CN119125532A - A high-throughput detection method for serum neutralizing antibodies in respiratory syncytial virus vaccines - Google Patents
A high-throughput detection method for serum neutralizing antibodies in respiratory syncytial virus vaccines Download PDFInfo
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Abstract
本发明属于高通量检测技术领域，尤其涉及一种呼吸道合胞病毒疫苗血清中和抗体高通量检测方法，包括使用稀释后的单克隆D25抗体与呼吸道合胞病毒稀释液的中和反应、中和后加入培养的细胞中进行吸附，弃去吸附液，洗涤，加入病毒培养液，将培养后的细胞进行固定，加入稀释后的单克隆101抗体反应后，再加入稀释后的FITC荧光二抗，拍照并分析计数，使用软件进行数据处理和结果判定，全程由仪器读数，人为的主观判断少，结果计算更加科学可靠，与金标准方法具有良好的相关性。
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
The invention belongs to the technical field of high-flux detection, and particularly relates to a high-flux detection method for neutralizing antibodies in respiratory syncytial virus vaccine serum.
Background
Respiratory syncytial virus (Respiratory Syncytical Virus, RSV) belongs to the genus pneumovirus of the family paramyxoviridae and has only one serotype, dividing into a/B2 subtypes. Is one of the most important viral pathogens causing acute lower respiratory tract infection of infants, immunocompromised population and old people, can cause respiratory tract infection of all age groups, is a main transmission source for virus infected patients and asymptomatic infected people, is a main transmission path by direct contact, and can be transmitted by aerosol under specific conditions.
Currently, 3 vaccine types are marketed worldwide, namely Arexvy for Gram (GSK), abrysvo for Pfizer (Pfizer), and mRNA vaccine for morgana (Moderna), for preventing lower respiratory tract disease caused by RSV infection in 60 and older people. In terms of anti-RSV mab, the united states food and drug administration (Food and Drug Administration, FDA) announced that a monoclonal antibody nisetum suitable for use in preventing RSV-related lower respiratory tract disease in all infant populations, prior to which only palivizumab was used in the prevention of severe lower respiratory tract infection in high-risk children with severe disease.
The current global RSV vaccine research and development types are mainly divided into recombinant protein vaccine, virus-like particle vaccine, nucleic acid vaccine, recombinant vector vaccine, virus chimeric vaccine, attenuated live vaccine and the like. In respiratory syncytial virus vaccine products, the challenge protection test is one of the main methods for evaluating vaccine efficacy, but it is costly to perform, requires operations in a specific animal laboratory, takes a long time, requires strict animal models, and is thus not suitable for early large-scale vaccine screening. Detection of the bound antibodies using ELISA methods failed to reflect the protective effect of the vaccine on the animals. Therefore, detecting the level of neutralizing antibodies produced by animals after immunization with a vaccine is one of the criteria for judging the efficacy of a vaccine. Conventional gold standard detection methods include a trace neutralization test by observing cytopathy (cytopathic effect, CPE), a plaque reduction neutralization test (plaque-reduction neutralization test, PRNT) by counting the number of plaques, and the like. However, the test period of the above method is long, for example, the plaque reduction neutralization test is to culture the virus for about 7 days, and calculate the neutralizing antibody titer after the plaque is sufficiently large to be seen with naked eyes. The micro neutralization test needs to manually judge Cytopathy (CPE), needs experience of experimenters, has large personnel difference, subjective consciousness of reading, and is easily influenced by cell states, side hole effect of long-term culture of cell culture plates and the like. And the detection flux of the two traditional methods is low, and the method belongs to labor-intensive projects.
Therefore, a rapid and standardized method for determining neutralizing antibodies generated by respiratory syncytial virus vaccine is developed to improve the detection throughput, greatly shorten the detection period of non-clinical and clinical samples in the vaccine development process, and provide more effective proof data.
Disclosure of Invention
In order to solve the problems of strong artificial subjectivity, overlong detection time, low calculation and flux and the like in the judgment of the neutralizing antibody result of the respiratory syncytial virus vaccine in the prior art, the invention provides the high-flux detection method for the neutralizing antibody of the respiratory syncytial virus vaccine, wherein the whole experimental result is read by an instrument, the artificial subjective judgment is less, the calculation of the result is more scientific and reliable, and the method has good correlation with a gold standard method.
