per well of the cell culture plate was added and cultured in a 37°C, 5% CO2 incubator for 24-48 h.

3. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The neutralization reaction in step 1 specifically includes mixing equal volumes of diluted antibodies and diluted viruses, and neutralizing them in a 37° C., 5% CO2 incubator for 1 hour; the virus control group is a mixture of equal volumes of diluted viruses and virus diluent, and the cell control directly uses the virus diluent.

4. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The adsorption reaction in step 2 specifically includes adding 100 μL of the neutralized sample to a 96-well cell culture plate and adsorbing it in a 37° C., 5% CO2 incubator for 1 hour; at the same time, a cell control group is set up, and 100 μL of virus dilution is added to each well of the cell control group.

5. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The fixative in step 3 is 80% acetone precooled at -20°C, added in an amount of 50 μL / well, and fixed at 4°C for 30 minutes.

6. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: In step 3, the photographing and analysis counting are performed using an ELISPOT analyzer.

7. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The data processing in step 4 includes: ① processing the fluorescence focus data using Excel, and calculating the mean of the same group of duplicate wells; ② calculating the difference between the detection value and the negative value, which is the true value; ③ using ELISA Calc software to process the original data, and drawing a four-parameter fitting curve with the logarithm of the sample dilution multiple as the horizontal axis and the corresponding number of fluorescence focus points as the vertical axis.

8. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: The result determination in step 4 includes: using ELISA Calc software to calculate the neutralizing antibody titer, taking the number of fluorescent foci of 1 / 2 of the virus control group as a reference, substituting it into the calculation formula, and using the software to calculate the dilution multiple of the sample to be tested that inhibits 50% of the virus, which is the neutralizing antibody titer of the sample; Calculation formula: ； Where, y: neutralizing antibody titer of the test sample; x: number of fluorescent spots; A: asymptote valuation on the curve; D: estimate of the asymptote under the curve; B: slope of the curve; C: Dose corresponding to half-maximal binding.

9. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 8, characterized in that: The logarithmic value of the sample dilution factor has a good linear relationship with the number of fluorescent foci, R 2 Greater than 0.

950.

10. The method for high-throughput detection of neutralizing antibodies in serum of respiratory syncytial virus vaccine according to claim 1, characterized in that: In step 2, the adsorption solution is discarded and then washed with RPMI-1640, and 100 μL / well of virus culture solution is added, and cultured in a 37° C., 5% CO 2 incubator for 24 to 48 hours.

Images