Development,verification and application of double antibody sandwich ELISA based on neutralizing antibody for determination of toxoid content in diphtheria vaccine
Objective To develop a double antibody sandwich ELISA method with neutralizing antibody for the determina-tion of content of functional antigen diphtheria toxoid(DTd),and to verify and preliminarily apply it to the quality analysis in the DTd production.Methods The concentration of anti-diphtheria toxin(DTx)coating antibody and enzyme-labeled anti-body in double antibody sandwich ELISA was determined by square titration,and a linear standard curve was established.The method was verified according to the requirements of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition),and was prelimi-narily applied to the detection of DTd stock solution.Results The optimal concentration of coating antibody was 5 µg/mL,the working dilution of enzyme-labeled antibody was 1:5 000 and the best linearity of dose-response curve was in a range of 0.000 781-0.012 5 Lf/mL(r＞0.99).This method showed no cross reaction with pertussis toxoid(PTd),filamentous haemagglutinin(FHA),pertactin(PRN),tetanus toxoid(TTd)and Sabin-strain inactivated poliomyelitis vaccine(sIPV)Ⅰ,Ⅱ,Ⅲ.For the DTd standard in the linear range,the coefficients of variation(CVs)of precision verification were 4.23％,2.98％,1.81％,6.61％and 1.82％,and the recoveries of accuracy verification were 90.67％,105.39％,102.11％,97.76％and 81.31％,respectively.The Pearson r was 0.638 0(P＜0.05)for the determination of toxoid content in 12 batches of DTd stock solution by this method and flocculation test.For the concentration of diphtheria toxoid less than 1 800 Lf/mL,the Pearson r was 0.899 2(P＜0.001).Conclusion A double antibody sandwich ELISA method for quantification of DTd antigen based on neutralizing antibody was successfully developed with good specificity,accuracy and precision,which provides an effective verification means for the production of DTd stock solution.
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