| Infectious bronchitis(IB) is an acute, highly contagious r espiratory disease that caused by Infectious bronchitis virus(IBV), which has brought serious economic losses to the poultry industry. As RNA virus, IBV has the characterization of constant mutation and recombination and has many kinds of serotypes. This makes the diagnosis of IB become more and more difficult. A sensitive and effective method detecting IBV has vital significance for the development of targeted comprehensive prevention and control program.In this study, the 6-8 week-old female BALB/c mice were immunized with purified IBV HH06 strain. By cell fusion technique, indect ELISA detection and limited dilution method, three hybridoma cell lines that stably secreted IBV McAbs were obtained and named as A8, B11 and H5 respectively. The ascites was produced by inoculation of BALB/c mice intraperitoneally. The ELISA titers of Mc Abs in ascites were higher than those of culture supernatants of hybridoma cells, which were 1: 8 192-1: 65 356 versus 1: 128-1: 1 024, respectively. In western blot, the specific band between 70-100 Ku was showed and this indicated the McAbs reacted with IBV, and more interesting thing was that the all three Mc Abs could specifically react with recombinant S1 protein of IBV. IFA test demonstrated that these Mc Abs could react with IBV-infected CEK cells. The subtypes of A8, B11 and H5 were Ig M. The average chromosome number of the hybridoma cells was 86-101. End-point neutralizing assay performed in chicken embro kidney(CEK) cells revealed that the neutralization titer of A8, B11 and H5 against IBV HH06 reached 1:4.45, 1:4.86 and 1:3.06, respectively. Three Mc Abs specifically reacted with IBV, and not with avian infectious laryngotracheitis virus(ILTV), infectious bursal disease virus(IBDV), avian influenza virus(AIV), avian leukosis virus(ALV) and Newcastle disease virus(NDV). This laid the foundation for the further development of diagnostic mthod for IB.Antigen capture ELISA is a rapid and efficient antigen detection method. An AC-ELISA was established for the detection of IBV, in which polyclonal antibody was used as capturing antibody and mouse-anti IBV Mc Ab of B11 as detecting antibody. Based on the optimization of every condition, the optimal conditions were determined: anti-IBV polyclonal antibody dilution as 1:4 000, the optimal working concentration of B11 Mc Ab was 5 μg/mL, and the enzyme-labeled antibody was diluted for 1:5 000. The coefficient of variation of reproducibility was less than 10%, and at least 0.0067 mg/m L IBV could be detectable. The result of ELISA showed no cross-reactions with other reference avian-derived virus. The established AC-ELISA was applied to detect IBV in kidney, lung and trachea of IBV M41-infected chickens at different time post infection. The results showed that the IBV titer and duration time in kidney was higher and longer compared with those in lung and trachea. The kidney could be selected as target organ when clinical samples were assayed. Because of the tissue tropism of IBV, trachea and lungs could be selected as auxiliary examination organs at the same time.In summary, the AC-ELISA developed in this study has good specificity and high sensitivity, and is a suitable method for rapid detection of IBV. |
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