Method for quantitatively detecting specific activity of SpA5 antigen in recombinant staphylococcus aureus vaccine or / and stock solution

Abstract

The invention discloses a method for quantitatively detecting specific activity of a SpA5 antigen in a recombinant staphylococcus aureus vaccine or / and a stock solution. The method comprises the following steps: S1, coating an elisa plate by taking SpA5-H3 as a capture antibody; s2, adding a sample to be detected into the coated elisa plate; s3, incubating an enzyme-labeled antibody SpA5-16, developing by using a TMB (Tetramethylbenzidine) developing solution, respectively detecting OD (Optical Density) values at 450 nm and 630 nm by using an enzyme labeling instrument, and calculating an OD (Optical Density) value of 450-630 nm; and S4, drawing a standard curve by using the SpA5 reference substance, and calculating the specific activity of the SpA5 antigen in the detected sample according to the standard curve. A sample with unknown antigen activity concentration is quantitatively detected by the method provided by the invention, so that the antigen activity concentration is obtained. The specific activity of the SpA5 antigen in a recombinant staphylococcus aureus vaccine or / and a stock solution can be quantitatively detected, and good stability and repeatability are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a method for quantitatively detecting the specific activity of SpA5 antigen in a recombinant Staphylococcus aureus vaccine or stock solution. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a "superbug" that is highly resistant to β-lactam antibiotics and resistant to most antibiotics such as aminoglycosides, macrolides, and fluoroquinolones. It has the characteristics of strong pathogenicity, wide transmission routes, easy outbreak and epidemic, and the development of multiple and high drug resistance. The global incidence of severe infections caused by MRSA has an increasing trend, and it has become a difficult problem in clinical treatment, posing a huge threat to human health.

[0003] So far, there are no effective antibody drugs or vaccine products for the treatment or prevention of Staphylococcus aureus infections in the world. The treatment of MRSA infections still mainly relies on antibiotic use. However, the speed of antibiotic research and development far lags behind the speed of bacteria developing drug resistance, and the pathogenic factors of MRSA are complex. Antigen-specific antibodies against Staphylococcus aureus have been proven to provide partial protection and may reduce the subsequent incidence of infections. An important mechanism by which Staphylococcus aureus uses to interfere with the protective humoral immune response is the polyclonal activation of B cells by staphylococcal protein A (SpA). Protein A can resist the invasion of the host immune system and is a highly virulent immune evasion factor, which can be used for the development of Staphylococcus aureus vaccines.

[0004] During the development of Staphylococcus aureus vaccines, in order to ensure the quality and effectiveness of Staphylococcus aureus vaccines, it is necessary to detect the specific activity of component proteins in the vaccines and stock solutions, and to ensure immunogenicity by monitoring the consistency between batches during production, and to use this as the release standard for detection. Therefore, it is particularly important to develop a method for quantitatively detecting the specific activity of SpA5 antigen in a recombinant Staphylococcus aureus vaccine or stock solution. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for quantitatively detecting the specific activity of SpA5 antigen in a recombinant Staphylococcus aureus vaccine or stock solution to solve the problem of the lack of a detection method for the specific activity of SpA5 antigen in the prior art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions: A method for quantitatively detecting the specific activity of SpA5 antigen in a recombinant Staphylococcus aureus vaccine or stock solution provided by the present invention includes the following steps: S1. Coat an ELISA plate with SpA5-H3 as the capture antibody; S2. Add the test sample to the coated ELISA plate; S3. Incubate with the enzyme-labeled antibody SpA5-16, develop color using TMB chromogenic solution, measure the OD values at 450 nm and 630 nm respectively with an ELISA reader, and calculate the OD 450-630nm value; S4. Draw a standard curve using the SpA5 reference product, and calculate the specific activity of the SpA5 antigen in the test sample according to the standard curve.

