Establishment and evaluation of DAS-ELISA for detecting bovine respiratory syncytial virus
10.3969/j.issn.1002-2694.2017.07.011
- VernacularTitle:牛呼吸道合胞体病毒双抗体夹心ELISA检测方法的建立
- Author:
Yanting LI
;
Xilin HOU
- Keywords:
bovine respiratory syncytial virus;
G protein;
polyclonal antibody;
monoclonal antibody;
DAS-ELISA
- From:
Chinese Journal of Zoonoses
2017;33(7):628-636
- CountryChina
- Language:Chinese
-
Abstract:
In order to develop a double antibody sandwich assay (DAS-ELISA) for detecting bovine respiratory syncytial virus (BRSV),New Zealand white rabbits and BALB/c mice were immunized with the purified G protein as an antigen to prepare anti-G protein polyclonal and monoclonal antibodies.The antibody concentration and reaction conditions of DAS-ELISA were optimized by square titration,and its sensitivity,specificity,and coincidence rate were validated.Five hybridoma were stably secreting Mab which subclass belonged to IgG1κ.Western blot and IFA test showed that PcAb and Mab could react specifically with G protein and BRSV.The PcAb and Mab as the capture antibody and detection antibody respectively,and their optimal working concentrations were determined to be 2.5 μg/mL and 10μg/mL,the critical value 0.22 and the detection limit of 1.43 μg/mL,batch,inter-assay coefficient of variation less than 10 ％.The DAS-ELISA had no cross-reaction with several pathogens which often caused bovine respiratory disease.When 45 nasal swabs of clinical samples were simultaneously detected by the DAS-ELISA and RT-PCR,the sensitivity,specificity and coincidence rate were 92.0 ％,100 ％,95.6 ％,respectively.It' s indicated that the established DAS-ELISA detection method can be used to detect a large number of clinical samples.It was the foundation of monitoring and quick diagnosis for BRSV.
