Development Of Colloidal Gold Immunochromatographic Assay Labeled Monoclonal Antibody For NDV Detection And Its Preliminary Application

Newcastle disease is a very important contagious disease in birds caused by Newcastle disease virus (NDV), which causes great economic losses to the poultry industry, especially to chicken industry.There are lots of different methods for detecting NDV. However most of them are still difficult practice in clinic. In this paper, a technology system for NDV detection was drawn up with the principle and technology of colloidal gold immuno-chromatographic assay.Firstly, rabbit antiserum against mouse IgG was made by rabbits immunized with the purified mouse IgG. Then the rabbit's serum was purified with saturated ammonium sulphate precipitation. The purity of rabbit IgG was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay. In addition, two MAbs ascites (NDV-F-6C4 and NDV-F-4D9) specific to the fusion protein of NDV were purified through protein G affinity chromatography column.Colloidal gold of 30nm was prepared by trisodium citrate method. The particles were detected under electronic microscope, which had good uniformity in shape and light refraction. The optimal conditions for preparing the colloidal gold conjugates with McAb to NDV-F 6C4 were compared and found that pH 8.7 and monoclonal antibody in 6.6μg/ml could get best results.Finally, a gold immuno-chromatographic assay (GICA) for detection of NDV was developed with that McAb to NDV-F 6C4 was conjugated with colloidal gold, which laid on a sample pad, McAb to NDV-F 4D9 and goat anti-mouse IgG antibody were used as the capture antibodies at the test line (T) and control line (C) respectively. The optimal concentrations were 30ul/cm for immunogold which diluted at the rate of 1:1.5 on the conjugate pad, 0.6ul/cm (3.0mg/ml) for T line and 0.6ul/cm (1.5mg/ml) for the C line on the membrane. The results can quickly be seen within 5 minutes.The sensitivity, specificity, repeatability, stability were studied with the strip test. Clinical examples from artificial infected chickens with NDV were verified for the method. The result showed that the GICA established in this experiment is a kind of rapid, sensitive, and specific method. This method was easy to be read, time-saving, differential and suitable for field test and epidemiologic investigation. The results concluded that our new GICA give a new way for the rapid diagnosis of ND clinically.