[0007] In the present invention, the SpA5-H3 comprises a heavy chain and a light chain. The amino acid sequence of the SpA5-H3 heavy chain is as shown in SEQ ID NO.1, and the amino acid sequence of the SpA5-H3 light chain is as shown in SEQ ID NO.2; Heavy chain FR1: KVQLLESGGGLVQPGRSLRLSCAAS FR2: MHWVRQAPGKGLEWVSG FR3: GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC FR4: WGQGTMVTVSS CDR1: GFTFDDYA CDR2: ISWNSGSI CDR3: AKDMSPWPDAFDI Light chain FR1: ELVLTQPPSASGTPGQRVTISCSGS FR2: VYWYQQLPGTAPKLLIY FR3: NQRPSGVPDRFSGSKSGTSASLAISGPRSEDEADYYC FR4: FGGGTKVTVLGQP CDR1: SSNIGNNY CDR2: RN CDR3: AAWDDSLRGPV In the present invention, the SpA5-16 comprises a heavy chain and a light chain. The amino acid sequence of the SpA5-16 heavy chain is as shown in SEQ ID NO.3, and the amino acid sequence of the SpA5-16 light chain is as shown in SEQ ID NO.4; Heavy chain R1: QVQLQESGGGLVQPGGSLRLSCAASGFTFS FR2: WVRQAPGKGLEWVS FR3: RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR FR4: WGQGTTVTVSS CDR1: GFTFSSYS CDR2: ISSSSSYI CDR3: ARDMITFGGVPVNGMDV Light chain FR1: ELVLTQPPSASGTPGQRVTISC FR2: WYQHLPGKPPKLIIY FR3: DLVATGVPDRFSGSKSGTSASLAISGLQSEDEADYYC FR4: FGGGTELTVLGQP CDR1: SSNIGDNT CDR2: YD CDR3: AAWDDSLNGWV Optionally or preferably, after coating the enzyme-linked immunosorbent assay (ELISA) plate with S1, incubate overnight at 4°C, wash the plate four times with PBST, incubate with 3% BSA blocking solution at 37°C for 1 hour, and wash the plate four times with PBST.

[0008] Optionally or preferably, after adding the test sample in S2 to the ELISA plate, incubate at 37°C for 1 hour and wash the plate four times with PBST.

[0009] Optionally or preferably, after adding the enzyme-labeled antibody SpA5-16 in S3 to the ELISA plate, incubate at 37°C for 1 hour, wash the plate four times with PBST, add TMB chromogenic solution for color development for 10 minutes, and add 2 M sulfuric acid termination solution after color development.

[0010] Optionally or preferably, the PBST is composed of 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

[0011] Optionally or preferably, in S4, the standard curve is constructed by taking the logarithm of the antigen concentration as the abscissa and the OD value as the ordinate to obtain a linear regression equation.

[0012] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: The present invention forms a "antibody-antigen-antibody" sandwich structure through a double-antibody sandwich. SpA5-H3 is used as a capture antibody for coating, specifically recognizing the SpA5 antigen. Through the obtained linear range, a sample with an unknown antigen activity concentration is quantitatively detected, thereby obtaining the relationship between the antigen protein concentration and the antigen activity concentration. It can quantitatively detect the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or the stock solution, and has good stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0014] Figure 1 is the standard curve of Example 2 of the present invention; Figure 2 is the optimized standard curve of Example 3 of the present invention; Figure 3 is the linear verification result diagram of Example 4 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] Preparation of antibodies SpA5-H3 and SpA5-16: 1. Expression and purification of wild-type (wSpA) and mutant (mSpA) proteins of Staphylococcus aureus protein A The process details can be found in the content of "Example 1" in Patent CN118480121B 2. Establishment of a human-specific anti-Staphylococcus aureus Fab antibody library The process details can be found in the content of "Example 2" in Patent CN118480121B 3. Screening of the human-specific anti-Staphylococcus aureus Fab antibody library (1) Bacterial purification and preparation of antibody phage The phage library was subjected to seven consecutive rounds of biopanning using Dynabeads™ M-280 streptavidin conjugated with biotinylated mSpA protein without phage amplification in between. The method involved incubating the phage library with mSpA-coated magnetic beads for 1 h. After adsorption by placing the tube on a magnetic stand, the supernatant in the tube was carefully removed; then, the magnetic beads were washed 10 - 15 times with 0.5% PBST; the bound phage particles were eluted with glycine-hydrochloric acid (pH 2.2), and the eluate was neutralized to pH 7.0 with 1 M Tris-HCl (pH 9.0) for the next round of panning. Phage particles were collected after seven rounds of panning, mixed with TG1 cells, plated, and colonies were picked. All clones were analyzed using phage ELISA and the DNA was sequenced.

