Avian Infectious Bronchitis Immune Colloidal Gold Diagnostic Test Strips Research

The foul infectious bronchitis caused by Infectious bronchitis virus ,due to the characters such as multi-serotype and multi-tissue tropism ,easy to vary and difficult to control this disease ,is becoming to be the main resistance of poultry industry. Therefore, people around the world attach much importance to it and do a lot of work on it. The diagnosis of IB is dependent on the isolation and characterization of IBV, serology analysis, molecular diagnosis and electronic microscope technique. Serology tests include HA, HI, NIT, AGP, NT, ELISA and IFT. However, these methods have the disadvantages of time consuming and expensive lab equipment cost ,which make them not suitable for clinical instant diagnosis. Therefore, it is necessary to establish a new fast diagnostic method. In this paper an instant method is successfully established to detect IB using Gold Immunochromatography AssayAvian infectious bronchitis virus subtype C strain in the allantoic fluid of chicken embryo was purificated by sucrose density gradient centrifugation,then the spleen cells of immunized mouse and SP2/0 cells were fused . The cell fusion rate of 68.3%was obtained. Two hybridoma cell lines were cloned through common hybridoma technique, designated as 4D4, 4D6, respectively, which can stably secrete monoclonal antibodies against infectious bronchitis virus(IBV). Both McAbs belong to IgG1,κisotype. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect monoclonal antibodies (McAbs) secreted by hybridoma cell lines. Monoclonal antibody only reacted specifically with the avian infectious bronchitis virus,and don't reacted with other common poultry virus antigen (AIV, NDV ,etc). Monoclonal antibodies play important role in slip of indicator paper.After long period immunization of rabbit with C subtype oil emulsion IB vaccine, high titer rabbit-anti-IBV polyclonal antibody is obtained which is proved by indirect ELISA and AGP. For the ELISA method the titer is above 1:100,000 and for the AGP method it is between 1:16 and 1:32. With these McAb and polyclonal antibody a fast Gold Immunochromatography Assay diagnostic method of IBV is established. Gold colloid particle of 30 nm diameter is prepared and the optimal stable marker concentration of the McAb 4D4 is determined. The result is 12ug/ml of the McAb in 100ml 0.01% gold colloid. Then gold marked anti-IBV McAb with stable and good immunologic reactivity characteristics is successfully prepared. It neither deposit nor have delamination after it is stored at 4℃for six months.The gold marked antibody and rabbit -anti-IBV IgG purified by caprylic-sulfate ammonium method were used in preparing Gold Immunochromatography Assay. In the system the gold marked antibody is fixed on the glass cellulose membrane as the source of color, and the rabbit -anti-IBV IgG is coated on nitrocellulose membrane as the capture line. The rabbit-anti-mouse IgG is fixed on the nitrocellulose membrane as the quality control line. After careful determination of the optimal quantity of spraying on each line the Gold Immunochromatography Assay strip is successfully assembled. In the process of diagnosing, the IBV antigen binds the gold marked antibody on the strip and move along the membrane. Then it binds the polyclonal antibody at the capture line on the nitrocellulose membrane displaying visible red line. The standard is as follows: if IBV antigen exists in the sample a red line will emerge at both the capture line and the quality control line; if IBV antigen does not exist in the sample a red line will only emerge at the quality line. The sensitivity of this strip is 5ng/ml. In this paper a different sandwich ELISA method is also established to detect IB using the McAb and polyclonal antibody with good linear range between 3.75ng/ml-500ng/ml in its standard curve , its sensitivity is 3.75ng/ml . The two methods have an accordance rate of over 90% in diagnosis.In this paper anti-IBV polyclonal antibody and McAb anti-IBV common epitope are successfully prepared and used in the diagnosis of IBV. Preliminarily, a Gold Immunochromatography Assay is established and compared with a sandwich ELISA method. A sandwich ELISA method is established as the control with anti-IBV polyclonal antibody. It is shown that IBV Gold Immunochromatography Assay srip has the advantages of high sensitivity, better stability and specificity, good repeatability and handling and on-line monitoring of the detecting process. Therefore, It is playing an important role in the diagnosis of IB.