Specifically, the high-throughput detection method of the respiratory syncytial virus vaccine serum neutralizing antibody comprises the following steps:
step 1, culturing cells in a 96-well cell culture plate, diluting a monoclonal D25 antibody in a gradient manner, diluting a respiratory syncytial virus subtype A strain, and mixing the diluted antibody with virus liquid for neutralization reaction;
Step 2, adding the neutralized sample in the step 1 into a 96-hole cell culture plate for adsorption reaction, discarding adsorption liquid, washing, and adding a virus culture liquid for culture;
Step 3, performing immunofluorescence experiments, namely discarding the culture solution in the cell culture plate in the step 2, adding a fixing solution for fixing, discarding the fixing solution, washing for 3 times, adding diluted monoclonal 101 antibody for reaction, adding diluted FITC fluorescent secondary antibody, photographing, analyzing and counting;
and 4, performing data processing and result judgment by using software.
In some embodiments, the culturing cells in step 1 specifically comprises adjusting the cell concentration to 2X 10 5 cells/mL after digestion and blowing of the Hep-2 cells grown into a monolayer, adding 100. Mu.L of the culture medium into a 96-well cell culture plate per well, and culturing in a culture box with 5% CO 2 at 37 ℃ for 24-48 hours.
In some embodiments, the neutralization reaction in step 1 specifically comprises mixing diluted antibodies with diluted viruses in equal volumes, neutralizing the diluted antibodies in a 37 ℃ and 5% CO 2 incubator for 1 hour, mixing the diluted viruses with the viruses in equal volumes, and directly using the viruses in the cell control.
In some embodiments, the adsorption reaction in step 2 specifically comprises adding 100. Mu.L of the neutralized sample to a 96-well cell culture plate, adsorbing for 1 hour in a 37 ℃ and 5% CO 2 incubator, and simultaneously setting a cell control group, wherein 100. Mu.L of virus diluent is added to each well of the cell control group.
In some embodiments, the fixative solution in step 3 is 80% acetone pre-chilled at-20 ℃ and added at 50 μl/well for 30min at 4 ℃.
In some embodiments, the photographing and analyzing counting in the step 3 are performed by using an ELISPOT analyzer.
In some embodiments, the data processing in the step 4 includes ① processing the fluorescence range data using Excel, performing mean value calculation on the same group of complex holes, ② calculating the difference between the detection value and the negative value, namely the true value, ③ processing the original data using ELISA CALC software, and drawing a four-parameter fitting curve by taking the logarithmic value of the dilution of the sample as the abscissa and the corresponding fluorescence range point number as the ordinate.
In some embodiments, the determination of the result in the step 4 comprises performing neutralizing antibody titer calculation by using ELISA CALC software, taking the number of fluorescence foci of 1/2 (i.e. 50% fluorescence foci) of the virus control group as a reference, and taking the number of fluorescence foci into a calculation formula, and calculating the dilution factor of the sample to be detected for inhibiting 50% of virus by using software to obtain the neutralizing antibody titer of the sample;
The calculation formula is as follows:
;
Wherein y is the neutralizing antibody titer of the test sample
X number of fluorescent spots
A asymptote estimation on A
D, estimating the asymptote under the curve
Slope of curve B
And C, the corresponding dosage when the maximum combination is half.
In some embodiments, the log value of the dilution of the sample is a good linear relationship with the number of fluorescence foci, and R 2 is greater than 0.950.
In some embodiments, the adsorption solution is removed in step 2, then the solution is washed with RPMI-1640, and the solution is added to 100. Mu.L/well of virus culture solution and cultured in a 37℃and 5% CO 2 incubator for 24 to 48 hours.
Compared with the prior art, the invention has the advantages and positive effects that:
The indirect immunofluorescence method of the invention detects serum after immunization of respiratory syncytial virus vaccine, successfully detects the neutralizing antibody titer of the serum, and carries out correlation analysis with a gold standard cytopathic method, and discovers that the indirect immunofluorescence method of the invention has higher sensitivity and specificity, short detection time, the whole experimental result is read by an instrument, less artificial subjective judgment is caused, and the result calculation is more scientific and reliable, and has good correlation with the gold standard method.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the description of the embodiments will be briefly described below.
FIG. 1 fluorescence foci generated by dilution factors of different respiratory syncytial virus subtype A strains;
FIG. 2 fluorescence foci generated by different dilution factors of bound antibody 101;
FIG. 3 shows the result of four-parameter fitting curve of the repeatability verification sample;
FIG. 4 shows the result of a four-parameter fitting curve of the intermediate precision verification sample;
FIG. 5 is a graph showing the result of linearly verifying the four-parameter fitting curve of the sample;
FIG. 6-101-2, D25, and AM22 four parameter fitting curve results;
FIG. 7 shows the results of an analysis of neutralizing antibody correlation for detecting respiratory syncytial virus vaccine by indirect immunofluorescence.