[0016] (2)Purification of phage Bacterial clones carrying recombinant phagemids were inoculated into 1 mL of SB medium (containing 50 μg / mL carbenicillin) and grown to an OD600 of 0.5 - 0.6. The culture was infected with helper phage at a cell-to-helper phage ratio of 1:20; incubated statically at room temperature for 30 min, then placed on a shaker at 200 rpm / min and incubated at 37 °C for 2 h. Then, 2 mL of SB medium (containing 50 μg / mL carbenicillin, 15 μg / mL tetracycline, 37.5 μg / mL kanamycin) was added and cultured overnight at 37 °C. The next day, the culture supernatant was collected by centrifugation at 2300×g for 5 min at room temperature; 4 volumes of 25 mM PEG-8000 and 2.5 M NaCl were added to the supernatant to precipitate phage particles. After the mixture was placed on ice for 1 h, it was centrifuged for 15 min. The obtained phage pellet was resuspended in sterile PBS buffer; the physical titer of phage was calculated using the absorbance values at 269 nm and 320 nm.

[0017] (3)Phage ELISA The physical titer of the resuspended phage was adjusted to 3 - 5×1012 virus / mL. 30 μL of the phage resuspension was added to an ELISA plate and incubated overnight at 4 °C, washed 3 times with 0.1% PBST buffer; blocked with 3% BSA blocking solution for 1 h, then washed 6 times with the washing solution; incubated with 10 μg / mL biotinylated mSpA protein for 1 h, then washed 6 times with the washing solution; finally, Streptavidin HRP was diluted 1:1000 with the blocking solution and then detected. Clones with high positivity were selected for further analysis.

[0018] 4. Cloning, expression, and purification of SpA5-H3 monoclonal antibody and SpA5-16 monoclonal antibody The selected positive Fab-display vectors were used as templates. Forward primers containing Kozak and Lonza specific leader sequences and homologous to the antibody sequences, and reverse primers homologous to the 3'-terminal regions of the heavy and light chain variable regions were used to clone the heavy and light chains into pCDNA3.4 to construct an expression vector for the fully human anti-SpA antibody. Then, the expression vector was transformed into DH5α competent bacteria and cultured overnight at 37°C on plates containing ampicillin. Ten single colonies were picked and subjected to PCR using specific primers. The reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 100 s, for 28 cycles; extension at 72°C for 5 min. 5 μL of the PCR product was detected by 1% agarose gel electrophoresis.

[0019] Antibody gene sequence determination and bioinformatics analysis: The PCR products of the antibody genes identified as positive by gel electrophoresis and with the heavy and light chains capable of pairing were purified using the Qiagen PCR product purification kit, and sequenced from both the forward and reverse directions. The IMGT online server (http: / / imgt.cines.fr / ) was used to analyze the antibody gene family, mutation rate, and CDR regions.

[0020] The vector plasmids in the obtained positive transformants were transformed into DH5α for large-scale amplification. After rapid extraction of the recombinant plasmids, they were co-incubated with the transfection reagent PEI at 37°C for 15 - 20 min and then transfected into HEK 293F cells, and cultured with shaking at 125 rpm / min in a 37°C shaker in a 5% CO2 incubator. After 5 days, the cell supernatant was collected by centrifugation at 3000 g for 30 min at 4°C and purified using protein G affinity chromatography; SDS-PAGE was used to examine the expression and purification of the antibody.

[0021] Finally, SpA5-H3 antibody and SpA5-16 antibody were obtained, and the sequences are shown in SEQ ID NO.1 - SEQ ID NO.4.

[0022] Example 1 Determination of the concentrations of the capture antibody and the detection antibody 1. Experimental method Dilute the capture antibody SpA5-H3 to 1, 2, 3, 4, 6, 8, 10, 12 μg / mL, coat it overnight at 4°C, wash it four times with PBST, block it with 3% BSA for 1 h, wash it four times with PBST. Add 100 μL of 1 μg / mL SpA5 antigen to each well in the experimental group, and add 100 μL of 1% BSA to the control group. Do three parallel wells for each group, incubate at 37°C for 1 hour, and wash it four times with PBST; The enzyme-labeled antibody SpA5-16, the original concentration of the SpA5-16 antibody is 3.40 mg / mL. Take out 100 μg, a total of 29.4 μL of the antibody is added to the HRP kit for enzyme labeling to obtain an enzyme-labeled antibody with a final volume of 200 μL. The concentration of the enzyme-labeled SpA5-16 antibody is 0.5 mg / mL. Dilute the enzyme-labeled antibody SpA5-16 to 500, 1000, 1500, 2000, 3000, 4000 times respectively, incubate at 37°C for 1 hour, wash it after incubation, add TMB chromogenic solution and develop color at 37°C for 10 minutes. Add 50 μL of 2M sulfuric acid termination solution to terminate the reaction, and detect the OD450nm and OD630nm values with an enzyme-labeled instrument to calculate OD 450-630nm value.

