Development and verification of double antibody sandwich ELISA method for determination of antigen content of recombinant SARS-CoV-2 vaccine based on spike glycoprotein receptor-binding domain
Objective To develop and verify a double antibody sandwich ELISA method for determination of the antigen content of recombinant SARS-CoV-2 vaccine based on the receptor-binding domain(RBD)of spike glycoprotein(S protein).Methods A double antibody sandwich ELISA was developed with GH4 human monoclonal antibody as coating antibody and CB6 human monoclonal antibody(CB6-HRP)labeled with HRP enzyme as detection antibody.The working concentrations of GH4(500,1 000 and 2 000 ng/mL)and CB6-HRP(3.906 25-500 ng/mL)were determined,and the linear range,accu-racy,precision,specificity and durability of the method were verified.The antigen content of three batches of recombinant SARS-CoV-2 vaccines(CHO cells)were detected by the developed method.Results The working concentrations of GH4 and CB6-HRP were 1 000 and 31.25 ng/mL,respectively.In the range of 0.488-125 ng/mL,the reference sample of stock solution showed agood linearrelationship with A450,and the values of R2 were not less than 0.99;The recoveries of accuracy veri-fication were between 87.2％and 120.5％;The relative standard deviations(RSDs)of repeatability and intermediate precision verification were not more than 20.0％;The error rate of specificity verification ranged from-16.6％to 2.1％;The RSDs of durability verification were all not more than 20.0％.The antigen content of three batches of vaccine stock solu-tion was 638.9,592.4 and 664.8 ng/mL,respectively,with the RSD of 5.8％.Conclusion The double antibody sand-wich ELISA based on S protein RBD has good accuracy,precision,specificity and durability,and can be used to detect the antigen content of recombinant SARS-CoV-2 vaccine,which lays a foundation of the quality control of the vaccine.
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