Development of uniform antigen content detection kit for recombinant SARS-CoV-2 protein vaccines / 中国生物制品学杂志

ABSTRACT

@#Objective To prepare and verify a uniform antigen content detection kit for recombinant protein vaccines against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). Methods Using goat anti-S protein polyclonal antibody prepared by National Institutes for Food and Drug Control(NIFDC) as coating antibody,one of four monoclonal antibodies(14C8,15F9,17A7 and 20D8)with high receptor-binding domain(RBD)binding activity and broad-spectrum resistance against major variants as HRP-labeled antibody,a uniform double-antibody sandwich ELISA kit for antigen content detection of recombinant SARS-CoV-2 vaccine was prepared,and the dilution of coating antibody(1∶125 ～ 1∶4 000)and enzymelabeled antibody(1∶250 ～ 1∶32 000)were optimized by chessboard titration. The specificity,linear range,accuracy,precision and durability of the kit were verified. The prepared uniform detection kits were distributed to 12 laboratories to detect15 batches of recombinant SARS-CoV-2 protein vaccine stock solutions(including 11 batches of stock solutions designed with WT strain and 4 batches of designed with Beta,Gamma and Delta variants as reference sequence)from different expression systems(CHO cells,Pichia pastoris,Sf9 cells or E. coli)and target proteins(RBD or S protein)prepared by each laboratory.Results Monoclonal antibody 20D8 was used as the enzyme-labeled antibody with the optimal dilution of 1 4 000,and the optimal dilution of coating antibody was 1 500. The uniform detection kit showed no cross reaction with recombinant S protein of severe acute respiratory syndrome(SARS)and Middle East respiratory syndrome(MERS). The first generation national standard for recombinant SARS-CoV-2 protein vaccine antigen(national standard for short)showed a concentration of 0. 16 ～ 2. 50 U/mL with a good linear relationship with A450/630,and the linear equation wasy = 0. 791 x-0. 100 4,R2= 0. 993 7;The recoveries of 0. 16 ～ 2. 50 U/mL national standard in 6 repeated tests were 95% ～ 104% and the coefficients of variation(CVs)were less than 15%;The CVs in 3 repeated tests by 2 experimenters at different time were 4. 4% ～6. 6% and the recoveries were in the range of 80% ～ 120% under different temperature and time conditions. The antigen content of 15 batches of recombinant SARS-CoV-2 protein vaccine stock solutions showed good parallelism with the national standard. Conclusion The uniform detection kit for antigen content developed in this study had good specificity,accuracy,precision and durability,and might be used for the detection of antigen content of recombinant SARS-CoV-2 protein vaccines.