[0023] 2. Experimental results Judgment criteria: (1)Calculate the OD ratio of each experimental well (OD 450-630nm value) to the control well (OD 450-630nm value); (2)The ratio of the experimental well (P) to the control well (N) > 2.1, select the maximum value of P / N as the preferred combination.

[0024] Table 1 P / N values of capture antibody and detection antibody

[0025] As shown in Table 1, the preferred concentration of the capture antibody SpA5-H3 is 3 μg / mL, and the enzyme-labeled detection antibody SpA5-16 is diluted 1:1000 as the combination.

[0026] Example 2 Establishment of standard curve 1. Experimental method Coat the capture antibody SpA5-H3 at a concentration of 3 μg / mL overnight at 4°C, wash it four times with PBST, block it with 3% BSA, incubate at 37°C for 1 h, wash it four times with PBST. Dilute the SpA5 antigen with a starting concentration of 10000 YU / mL in a 2-fold dilution, incubate at 37°C for 1 hour, wash it four times with PBST. Dilute the enzyme-labeled antibody SpA5-16 at a ratio of 1:1000, incubate at 37°C for 1 hour, wash it four times with PBST, add TMB to develop color for 10 minutes, and terminate the color development with 2M sulfuric acid termination solution. Detect the OD450nm and OD630nm values with an enzyme-labeled instrument to calculate OD 450-630nmValue. Taking the logarithm of the reference product concentration with base 2 as the abscissa and the OD value as the ordinate, a linear regression equation is constructed, and the correlation coefficient is obtained.

[0027] Regarding YU: The specific activity determination of the stock solution refers to the method for determining the specific activity of the stock solution of the recombinant Escherichia coli hepatitis E vaccine recorded in "Quality Control and Evaluation of Vaccines" edited by Wang Junzhi. It is stipulated that 1 ng = 1 YU for the activity unit of the reference product stored at -80°C. Through the double antibody sandwich enzyme-linked immunosorbent assay, taking the logarithm of the reference product activity concentration (YU / mL) as the abscissa and the OD value as the ordinate, a linear regression equation is constructed. According to the standard curve equation, the activity concentration (YU / mL) of the diluted sample is calculated, and then the specific activity of the sample is calculated according to the formula. Sample specific activity = diluted sample activity concentration (YU / mL) × dilution factor / sample protein concentration (mg / mL).

[0028] 2. Experimental results Table 2 Data for establishing the standard curve

[0029] As shown in Table 2, Figure 1 when the reference product concentration is 4.88 - 78 YU / mL, R 2 = 0.9924, showing good linearity and the linearity holds.

[0030] Example 3 Optimization of the standard curve 1. Experimental method Coat the capture antibody SpA5-H3 at a concentration of 3 μg / mL overnight at 4°C, wash four times with PBST, block with 3% BSA, incubate at 37°C for 1 h, wash four times with PBST. Dilute the SpA5 standard product to 200, 100, 80, 60, 40, 20, 10, 5, 2.5, 1.25, 0 YU / mL, add 100 μL per well, incubate at 37°C for 1 hour, wash four times with PBST. Dilute the enzyme-labeled antibody SpA5-16 at 1:1000, incubate at 37°C for 1 hour, wash four times with PBST, add TMB for color development for 10 minutes, terminate the color development with 2M sulfuric acid termination solution, and detect the OD 450nm and OD 630nm values, and calculate the OD 450-630nm value. Taking the logarithm of the antigen concentration with base 2 as the abscissa and the OD value as the ordinate, a linear regression equation is constructed, and the correlation coefficient is obtained.

