Studies On Development And Application Of Method For Rapid Detection Of Antibody Against Avian Main Viral Diseases

Avian influenza (AI), Newcastle disease (ND), avian infectious bronchitis (IB), infectious bursal (IBD) and Mycoplasma gallisepticum (MG) are important respiratory diseases harmful to poultry industry, which could lead to death of chicken, degradation of performance, and great economic losses of poultry industry in this country. Currently, vaccination is still the primary ways of disease prevention, but the outbreaks of chicken respiratory diseases still occur frequently because of extensive areas and diversified raising model. This may be a pathogenic mutation, vaccines could not resist the infection of variant strains, also caused by the immune failure, or antibody titers of the chickens vaccinated could not cope with infection. A quick and easy method is required to monitor antibody titers in chickens vaccinated, because the HI test, Agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) is also time-consuming and requires trained personnel and special equipments. Although the serum plate agglutination test and latex agglutination test easy to be used, but there is a certain lack of the reading results.Colloidal gold immunochromatography is a new immunochromatographic technique in which a cellulose membrane used as the carrier, and a colloidal gold conjugented antigen or antibody used as the tracer, using the principle of capillary action. It's simple, rapid and cheap the result of which is clear and easy to analyze without any equipment/facilities and skilled technicians. Since it is very suitable for spot, rapid detection and automatic detection, the method has been widely used for rapid detection in human medicine, used for rapid detection in veterinary medical analysis presently.In view of the shortage of the present methods used in detecting respiratory disease antibody in poultry, a technology system for rapid detecting antibody of respiratory disease in poultry were drawn up using the principle and technology of colloidal gold immunochromatographic assay. This study focused on developing and evaluating the method for rapidly detecting antibody of avian influenza virus, Newcastle disease virus, infectious bronchitis, infectious bursal bisease virus, mycoplasma gallisepticum。The results of research as follow.1. AIV-HA1, NDV-HN, IBV-N, IBDV-VP2, and MG-V1hA1.2 expression and purification of recombinant proteinUsing the expression vector with the GST label, high level expression of AIV-HA1, NDV-HN, IBV-N, IBDV-VP2 and MG-V1hA1.2 recombinant protein was developted. Purified recombinant protein obtain by affinity chromatography were proved to be suitable for preparation of strips. The results of western blot indicate these five recombinant proteins have ideal immunocompetence.2. Preparation monoclonal antibodies against the Fc fragment of chicken IgGResuscitation BDRPDP cells producted monoclonal antibodies against the Fc fragment of chicken IgG (mFc-Ab), obtained a high titer ascties which was purified using actanoic acid and ammonium sulfate, then received dialysis in ord to desalination and purification and obtain a high purity monoclonal antibodies.3. Preparation colloidal gold and gold-labeled antibodiesThe conditions of colloidal 20nm gold prepared were successfully established using sodium citrate reduction method. Studies were progressed on the optimal conditions about preparing the colloidal gold conjugated mFc-Ab. The optimal conditions were used 0.1mol/L K2CO3 per 1ml colloidal gold and the optimal mount of monoclonal antibody is 12μg/mL. The mFc-Ab was conjugented with colloidal gold in the optimal conditions.4. Development of test strips for rapid detection of serous antibody against AIV, NDV, IBV, IBDV, and MGThe strip was colloidal gold conjugated mFc-Ab as a tracer, coating antigen (AIV-HA1 or NDV-HN or IBV-N or IBDV-VP2, or MG-V1hA1.2 protein) in test line, coating the rabbit anti-mouse IgG. Using the indirect method, test strips for rapidly detection of serous against AIV HA1, test strips for rapidly detection of serous against NDV, test strips for r rapidly detection of serous against IBV, test strips for rapidly detection of serous against IBDV and test strips for rapidly detection of serous against MG were developed. Compared with other methods, this five test strips show a well specitivity and sensitivity. On this basis, developed the test strips for rapidly detection of serous antibody against AIV, IBV, and IBDV antibodies. Results of this study demonstrated that the ICS developed was highly sensitive and specific for detecting AIV, IBV, and IBDV antibodies in serum samples from chickens.5. Development of the test strips for detecting the antibody titer against IBVIn this study, colloidal gold conjugated monoclonal antibodies against the Fc fragment of chicken IgG as a tracer, using three testing line coating antigen by three different concentrations of IBV-N protein, using the indirect method, successfully established a immnunochromatographic strip for rapidly detecgting anti-IBV antibody titers. Testing AIV H5 subtype positive serum, the AIV H9 subtype positive serum, NDV-positive serum, IBV-positive serum, IBDV-positive sera, IBDV-positive serum and MG sera by positive made of strip shows its specificity. Different IBV antibody titer testing to determine the serum titer of greater than 8log2 three test line appears, between 8log2 and 5log2 occur two test lines, between 5log2 and 4log2 occur a test line, less than 4log2 test line does not appear. Experimental results show that this method is specific, and simple, intuitive determination, and can be used for IBV antibody titer determination.