Development and verification of indirect immunofluorescence assay for detection of baculovirus titer
10.3760/cma.j.issn.0254-5101.2018.09.009
- VernacularTitle:免疫荧光法检测杆状病毒滴度的建立及验证
- Author:
Zhandong ZHANG
1
;
Yuanyuan LI
;
Qingsheng WU
;
Chunping YAO
;
Yichi ZHANG
Author Information
1. 1101111,北京生物制品研究所有限责任公司第七研究室
- Keywords:
Baculovirus;
Virus titer;
Indirect immunofluorescence assay ( IFA)
- From:
Chinese Journal of Microbiology and Immunology
2018;38(9):702-709
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop, optimize and preliminarily verify an indirect immunofluores-cence 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 es-tablished assay were verified. And the results were compared with those of baculovirus rapid titer kit. Re-sults The optimal cell concentration for coating was 0. 6×106 cell/ml, and the optimal reaction time be-tween 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. Spe-cific 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 reproduc-ibility and intermediate precision were less than 10％. Conclusion IFA for the detection of baculovirus ti-ter was established with good specificity, accuracy, reproducibility and intermediate precision.
