Development Of ELISA Antibody Test Kit Of Newcastle Disease

Using the antigen of Clone 30 strain for Newcastle disease virus, a detecting kit was developed by determining the optimal reaction conditions of EL1SA.By exploring the various factors of ELISA ,the optimal reaction conditions were determined . It was shown that the optimal concentration of NDV for coating of plate was 1.086ug/ml,the optimal coating condition of NDV was 4℃ overnight ; the dilution of serum sample was l:100;the working concentration of enzyme -labeled rabbit anti-chicken IgG was 1:800, the optimal reaction time of serum and enzyme-labeled rabbit anti-chicken IgG was 30minutes at 37℃, the optimal reaction time of substrate was 15minutes at 37℃. Following the determined conditions of ELISA, 14 serum samples which were negative by the HIPRA,S.A. ND Antibody Test Kit were used for the determination of threshold value of ELISA. Based on the formula of the threshold value=Average ODneg+3SD, threshold value of ELISA was 0.34. The blocking test for the reaction between the NDV positive and the Clone 30 strain of NDV was carried out by using the Lasota stain of NDV. The results showed that the Lasota stain of NDV could block the reaction completely. It was confirmed that there was no cross reaction between the antibodies against E.coli, AI H9,EDS76,IBD and coating NDV. The results revealed that the indirect ELISA by the purified NDV had good specificity for the detection of NDV antibody in serum. 64 serum samples from chicken were detected for the antibody to NDV by using the developed ELISA and the HIPRA,S.A.ND Antibody Test Kit simultaneously. It was found that the specificity and sensitivity of the developed ELISA were 90% and 93.2% respectively. 80 serum samples from chicken were detected for the antibody to NDV by using the developed ELISA and HI. It was found that the ELISA titers regressed on the HI titers. The correlational coefficient was 0.91. The storage conditions of the kit were explored. It was shown that the kit could be stored for 3 months at 4℃.The study have developed an inexpensive, rapid, specific diagnostic assay, which provides a available technique for the detection and serological survey of ND and a good fundament for the further development and commercialization of the kit.