[0031] 2. Experimental results Table 3 Data for optimizing the standard curve

[0032] As shown in Table 3, Figure 2As shown, when the concentration of the reference product is 2.5 - 100 YU / mL, R 2 = 0.995, showing good linearity and the linearity holds.

[0033] Example 4 Method Validation 1. Specificity Validation (1) Experimental Method The recombinant Staphylococcus aureus vaccine is composed of a mixture of four antigens, HI, SpA5, MntC, and mSEB. To evaluate the specificity of the established method, a mixture of HI + MntC + mSEB proteins, PBST buffer, and the desorbed recombinant Staphylococcus aureus vaccine were used as test samples to verify the specificity of the method.

[0034] The mixture of HI + MntC + mSEB proteins, PBST buffer, and the desorbed recombinant Staphylococcus aureus vaccine were used as test samples respectively. Using the concentration range of 2.5 - 100 YU / mL of the reference product as the standard curve, the specificity validation was carried out according to the steps listed in Example 3. Each group of test samples was subjected to three parallel well experiments.

[0035] (2) Experimental Results As shown in Table 4: There was no difference in the OD 450-630nm values between the group of the mixture of HI + MntC + mSEB proteins and the PBST buffer group, and no specific activity values were detected in the experimental results. The specific activity of the desorbed Staphylococcus aureus vaccine SpA5 protein was 1.12 * 10 6 YU / mg, indicating that the method has good specificity for specifically recognizing the SpA5 antigen.

[0036] Table 4 Specificity Validation Results

[0037] 2. Intermediate Precision Validation (1) Experimental Method According to the standard curve and operating steps established in Example 3, the intermediate precision was detected. That is, for the same sample, person 1 repeated the detection 3 times in the first 3 days, and person 2 repeated the detection 3 times in the next 3 days. A total of 6 groups of data were detected by 2 people to calculate the relative standard deviation (RSD).

[0038] (2) Experimental Results The relative standard deviation was within 10% (RSD ≤ 15%), indicating that the method in the present invention has relatively high intermediate precision, as shown in Table 5.

[0039] Table 5 Intermediate Precision Validation Results

[0040] 3. Linearity Validation (1) Experimental method According to the standard curve and operation steps established in Example 3, the reference product was diluted to 100, 80, 60, 40, 20, 10, 5, 2.5, 0 YU / mL for linear verification. The logarithm of the reference product concentration with base 2 was used as the abscissa, and OD 450-630nm value was used as the ordinate to construct a function, and R 2 .

[0041] (2) Experimental results Table 6 Linear verification results

[0042] As Figure 3 shown, the Rs 2 of the six experiments were 0.9907, 0.9900, 0.9985, 0.9942, 0.9932, and 0.9904 respectively. The Rs 2 were all greater than 0.99, indicating that the method had good linearity.

[0043] 4. Accuracy verification (1) Experimental method According to the standard curve and operation steps established in Example 3, reference products of low concentration 10 YU / mL, medium concentration 50 YU / mL, and high concentration 80 YU / mL SpA5 were selected for accuracy verification. Each concentration was measured three times repeatedly, and the recovery rate of the three detections at the same concentration was calculated.

[0044] (2) Experimental results As shown in Table 7, when the double antibody sandwich ELISA method established was used to detect the accuracy of low, medium, and high SpA5 reference products, the recovery rates of the three detections at the same concentration were between 90 - 115%, indicating that the method had high accuracy.

[0045] Table 7 Accuracy verification results

[0046] Example 5 Detection of SpA5 specific activity in multi - batch vaccine products and stock solutions (1) Experimental method According to the standard curve and operation steps established in Example 3, the specific activities of three batches of vaccine products and three batches of SpA5 stock solutions were detected.

[0047] (2) Experimental results If the specific activity value of the test sample is less than 0.5 * 10 6If it is less than 10 YU / mg, the sample is determined to be unqualified. For each batch of the finished vaccine, three groups of repeated experiments are conducted and the average value is taken. The SpA5 antigen specific activity tests of three batches of the finished vaccine are all qualified, and the RSD is less than 10%, indicating that the processes of different batches of the finished product are stable, as shown in Table 8. For each batch of the stock solution sample, three groups of repeated experiments are conducted and the average value is taken. The SpA5 protein specific activity tests of three batches of the stock solution are all qualified, and the RSD is less than 10%, indicating that the processes of different batches of the stock solution are stable, as shown in Table 9.