Detailed Description
The invention is further illustrated by the following description of specific embodiments:
The experimental methods in the following examples, which are conventional methods unless otherwise specified, are provided for better understanding of the present invention, but are not limited thereto. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Embodiments of the invention are as follows:
1 neutralization test
(1) After the Hep-2 cells growing into a monolayer are digested and blown uniformly, the cell concentration is adjusted to be 2X 10 5 cells/mL, 100 mu L of each well is added into a 96-well cell culture plate, and the mixture is cultured for 24 to 48 hours in a culture box containing 5 percent CO 2 at 37 ℃.
(2) And diluting the monoclonal D25 antibody by a certain multiple and gradient for later use.
(3) Respiratory syncytial virus subtype A strain is diluted by a certain multiple by using virus diluent for later use.
(4) Diluted antibodies were mixed with diluted virus in equal volumes, and neutralized for 1 hour in 37 ℃ 5% co2 incubator. The virus control group is prepared by mixing diluted virus with virus diluent in equal volume, and the cell control is directly used with virus diluent.
(5) 100. Mu.L of the neutralized sample was added to a 96-well cell culture plate and adsorbed in a 37℃and 5% CO2 incubator for 1 hour. A cell control group was also set up, and 100. Mu.L of virus dilution was added to each well of the cell control group.
(6) The adsorbed solution was discarded, washed with RPMI-1640, and then cultured in a 37℃5% CO2 incubator for 24 to 48 hours with 100. Mu.L/well of the virus culture solution.
2 Immunofluorescence experiments
(1) The 96-well cell culture plate was removed, the culture broth was discarded, and 50. Mu.L/well of 80% acetone pre-chilled at-20℃was used for fixation at 4℃for 30min.
(2) The fixative was discarded and washed 3 times with 1 XPBS solution, 200. Mu.L/well.
(3) 50. Mu.L/well of the diluted monoclonal 101 antibody was added, and the mixture was left in a 37℃incubator for 1 hour.
(4) The solution was discarded and washed 3 times with 1 XPBS solution, 200. Mu.L/well.
(5) 50. Mu.L/well of diluted FITC secondary fluorescent antibody was added, and the mixture was left in a 37℃incubator for 1 hour.
(6) The solution was discarded and washed 3 times with 1 XPBS solution, 200. Mu.L/well.
(7) The foci were photographed with an ELISPOT analyzer and counted.
3, Calculating the result:
(1) Data processing
① Processing the fluorescence range data by using Excel, and calculating the average value of the same group of compound holes;
② Calculating the difference between the detection value and the negative value to obtain a true value;
③ The original data are processed by ELISA CALC software, and four-parameter fitting curves are drawn by taking the logarithmic value of the dilution of the sample as an abscissa and the corresponding fluorescence range point number as an ordinate.
(2) Result determination
① The logarithmic value of the dilution of the sample and the number of fluorescent foci form a good linear relation, and R2 is more than 0.950;
② Calculating the titer of the neutralizing antibody by using ELISA CALC software, taking the number of the fluorescent foci of 1/2 (namely 50% of the fluorescent foci) of a virus control group as a reference, and using the software to calculate the dilution factor of the sample to be detected for inhibiting 50% of the virus to obtain the titer of the neutralizing antibody of the sample.
According to the four-parameter fitting principle and the operation of software, the formula is calculated:
;
Wherein y is the neutralizing antibody titer of the test sample
X number of fluorescent spots
A asymptote estimation on A
D, estimating the asymptote under the curve
Slope of curve B
And C, the corresponding dosage when the maximum combination is half.
③ In the precision verification, the coefficient of variation of each biological measurement method is larger than that of the physicochemical measurement method, and the variation of the biological measurement methods of different products is also different. The Relative Standard Deviation (RSD) of the detection method is less than or equal to 40% on the basis of the general principle of biological product quality control analysis method verification technology review issued by the drug review center of the reference national drug administration and the combination of the detection method.
The following establishes, verifies and initially applies the technical scheme of the invention in detail by combining a specific immunofluorescence method for detecting the anti-RSV neutralizing antibody.
