兽用狂犬病灭活疫苗效力评价的双抗体夹心ELISA方法的建立与应用OA

This study successfully established a double-antibody sandwich ELISA method based on monoclonal antibodies against rabies virus(RABV)glycoprotein(G protein)for the rapid and quantitative detection of potency in veterinary inactivated rabies vaccines,aiming to replace the traditional NIH mouse challenge test.By systematically optimizing key parameters including the concentrations of capture and detection antibodies,coating time,blocking conditions,antigen-antibody reaction time,and chromogenic time,a stable and reliable detection system was constructed.The experimental results demonstrated excellent analytical performance of the method:the standard curve exhibited good linearity(R2=0.9929),high sensitivity(limit of detection=0.0035 IU/mL),and no crossreactivity with common canine pathogens such as canine parvovirus,canine parainfluenza virus,canine adenovirus,and canine distemper virus.Both intraassay and interassay coefficients of variation were below 10%,and the average recovery rate was 96.1%.Comparison with the NIH method showed good agreement between ELISA and NIH results across seven vaccine samples(The relative deviation between the ELISA and NIH results was within±20%).Bland-Altman analysis indicated an overall bias of 0.3257 IU/mL,with 95%limits of agreement from 1.140 to 0.4882 IU/mL,confirming that the method reliably reflects vaccine immunogenicity.In conclusion,the established doubleantibody sandwich ELISA method is rapid,sensitive,specific,and reproducible.It can serve as a potential alternative or an important supplementary in vitro assay for potency evaluation of veterinary inactivated rabies vaccines,providing new technical support for vaccine quality control and facilitating the implementation of the "3R" principles.