Development and verification of indirect immunofluorescence assay for detection of baculovirus titer

Objective To develop, optimize and preliminarily verify an indirect immunofluorescence assay (IFA) for detecting the titer of recombinant baculovirus. Methods Conditions for performing IFA, including cell concentration, co-incubation time, reaction temperature, dilution ratio, reaction time and types of fixative solution, blocking liquid and antibodies, were optimized to establish an IFA method for the detection of baculovirus titer. Specificity, accuracy, reproducibility and intermediate precision of the established assay were verified. And the results were compared with those of baculovirus rapid titer kit. Results The optimal cell concentration for coating was 0.6×106 cell/ml, and the optimal reaction time between viruses with cells was 3 d. The optimal conditions for conducting IFA were as follows: formaldehyde buffered acetone (-20℃) was used as fixative, normal goat serum was used as blocking liquid and the first and second generation antibodies at a dilution of 1∶200 were incubated at 37℃ for 1 h, respectively. Specific fluorescence was observed only in baculovirus but not in others by using the method. No significant difference in virus titers was observed between the established IFA and baculovirus rapid titer kit. The two methods showed a good linear correlation (R2=0.996). Coefficients of variation for evaluating the reproducibility and intermediate precision were less than 10%. Conclusion IFA for the detection of baculovirus titer was established with good specificity, accuracy, reproducibility and intermediate precision. Key words: Baculovirus; Virus titer; Indirect immunofluorescence assay (IFA)