Reagent information:
Instrument information:
Preparation of monoclonal 101 antibody:
the expression plasmids (pcDNA3.4-H101H/pcDNA3.4-H101L, pcDNA3.4-m 101H/pcDNA3.4-m 101L) 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 with PEI. The day prior to transfection, the epi 293 cells were passaged at a cell density of 3X 10 6 cells/mL to a cell density of 4.5-5.5X10 6 cells/mL on the day of transfection. Cells were diluted to 3.0X10 6 cells/mL with fresh medium that had been pre-warmed. Preparing a polypropylene centrifuge tube, firstly adding MEM culture medium with the final culture volume of 8% as diluent, then adding DNA (the ratio of heavy chain to light chain is 1:1) below the liquid surface, and uniformly mixing for 4-5 times in an upside-down way. The transfection Reagent was added to the DNA dilution and mixed immediately, and then the Reagent-DNA mixture was allowed to stand at room temperature for 15-20min. The Reagent-DNA mixture after incubation was slowly added dropwise to the shake flask. At the time of addition, shake flask was gently swirled to aid in the homogenization of the cell suspension and transfection mixture. 18-22 hours after transfection, 293-ProFeed% of the initial culture volume was added, then culture was continued, and 293-ProFeed 5% of the initial culture volume was again added on day 2 post-transfection. Cell supernatants were collected by centrifugation 4-5 days after transfection, and purified to obtain humanized monoclonal antibody 101 (h 101) and murine monoclonal antibody 101 (m 101), the amino acid sequences of the monoclonal antibody 101 were as shown in SEQ ID NO. 2 (heavy chain of h 101)/SEQ ID NO. 4 (light chain of h 101) and SEQ ID NO. 6 (heavy chain of m 101)/SEQ ID NO. 8 (light chain of m 101).
The information on the reagents used in the preparation process is as follows:
Heavy chain nucleotide sequence of h101 (SEQ ID NO: 1)
ATGGAATTTGGTCTAAGTTGGCTATTTCTAGTGGCCATCCTAAAGGGAGTGCAGTGCCAGGTGACCCTGAAAGAGTCTGGCCCCGGCATCCTGCAACCCAGCCAAACACTGTCCCTGACCTGCTCTTTCAGCGGCTTTAGCTTGTCCACATCTGGCATGGGCGTGTCCTGGATTAGACAGCCATCTGGAAAGGGCCTGGAATGGCTGGCCCACATCTACTGGGACGACGACAAGCGGTACAACCCCTCTCTGAAGTCCAGGCTGACCATCTCCAAGGACACCTCCCGGAACCAGGTGTTCCTGAAGATCACCTCTGTCGATACCGCCGACACCGCTACCTACTACTGCGCCAGACTGTATGGCTTCACCTACGGCTTCGCCTACTGGGGCCAGGGCACCCTGGTCACCGTGTCTGCTGCTTCTACAAAAGGCCCTTCTGTGTTTCCTCTGGCTCCTTGCTCCCGGTCCACCTCCGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCTGTGACCGTGTCCTGGAACTCCGGCGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGTCCAGCGGACTGTACTCCCTCTCCTCCGTGGTTACCGTGCCTTCTTCCTCTCTGGGAACCAAGACCTATACCTGCAACGTGGACCACAAGCCTAGCAACACCAAAGTGGACAAGAGAGTGGAATCTAAGTACGGCCCGCCTTGCCCTCCTTGTCCTGCTCCTGAGTTCCTGGGCGGCCCAAGTGTGTTTCTGTTCCCACCTAAGCCCAAGGATACACTGATGATCTCTAGAACCCCTGAAGTGACCTGCGTGGTGGTCGATGTGTCCCAAGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCATAATGCCAAGACCAAGCCTAGAGAAGAGCAGTTCAACTCTACATACAGAGTGGTGTCTGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCTCCATCGAGAAGACAATCTCCAAGGCCAAAGGCCAGCCTCGGGAGCCTCAGGTGTACACTCTGCCTCCTTCTCAGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGACATCGCCGTCGAGTGGGAGTCCAATGGACAGCCTGAGAACAACTACAAAACCACCCCACCTGTGCTGGACTCCGATGGCTCCTTCTTCCTGTACAGCCGGCTGACAGTGGATAAGTCTAGATGGCAGGAAGGCAACGTGTTCTCCTGTAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGA
Heavy chain amino acid sequence of h101 (SEQ ID NO: 2)
MEFGLSWLFLVAILKGVQCQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*
> Light chain nucleotide sequence of h101 (SEQ ID NO: 3)
