| For the sake of high nutrition of quail and its egg contains rich protein, nowadays, the breeding scale of quail just follow chicken’s it also has. At same time, various diseases of quail impeded the development of the poultry industry and caused a huge economic loss. So it is important of the prevention, monitoring and control these diseases. For diagnosis, multiple methods are required, especially the detection of antibody levels of various pathogens.70%to80%antibody in serum is IgG. It plays a dominant role in the resistance to the invasion of different pathogen. The certain IgG against the certain pathogen in the serum is the commonly used for clinical diagnosis and research. Commercial antibody detection kits are mainly used to detect chicken’s diseases; however, there is no report about monoclonal antibody against quail IgG. In this study we developed anti-quail IgG monoclonal antibody and we established three methods to detect antibody against NDV in quail serum based on this antibody, i.e. IFA, indirect-ELISA and a test strip. The results showed that both the sensitivity and specificity of the monoclonal antibody (McAb) was good. Thus, it is probable to develop quail diseases detection kits with the McAb.1.Parperation and identification of McAbs against quail IgGThe IgG protein which was purified from quail serum by saturated ammonium sulfate method immunized Balb/c mice three times. Then, the spleen cells from Balb/c mouse were fused with SP2/0myeloma cells. According to the results of indirect-ELISA, the positive hybridoma cells were subcloned twice by limited dilution method. At last, four hybridoma cell lines secreting anti-quail IgG McAbs were selected and named as QIgG-5F11, QIgG-5G2, QIgG-5G6and QIgG-3D4. By analysis with a commercial capture-ELISA kit, the subtypes of QIgG-5F11and QIgG-5G2were IgG1,the QIgG-5G6and QIgG-3D4were IgM, all the McAbs’light chain type were κ. The titers of the supernatants of the four McAbs were1:2048,1:4096,1:2048and1:1024respectively, and the titers of ascites were1:409600,1:204800,1:409600and1:102400. The results of Western-blot indicated that QIgG-5F11and QIgG-5G2recognized the light chain of quail IgG while QIgG-5G6and QIgG-3D4recognized the heavy chain. The cross-reaction of QIgG-5F11and QIgG-5G2was analysed by indirect-ELISA and Western-blot. The result showed that QIgG-5F11and QIgG-5G2just specifically reacted with quail IgG and no cross-reactivity with any other animals IgG. The above experimental results suggested that QIgG-5F11and QIgG-5G2had good specificity. In brief, both of the McAbs could be candidates of universal secondary antibodies for development detection kit.2. Application of the McAbs against quail IgGIn this study, QIgG-5F11was chose for application research. Three methods for detection NDV antibody in quail serum were established. They were IFA, indirect-ELISA and test strip. CEF were infected with LaSota and fixed as the antigen of IFA, quail serum was used as first antibody, McAb QIgG-5F11was used as the second antibody and FITC labeled goat anti-mouse IgG as third antibody. Indirect-ELISA was established and the purified NDV was used as coated antibody, quail serum as first-antibody, QIgG-5F11conjugated with HRP as secondary-antibody. This study also established a test strip in which the colloidal gold labeled QIgG-5F11monoclonal antibody was dispensed onto the conjugate pad, the purified NDV and rabbit serum anti-mouse IgG were dispensed on the bottom of the NC membrane as test line and control line. The results of the three detection methods were compared with HI. The coincidence rates of the three methods were69.8%,94.1%and95.8%respectively, which indicated that these methods have been achieved the desired effect. Through the establishment and application of the three methods QIgG-5F11was proved to be feasible to be used as secondary antibody. In this study we just established test method to detect antibody against NDV, we could also establish detection methods to detect antibody against other pathogen. |
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