[0048] Table 8 Test Results of SpA5 Antigen Specific Activity of the Finished Vaccine

[0049] Table 9 Test Results of SpA5 Stock Solution Antigen Specific Activity

[0050] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for quantitatively detecting the specific activity of SpA5 antigen in a recombinant Staphylococcus aureus vaccine or its stock solution, characterized in that, It includes the following steps: S1. Coat an enzyme-linked immunosorbent assay (ELISA) plate with SpA5-H3 as the capture antibody; the SpA5-H3 includes a heavy chain and a light chain, and the heavy chain and the light chain respectively contain three complementarity-determining regions (CDRs) and four framework regions (FRs). Among them, the heavy chain CDR1 is GFTFDDYA, the heavy chain CDR2 is ISWNSGSI, the heavy chain CDR3 is AKDMSPWPDAFDI, the heavy chain FR1 is KVQLLESGGGLVQPGRSLRLSCAAS, the heavy chain FR2 is MHWVRQAPGKGLEWVSG, the heavy chain FR3 is GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC, the heavy chain FR4 is WGQGTMVTVSS; the light chain CDR1 is SSNIGNNY, the light chain CDR2 is RN, the light chain CDR3 is AAWDDSLRGPV, the light chain FR1 is ELVLTQPPSASGTPGQRVTISCSGS, the light chain FR2 is VYWYQQLPGTAPKLLIY, the light chain FR3 is NQRPSGVPDRFSGSKSGTSASLAISGPRSEDEADYYC, the light chain FR4 is FGGGTKVTVLGQP; S2. Add the sample to be measured into the coated ELISA plate; S3. Incubate with the enzyme-labeled antibody SpA5-16, develop color using the TMB chromogenic solution, detect the OD values at 450 nm and 630 nm respectively with an enzyme-labeled instrument, and calculate the OD 450-630nm value; the SpA5-16 includes a heavy chain and a light chain, the heavy chain and the light chain respectively contain three CDRs and four FRs, wherein the heavy chain CDR1 is GFTFSSYS, the heavy chain CDR2 is ISSSSSYI, the heavy chain CDR3 is ARDMITFGGVPVNGMDV, the heavy chain FR1 is QVQLQESGGGLVQPGGSLRLSCAASGFTFS, the heavy chain FR2 is WVRQAPGKGLEWVS, the heavy chain FR3 is RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR, the heavy chain FR4 is WGQGTTVTVSS; the light chain CDR1 is SSNIGDNT, the light chain CDR2 is YD, the light chain CDR3 is AAWDDSLNGWV, the light chain FR1 is ELVLTQPPSASGTPGQRVTISC, the light chain FR2 is WYQHLPGKPPKLIIY, the light chain FR3 is DLVATGVPDRFSGSKSGTSASLAISGLQSEDEADYYCYYC, the light chain FR4 is FGGGTELTVLGQP; S4. Use the SpA5 reference product to draw a standard curve, and calculate the specific activity of the SpA5 antigen in the sample to be measured according to the standard curve.

2. The method for quantitatively detecting the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, After coating the ELISA plate in S1, refrigerate it overnight, wash the plate four times with PBST, incubate it with 3% BSA blocking solution at 37 °C for 1 hour, and wash the plate four times with PBST.

3. The method for quantitatively detecting the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, After adding the sample to be measured into the ELISA plate in S2, incubate it at 37 °C for 1 hour, and wash the plate four times with PBST.

4. The method for quantitatively detecting the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, After adding the enzyme-labeled antibody SpA5-16 into the ELISA plate in S3, incubate it at 37 °C for 1 hour, wash the plate four times with PBST, then add the TMB chromogenic solution for color development for 10 minutes, and add 2 M sulfuric acid termination solution after color development.

5. The method for quantitatively detecting the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or the stock solution according to any one of claims 2 to 4, characterized in that, The PBST is composed of 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, and 0.05% v / v Tween-20.

6. The method for quantitatively detecting the specific activity of SpA5 antigen in the recombinant Staphylococcus aureus vaccine or stock solution according to claim 1, characterized in that, In S4, the standard curve is constructed by taking the logarithm of the antigen concentration as the abscissa and the OD value as the ordinate to obtain a linear regression equation.

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