ATGGATATGCGAGTGCCAGCACAACTACTAGGACTGCTGCTGCTGTGGTTTCCCGGATCTCGGTGCGACATCGTGCTGACCCAGAGCCCAGCTTCTCTGGCCGTGTCCCTGGGCCAACGGGCTACCATCTTCTGCAGAGCCTCTCAGTCCGTGGATTATAATGGAATCTCCTACATGCACTGGTTCCAGCAGAAGCCTGGCCAGCCTCCTAAGCTGCTGATCTACGCTGCTTCCAACCCCGAGTCCGGCATCCCTGCCAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGCCGCCACCTACTACTGCCAGCAGATCATTGAGGATCCTTGGACCTTCGGCGGCGGCACCAAACTGGAAATCAAGCGGACCGTCGCCGCTCCTAGCGTGTTCATCTTTCCTCCATCCGACGAACAGCTGAAGTCTGGCACAGCTAGCGTCGTGTGTCTGCTGAACAACTTCTACCCTAGAGAGGCCAAGGTGCAGTGGAAAGTGGACAACGCCCTGCAGTCTGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCCACCTACTCCCTCAGCTCTACCCTGACACTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACATCAGGGCCTGTCCTCTCCTGTGACCAAGTCCTTCAACAGAGGCGAGTGTTGA
> Light chain amino acid sequence of h101 (SEQ ID NO: 4)
MDMRVPAQLLGLLLLWFPGSRCDIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*
Heavy chain nucleotide sequence of m101 (SEQ ID NO: 5)
ATGGAATTTGGTCTAAGTTGGCTATTTCTAGTGGCCATTCTGAAGGGCGTGCAGTGTCAGGTCACACTGAAAGAATCTGGCCCTGGAATCCTGCAGCCCTCTCAGACACTGTCTCTGACTTGTTCCTTCTCTGGATTCTCCTTAAGCACCTCTGGCATGGGCGTGTCCTGGATCCGGCAGCCTAGTGGAAAAGGCCTGGAATGGCTGGCCCACATCTACTGGGACGATGACAAGCGGTACAACCCTTCCCTGAAGTCTCGCCTGACCATCTCTAAGGACACCTCCCGGAACCAGGTGTTCCTGAAGATCACCTCCGTGGATACCGCCGACACCGCTACCTACTACTGCGCAAGACTGTACGGCTTTACCTATGGCTTCGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCTGCCGCTAAGACCACCCCACCTTCCGTGTACCCTCTGGCTCCTGGCTCTGCCGCCCAGACCAACTCCATGGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTTCCTGAGCCTGTGACCGTGACATGGAACTCCGGCTCCCTGTCCTCCGGTGTGCACACCTTTCCCGCCGTGCTGCAGTCCGACCTGTACACCCTGTCCTCTAGCGTGACCGTGCCCAGCTCTACCTGGCCTTCTGAGACAGTGACCTGCAACGTGGCTCATCCTGCTAGCAGCACCAAAGTGGATAAGAAGATCGTGCCTCGGGACTGTGGATGCAAGCCTTGCATTTGCACCGTGCCAGAGGTGTCCTCTGTGTTCATCTTCCCTCCTAAGCCCAAGGATGTGCTGATGATCACCCTGACACCTAAGGTCACCTGTGTGGTGGTCGATATCTCCAAGGACGATCCCGAAGTGCAGTTCTCTTGGTTCGTGGACGACGTGGAAGTGCACACAGCTCAGACCCAGCCTAGAGAGGAACAGTTCAACTCCACATTCAGATCCGTGTCCGAGCTGCCTATCATGCACCAGGACTGGCTGAACGGCAAGGAATTCAAGTGCAGAGTGAACTCTGCTGCTTTCCCAGCCCCCATCGAGAAGACCATCTCCAAAACCAAGGGCAGACCTAAGGCCCCTCAAGTGTACACCATCCCTCCACCTAAAGAGCAGATGGCCAAGGACAAGGTCTCCCTGACCTGCATGATCACCGACTTCTTCCCCGAGGACATCACTGTCGAGTGGCAGTGGAATGGCCAACCCGCCGAGAACTATAAGAACACCCAGCCTATCATGGACACGGACGGCTCCTACTTCGTCTACTCCAAGCTGAATGTTCAAAAGTCCAACTGGGAGGCCGGCAACACCTTTACCTGCTCTGTGCTGCACGAGGGCCTGCATAACCACCACACCGAGAAGTCACTGTCTCACAGCCCTGGCAAGTGA
Heavy chain amino acid sequence of m101 (SEQ ID NO: 6)
MEFGLSWLFLVAILKGVQCQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLMITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK*
Light chain nucleotide sequence of m101 (SEQ ID NO: 7)
ATGGATATGCGAGTGCCAGCACAACTACTAGGCCTGCTGCTGCTGTGGTTCCCCGGCTCCAGATGCGACATCGTGCTGACCCAGTCTCCTGCCTCTCTGGCCGTGTCTCTGGGCCAACGGGCTACCATCTTCTGCAGAGCCTCTCAGTCCGTGGACTACAACGGCATCTCCTACATGCACTGGTTCCAGCAGAAGCCTGGCCAGCCTCCTAAGCTGCTGATCTACGCCGCCTCCAACCCCGAGTCTGGCATCCCTGCTAGATTCACCGGATCCGGCAGCGGAACAGATTTTACCCTGAACATCCATCCTGTGGAAGAAGAGGACGCTGCTACCTACTACTGTCAACAGATCATCGAGGATCCTTGGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGGCCGACGCCGCTCCTACCGTGTCCATCTTTCCTCCAAGCTCTGAGCAGCTGACCTCTGGCGGCGCTTCTGTGGTGTGCTTCCTGAACAACTTCTACCCCAAAGATATCAACGTGAAGTGGAAGATCGACGGCTCTGAAAGACAGAACGGCGTGCTGAATTCCTGGACCGACCAGGACTCCAAGGACAGCACCTATTCCATGAGCTCCACACTCACACTGACAAAAGACGAGTACGAGCGGCACAATTCCTACACCTGCGAGGCCACCCACAAGACCTCCACCAGCCCAATTGTCAAGTCCTTCAACCGGAACGAGTGTTGA
Light chain amino acid sequence of m101 (SEQ ID NO: 8)
MDMRVPAQLLGLLLLWFPGSRCDIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIEDPWTFGGGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* ;
Example 1 method set up:
in the indirect immunofluorescence method, the main factors influencing the detection result include the culture time of effector cells, the virus titer of a neutralized sample and the working concentration of a bound antibody.
