Research Of Indirect Immunofluorescence Assay For Rotavirus

Currently, the only licensed rotavirus vaccine in China was developed by Lanzhou Institute of Biological Products Co. Ltd. in 2000. The rotavirus vaccine was detected with the log 50% cell culture infectious dose–enzyme-linked immunosorbent assay(lgCCID₅₀–ELISA), but experiments show that this method is limited on the time spent and experimental repeatability in quantifying rotaviral titers. In this experiment, we established an indirect immunofluorescence assay（IFA） to determine the infectious titer of Lanzhou lamb rotavirus（LLR） vaccine produced by Lanzhou Institute of Biological Products Co. Ltd, and we also compared the IFA and lgCCID₅₀–ELISA methods for assaying rotavirus vaccines.This article focus on optimizing the experimental conditions for IFA, including the optimal cell density for MA104 cells, the optimal trypsin concentration required to activate rotavirous, the best time to fluorescently stain the infected MA104 cells and so on. The research we find the optimum conditions of IFA was as follows: the cell density was 2 ′ 104 cells/well; Dilute the sample use virus diluents which comprise 1 μg/ml trypsin; The best time of virus infection for 18h; the 80% cold acetone as the stationary liquid and a fixation time of 10min; The working concentration of a rabbit anti-SA11 polyclonal antibody as the primary antibody and Alexa-488 labeled the goat anti-rabbit antibody as the secondary antibody were 1:800 and 1:500, and the time for bonding were all 60 min. Through exploring the optimum conditions of IFA, the test was established for assaying the titer of RV.Using IFA, it’s easy to detect a sample when the viral titer was diluted from 1 ′ 106 FCFU/ml to 10 FCFU/ml. This result showed that IFA was sufficiently sensitive to detect rotavirus at 10 FCFU/ml; The results were positive in MA104 cells infected with RV and were negative in MA104 cells infected with rabies virus, influenza virus, varicella virus. It showed that the IFA was specificity; To determine the reproducibility of IFA, the experiment using Lanzhou lamb rotavirus vaccine was repeated nine times. The coefficient of variation（CV） for the viral titers of the rotavirus vaccine was 2.3%. In summary, it is evident that this IFA is a sensitive, specific and repeatable method for the detection of RV in MA104 cells. This method provided a reliable basis for the qualitative and quantitative analysis of rotavirus and certifying the production of rotavirus vaccine.