Development and verification of a double antibody sandwich ELISA for quantitative detection of recombinant trivalent poliomyelitis vaccine antigen
10.13200/j.cnki.cjb.004672
- VernacularTitle:重组三价脊髓灰质炎疫苗抗原双抗体夹心ELISA定量检测方法的建立及验证
- Author:
Fangfang SHA
1
Author Information
1. CanSino Biologics Inc., Tianjin 300301, China
- Publication Type:Journal Article
- Keywords:
Poliomyelitis vaccine;
Poliovirus(PV);
Double antibody sandwich ELISA;
Virus-like particles(VLPs)
- From:
Chinese Journal of Biologicals
2026;39(03):331-341+349
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo develop a double antibody sandwich ELISA method for the detection of types Ⅰ, Ⅱ and Ⅲ antigen content of recombinant trivalent poliomyelitis vaccine, and to optimize, verify and preliminarily apply the method, in order to provide a quality control method for vaccine development.MethodsMale New Zealand white rabbits were immunized with types Ⅰ, Ⅱ and Ⅲ recombinant poliomyelitis vaccine antigens, and the corresponding polyclonal antibodies were prepared.The polyclonal antibodies were used as coating antibodies and HRP-labeled antibodies as detection antibodies to establish a double antibody sandwich ELISA method for detecting the content of three types of antigens in recombinant trivalent poliomyelitis vaccine. The checkerboard titration method was used to determine the coating antibody concentration and the detection antibody dilution. The single factor experiments were used to optimize the types of blocking solution, antibody lyophilization time, enzyme-labeled antibody diluents and chromogenic solution formulations. The established method was verified for linear range, accuracy, specificity, precision, lower limit of quantification, robustness and stability, and was used to detect the content of types Ⅰ, Ⅱ and Ⅲ antigens in recombinant trivalent poliovirus vaccine.ResultsThe optimal coating antibody concentration was 5 μg/mL, and the optimal dilutions of enzyme-labeled antibodies were 4 000, 9 000 and 5 000, respectively,for types Ⅰ, Ⅱ and Ⅲ antigens. The optimal conditions were as follows: blocking solution of 1% BSA solution, lyophilization time of 2 h, enzyme-labeled antibody dilution of 1% BSA + 1% sucrose + 1% trehalose + 0. 01% sodium thimerosal, and chromogenic solution of recipe 2 [Solution A: 13. 6 g sodium acetate, 1. 6 g citric acid, 0. 3 mL of 30% hydrogen peroxide,adding distilled water to a total volume of 500 mL. Solution B: 0. 2 g disodium ethylenediaminetetraacetate, 0. 95 g citric acid, 50 mL glycerol, 0. 15 g TMB(dissolved in DMSO before slowly adding to distilled water), adding distilled water to a total volume of 500 mL]. TypesⅠ and Ⅱ antigens showed a good linear relationship with A_(450)in the concentration range of0. 78-25 DU/mL, and type Ⅲ antigen exhibited a good linear relationship with A_(450)in the concentration range of 1. 56-50 DU/mL,each R~2> 0. 99. The recovery rates of spiked samples at high, medium and low concentration of the three types of antigen content detection methods were all between 80% and 120%. All three types of antigen detection methods detected their corresponding specific antigens, and there was no cross-reaction with the other two antigens. The lower limits of quantification of types Ⅰ, Ⅱ and Ⅲ antigen detection methods were 1. 56, 1. 56 and 3. 13 DU/mL, respectively. The CVs of precision and robustness verification were both less than 20%. The antibody-coated plate, detection antibody working solution and chromogenic solution were stored stably at 4 ℃ for six months, and the recovery rates of the three types of antigens were all within the range of 80%-120%. The CVs of harvest solution, clarified solution, concentrated solution, ion exchange chromatography solution, recovery solution and bulk solution samples in the vaccine process were all not more than 15% by the established method.ConclusionThe established double antibody sandwich ELISA quantitative detection method has good specificity,accuracy, precision, robustness and stability, and can be used to detect the content of typesⅠ, Ⅱ and Ⅲ antigens in the development of recombinant trivalent poliovirus vaccines.