1.1 Determination of effector cell culture time:
After the Hep-2 cells growing into a monolayer are digested and blown uniformly, the cell concentration is adjusted to be 2X 105 cells/mL, 100 mu L of each well is added into a 96-well cell culture plate, the cell inoculation density is 2X 104 cells/well, the cells are cultured in a 37 ℃ and 5% CO2 incubator for 24 to 48 hours, and the cell morphology and density are observed under a microscope.
1.2 Validation determination of dilution fold with respiratory syncytial virus subtype a strain:
Respiratory syncytial virus subtype A strain (4.6X105 PFU/mL) was diluted with virus dilutions at 300/500/1000/1500/2000 PFU/well, respectively, to determine the optimal dilution of the virus.
1.3 Determination of dilution fold of monoclonal 101 antibody:
monoclonal 101 antibody (h 101) was diluted with PBS at 1:100, 1:200, 1:400, 1:800, respectively, to determine the optimal dilution factor for monoclonal 101 antibody.
Example 2 method validation:
2.1 specificity refers to the ability of an assay to accurately determine the analyte when the product contains other components (e.g., impurities, degradation products, excipients, etc.) that may be present.
(1) In order to evaluate the cross-reactivity of the established method, 1 XPBS solution, m101 antibody, negative serum and monoclonal D25 antibody were used as the test products, and the specificity of the method was verified.
(2) Experimental procedure
① Taking 1 XPBS solution, m101 antibody, negative serum and monoclonal D25 antibody as a to-be-detected product, and detecting according to the establishment steps;
② Fluorescence foci counting the specificity verification samples were subjected to fluorescence foci counting with an ELISPOT analyzer.
(3) And (5) analyzing results, namely performing data processing and analysis according to a calculation method.
(4) Analysis method for verifying evaluation index
The specificity is that the fluorescence focus of PBS solution and negative serum is equivalent to that of a cell control group and is far smaller than that of monoclonal D25 antibody.
2.2 Precision refers to the approach degree of a series of detection results by sampling the sample to be detected for multiple times under the specified condition, and the precision can be considered from three aspects, namely repeatability, intermediate precision and reproducibility.
2.2.1 Repeatability means the precision of the results obtained by the same experimenter under the same conditions.
(1) The method aims at detecting the monoclonal D25 antibody by the same operator on the same date, repeating for three times, and carrying out statistical analysis on results by double-hole.
(2) Experimental procedure
① Repeatedly detecting the monoclonal D25 antibody for three times, and detecting according to the establishment steps;
② Fluorescent foci counting fluorescent foci were counted using an ELISPOT analyzer.
(3) And (5) analyzing results, namely performing data processing and analysis according to a calculation method.
(4) Analysis method for verifying evaluation index
Repeatability, the RSD of the detection result of the same sample and the same date is less than or equal to 40 percent.
2.2.2 Intermediate precision-precision between results obtained by different laboratory workers under the same conditions.
(1) The method aims at detecting the monoclonal D25 antibody by different operators on the same date, repeating for three times, and double-hole, and calculating the intermediate precision.
