Development, optimization and verification of double antibody sandwich ELISA for antigen content detection in recombinant pneumococcal protein vaccine
10.13200/j.cnki.cjb.004409
- VernacularTitle:重组肺炎球菌蛋白疫苗抗原含量双抗体夹心ELISA检测方法的建立、优化及验证
- Author:
SHA Fangfang
- Publication Type:Journal Article
- Keywords:
Recombinant pneumococcal protein vaccine;
Pneumococcal surface protein A(PspA);
Double antibody sandwich ELISA;
Antigen content
- From:
Chinese Journal of Biologicals
2025;38(01):80-88
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop double antibody sandwich ELISA methods for the determination of P3296, P5668 and PRX1 antigen components in recombinant pneumococcal protein vaccine based on pneumococcal surface protein A(PspA), and to optimize,verify and preliminary apply it, in order to provide a reliable detection method for the quality monitoring of the vaccine.Methods The male New Zealand white rabbits were immunized with P5668, P3296 and PRX1 purified proteins. The immunized serum was purified by Protein A-Sephaorse 4B affinity chromatography, and P5668, P3296 and PRX1 polyclonal antibodies were obtained. Using the polyclonal antibodies as the coating antibodies and the corresponding HRP-labeled monoclonal antibodies as the enzyme-labeled antibodies, the double antibody sandwich ELISA methods were developed. The concentration of coating antibodies(all three polyclonal antibodies diluted to 2, 4 and 8 μg/mL), the dilution of enzyme-labeled antibodies(HRP-labeled P5668 monoclonal antibody diluted at 1∶4 000 and 1∶8 000, HRP-labeled P3296 monoclonal antibody diluted at 1∶40 000 and 1∶60 000, HRP-labeled PRX1 monoclonal antibody diluted at 1∶12 000 and 1∶24 000),the sealing liquid type(1% BSA, 1% fish skin gelatin, 1% skimmed milk powder and 1% casein), the diluent type(purified water, 1 × PBS, 2 × PBS), and the diluent pH(6. 4, 7. 4, 8. 4) were optimized. The linear range, specificity, accuracy, precision, and robustness of the methods were verified. The developed methods and Lowry method were used to detect purified proteins of P5668, P3296 and PRX1(20 batches each), and the correlation between the results of the methods was analyzed. Three batches of P5668, P3296 and PRX1 mixed vaccines were desorbed by propanesulfonic acid internal salt, and the antigen contents of P3296, P5668 and PRX1 were detected by the developed methods. Results The protein concentrations of purified P5668,P3296 and PRX1 polyclonal antibodies were 1. 27, 2. 20 and 1. 53 mg/mL, respectively. The optimal coating concentration of P3296, P5668 and PRX1 polyclonal antibodies was all 4 μg/mL, and the optimal dilution of HRP-labeled P5668, P3296,and PRX1 monoclonal antibodies was 1∶4 000, 1∶60 000, and 1∶12 000, respectively. The optimal sealing liquid was 1% BSA, the diluent was 1 × PBS, and the diluent pH was 7. 4. Three reference materials of P5668, P3296 and PRX1 in the range of 3. 125-100 ng/mL showed a good linear relationship with A_(450), with R~2 values more than 0. 99. The spike recovery rates of three antigens with high, medium and low concentration were all in the range of 80%-120%. All the three methods detected the corresponding specific antigen, with no cross-reaction to the other two antigen proteins. The CVs of repeatability and intermediate precision verification were less than 20%, and the CVs of detection results of the same sample under different conditions were also less than 20%. The R of ELISA and Lowry methods for the determination of P5668, P3296 and PRX1 antigens was 0. 984 6, 0. 997 0 and 0. 990 9(each P < 0. 000 1), respectively, and the Lowry method exhibited a positive correlation with the developed methods. Three batches of P3296, P5668 and PRX1 mixed vaccines were detected for the antigen contents by the developed method, and the coincidence rate between the results and theoretical values was 87%-114%.Conclusion The developed double antibody sandwich ELISA methods have good specificity, precision, accuracy and robustness, and can be used for the determination of P3296, P5668 and PRX1 antigens in recombinant pneumococcal protein vaccines.
