Development and application of in vitro antiviral neutralizing activity detection method based on Vero cell line stably expressing firefly luciferase
Objective To develop a high-throughput detection method based on Vero cell line stably expressing firefly luciferase(flue)to evaluate the antiviral neutralizing activity of samples in vitro.Methods Based on CRISPR/Cas9 targeted gene editing technology,a Vero cell line stably expressing fluc was constructed,and a cell viability detection method for poxvirus and respiratory syncytial virus(RSV)infection was developed.This method was used to evaluate the neutralizing activity of serum immunized by vaccinia virus and RSV monoclonal antibodies,and the result was compared with that by plaque suppression method and cell viability(adenosine triphosphate,ATP)assay.Results Vero-fluc cell line was successfully constructed,and there was a good correlation between the cell quantity and the expression of fluc(R2=0.994 8).Vero-fluc cells were used as infected cells to test the neutralizing activity of poxvirus,which had good stability and reliability[signal/background ratio(S/B)=25],and met the requirements of high-throughput detection(Z'=0.9).Compared with traditional plaque suppression method,the consistency of this method was stronger(R2=0.76),and the ID50 value of immune serum was twice higher than that of plaque suppression method on average.The neutralizing activity of anti-RSV monoclonal antibodies detected by the developed method was consistent with that by ATP activity kit.The inhi-bition rate of Vero-fluc cell method was significantly higher than that of ATP method at the titer level of 1∶103 to 1∶105.Conclusion The antiviral neutralizing activity detection method based on Vero-fluc cells has good correlation with tradi-tional methods,and has higher sensitivity and detection throughput with reliable results,which is a universal in vitro antiviral neutralizing activity evaluation method.