(2) Experimental procedure
① Operators A and B respectively detect the monoclonal D25 antibody for three times on the same date, double-hole and double-hole, and detect according to the establishment steps;
② Fluorescent foci counting fluorescent foci were counted using an ELISPOT analyzer.
(3) And (5) analyzing results, namely performing data processing and analysis according to a calculation method.
(4) Analysis method for verifying evaluation index
The intermediate precision is that the RSD of the detection results of the operator A and the operator B of the same sample is less than or equal to 40 percent.
2.3 Linearity means the degree that the detection result and the concentration of the sample are in a linear relationship within a set range.
(1) Purpose(s)
The monoclonal D25 antibody was repeated at least three times with R2 of each standard curve greater than 0.950.
(2) Experimental procedure
① Taking a monoclonal D25 antibody, and repeatedly detecting for 6 times according to the establishment steps;
② Fluorescent foci counting fluorescent foci were counted using an ELISPOT analyzer.
(3) And (5) analyzing results, namely performing data processing and analysis according to a calculation method.
(4) Analysis method for verifying evaluation index
The linearity is that 6 standard curves R2 is more than or equal to 0.950.
Example 3 experimental results:
3.1 determination of the time to culture effector cells
The effector cells are cultured for 24 to 48 hours in a culture box with the inoculation density of 2 multiplied by 10 4 per hole and the temperature of 37 ℃ and the concentration of 5 percent CO 2, the confluence of the cells is good and the growth form of the cells is good when the cells are observed under a microscope after 24 hours of inoculation, the cells are too compact when the cells are observed under the microscope after 48 hours of inoculation, the cells grow to a single layer, the shedding phenomenon does not occur, and the round shrinkage of part of the cells occurs. Based on the effector cell growth characteristics, the effector cell seeding density was thus determined to be 2×10 4 per well and 24 hours of growth was available for the experiment.
3.2 Determination of dilution factors of respiratory syncytial Virus subtype A strains
In the establishment process of the detection method, monoclonal D25 antibody is used, the dilution factor is set to 800 times, 4 times of serial dilution, 8 gradients are used, the dilution factors of respiratory syncytial virus A subtype strains are respectively 300/500/1000/1500/2000 PFU/hole dilution, and the dilution factor of monoclonal 101 antibody (h 101) is 1:100. The dilution factors of respiratory syncytial virus subtype A strains are determined according to established detection methods with different dilution factors of viruses.
When the virus is 2000PFU, the result shows that the counting background is dark, the fluorescence intensity is high, and the number of the fluorescent foci is better than the rest dilution factors, so that the dilution factors are selected as the dilution factors of respiratory syncytial virus subtype A strains. The experimental results are shown in Table 1, the results of the fluorescent cooker are shown in FIG. 1, and the dilution factors A (2000), B (1500), C (1000), D (500) and E (300) in FIG. 1 are shown in the specification.
TABLE 1 dilution ratio of fluorescence foci of different respiratory syncytial virus A strains
3.3 Determination of dilution fold of monoclonal 101 antibody
In the establishment process of the detection method, monoclonal D25 antibody is used, the dilution factor is 800 times, 4 times serial dilution is carried out, 8 gradients are carried out, the respiratory syncytial virus A subtype virus is 2000PFU, the dilution factor of monoclonal 101 is 1:100, 1:200, 1:400 and 1:800, and the dilution factors of the binding antibody are different, so that the dilution factors of the binding antibody are determined according to the established detection method.
When the dilution factor of the monoclonal antibody 101 is 1:100, the antibody is combined stably, the outline of the fluorescence range is clear, and the counting is accurate. Thus 1:100 was chosen as dilution factor for the bound antibody 101. The experimental results are shown in Table 2, the fluorescent range results are shown in FIG. 2, and the dilution ratio A (1:100), the dilution ratio B (1:200), the dilution ratio C (1:400) and the dilution ratio D (1:800) in FIG. 2 are shown in the following.
TABLE 2 dilution of different binding antibodies 101 fluorescence intensity
3.4 Specific results
The number of fluorescence foci of the 1 XPBS solution and negative serum is equivalent to that of the cell control group and is significantly smaller than that of the antibody.
The test result shows that the number of fluorescence foci of the negative serum and the 1 XPBS solution is equivalent to that of the cell control group and is obviously smaller than that of the antibody sample and the virus control. Indicating that there is no neutralizing antibody against RSV, negative serum and 1 x PBS solution were determined to be unresponsive and specifically pass in this assay. The specific verification sample measurement results are shown in tables 3 and 4.
TABLE 3 specificity verification of sample measurement results (inhibition ratio)
TABLE 4 specificity verification of sample measurement results
3.5 Reproducible results
The standard is acceptable, that is, the RSD of the detection result of the same person on the same date is less than or equal to 40% of that of the same sample.
