| Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), which can lead to great losses of poultry industry in China. Numerous IBV serotypes have been described in previous studies, more and more newly emerging serotypes are being identified gradually. As demonstrated in those studies, different IBV serotypes have no immune cross protection or only partial cross protection. At present, the poultry respiratory diseases in our country are complicated. It is hard to distinguish them by atypical clinical symptoms. In case of mixed infection, differential diagnosis gets even harder. The traditional diagnostic methods for IB are regularly based on the isolation and identification of IBV, more sensitive methods (serology and molecular assays) are also applied for rapid detection. However, the methods mentioned above are often complicated and time-consuming and must be performed by the means of professional lab assistants and valuable equipments. To meet the needs for accurate, sensitive and specific detection of IBV, a rapid immunochromatographic assay was developed to detect the IBV antigens within 10 min. The colloidal gold-based immunochromatographic strips are easily made and performed for detection tests.IBV N-protein appears conserved in evolution and contains a large number of epitopes, which is able to induce cross-protective antibodies. Despite being a little less immunogenic than S protein, N-protein can be developed to detect different IBV serotypes for early infection relying to group-specific antibodies against N protein.In the present study,25nm colloidal gold beads were primarily prepared with good uniformity in density using citrate reduction reaction assay. Then, the monoclonal antibodies of V1 and V5 against IBV-N protein and goat anti-mouse IgG were purified to meet the needs for making sensitive and specific detection strips. Finally, the optimal pH value was tested to be 8.0 and the optimal application amount of monoclonal antibody was 8.4μg/mL. The coating concentrations of monoclonal antibody V5 and the goat anti-mouse IgG and jetting rate were also determined. Then, the NC membrane was coated with anti-IBV-NP monoclonal antibody V5 as the test line (T) and goat anti mouse IgG as the control line (C). The test strips were finally successfully constructed with high sensitivity, specificity and repeatability. Specificity tests showed that our strips had no cross-reaction with AIV, NDV, IBDV, ILTV and EDSV-76. Our strips can simultaneously detect the various IBV isolates including strains H-120 4-91, Ma5 and M41. Sensitivity tests on IBV strain 4-91 indicated the detection limit of EID50=102. Storage conditions of strip detection kit were also determined with the temperature of 4℃and the duration of 5 months.To compare the detection limits of our test strips and ELISA assay, we simultaneously used both of the assays to detect the samples of throat swab and cloacal swab. The positive rate of our test strips was tested to be 86.67%, and ELISA assay was 80.00%. Our strip assay appeared to be more sensitive than ELISA assay, and the coincidence rate was 93.33%.In our study, the colloidal gold-based immunochromatographic strip assay was successfully established, which provides an excellent tool for the early diagnosis and prevention of IBV infection. |
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