The reproducibility of the method was verified by repeating the test for the monoclonal D25 antibody for verification three times on the same date by the same laboratory staff.
The test results show that the same person can test the same sample on the same date, the RSD is less than 40%, and the repeatability is passed. The data of the repeatability results are shown in Table 5, and the conditions of four-parameter fitting curves of the repeatability verification samples are shown in FIG. 3 (corresponding to No.1, no.2 and No.3 in sequence from left to right).
TABLE 5 results of determination of repeatability verification samples
3.6 Intermediate precision results
The middle precision can be accepted as standard, the RSD of the detection results of the personnel A and the personnel B of the same sample is less than or equal to 40 percent;
Different laboratory workers used the set-up method to perform three replicates of the monoclonal D25 antibody used for validation, thereby validating the intermediate precision of the method.
The test result shows that the RSD value of the same sample is less than 40% after the measurement of the personnel A and the personnel B, and the intermediate precision is passed. The data of the intermediate precision results are shown in Table 6, and the conditions of four parameter fitting curves of the intermediate precision verification samples are shown in FIG. 4A (corresponding to the staff A from left to right in sequence No.1, no.2 and No. 3) and FIG. 4B (corresponding to the staff B from left to right in sequence No.1, no.2 and No. 3).
TABLE 6 intermediate precision validation of sample measurements
3.7 Linear results
Acceptable standard, at least 3 standard curves R2.gtoreq.0.950
The experimenter uses the established method to detect the monoclonal D25 antibody for verification, thereby verifying the linear result of the method.
The test results show that R2 of each standard curve is greater than 0.950 and passes linearly through the same sample measured by using the established detection method. The linear results are detailed in Table 7. The linear verification sample four-parameter fitting curve is shown in table 7 and fig. 5, wherein a (corresponding to a first curve, a second curve and a third curve from left to right) and B (corresponding to a fourth curve, a fifth curve and a sixth curve from left to right) are shown in fig. 5.
TABLE 7 Linear validation of sample four parameter fitting curve results
3.8 Preliminary detection sample results
After the detection method is initially established, the anti-RSV antibody 101-2, the monoclonal D25 antibody and the AM22 are respectively detected to judge the stability of the detection method.
The experimental result shows that the anti-RSV antibody 101-2 (m 101), the monoclonal D25 antibody and the AM22 can be detected by the established immunofluorescence method, and R2 of each standard curve is larger than 0.950 by drawing four-parameter fitting curves, so that the established detection method is stable, and the fluorescence range is clear and distinguishable. The four parameter fitting curves are detailed in table 8 and fig. 6.
Tables 8 101-2, D25 and AM22 plot four parameter fitting curve conditions
3.9 Positive control results Range
And analyzing the detection data of the monoclonal D25 antibody in the verification process, taking the average value of the titer of the neutralizing antibody which is repeatedly detected for 18 times + -2 SD as the range of a positive control, wherein after the positive control result Lg, the result is 5.26 + -0.46, and the result can be used as a judging standard of the effectiveness of the detection method.
3.10 Correlation analysis with micro neutralization test
The immune serum after immunization of the respiratory syncytial virus vaccine is used, and the established indirect immunofluorescence method is used for comparing the neutralizing antibody result of the respiratory syncytial virus vaccine detected by the micro-neutralization experiment, so that the correlation coefficient R= 0.8735 (the correlation coefficient is 0.8-1.0 extremely strongly correlated, 0.6-0.8 strongly correlated, 0.4-0.6 moderately correlated and 0.2-0.4 weakly correlated) is shown as the result of the figure 7, and the immune serum has good correlation.
In summary, the invention establishes a high-flux detection method for the serum neutralization antibody of the respiratory syncytial virus vaccine based on an indirect immunofluorescence technology, and the optimal conditions of the method are that Hep-2 cells are used for culturing for 24 hours, the neutralized immune serum is added for culturing for 48 hours, acetone is used for fixation, and diluted monoclonal 101 antibody (1 mg/ml) (1:100) and FITC fluorescent secondary antibody (1:800) are added. The method has the advantages of being quick and efficient in detection of the serum neutralizing antibodies of the respiratory syncytial virus vaccine, small in interference of artificial factors in result judgment and the like, has good correlation with the traditional gold standard, and can be used for large-scale preclinical vaccine screening of the respiratory syncytial virus vaccine.
The above examples are given for clarity of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
While certain specific embodiments of the 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 invention. Those skilled in the art will also appreciate that many modifications may be made to the embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.
Claims (10)
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