| Newcastle Disease(ND), commonly called as asian roup, is a highly contagious disease of birds caused by avian paramyxovirus serotype 1(APMV-1). In recent years, traditional diagnosis method would no longer effective in clinical along with the rising rate of non-typicle ND, a kind of ND which has no typical symptom and pathological changes. We prepared three lines of monoclonal antibodies(McAbs) and developed a McAbs sandwich enzyme-linked immunosorbant assay(ELISA) which could satisfy the clinical demand in this study. The paper included two parts. The destiny of the former was to get the McAbs against Newcastle Disease Virus(NDV) which would be used in the sandwich ELISA. In the second part, we developed the sandwich ELISA based on two lines of the McAbs and optimized the terms of the assay.It is very effective to concentrate virions by high speed centrifugation through sucrose cushions. First of all, we inoculate NDV F48E9 strain in 10 to 11-day-old embryos and harvested 8mL allantoic fluids average. Then the concentrated viral stock was overlaid on 20～60%,20% distance sucrose gradient. Last, harvested the fractions between 40～60% gradient. After purifying, the hemagglutination (HA) titers of NDV was 215, much higher than the allantoic fluids, 28～29, and the concentration was 1 mg/mL by Folin phenol method.An indirect ELISA procedure was established and optimized to screen positive hybridoma. The optimal coating quantity was 47 ng per well and the 4D5-HRP should be diluted by 800 times. Serum samples from immunized and healthy mice were measured as positive and negative control respectively. Through immuning Balb/C mouse with immunogen that is virulent NDV F48E9 strain purified by the method of sucrose density centrifuge, cellular fusion, clone and detection, we obtained three lines of hybridoma cells named 1F9,1C6,4D5 that could secrete McAbs. The fusion rate was 67.9%(391/576) and the positive rate was 6.9%(40/576). Hybridoma that producing McAbs anti-NDV was prepared by fusion of SP2/0 murine myeloma cells with spleen cell isolated from the immunized mouse.McAbs were produced in vivo by mouse ascites method and 4～5 mL ascites per mouse could be harvested. The ascites was purified by C8H16O2-(NH4)2SO4 and the recovery rate of target protein was 28.2～31.4%.Some of the biology characteristics of the McAbs cell lines were verified and the results demonstrated that all of them had high ELISA titers(1.5×104～1×105). 1F9 and 4D5 had high haemagglutination inhibition (HI) titers(29,28) also, but the virus neutralisation(VN) tiers were lower than 10. While 1C6 had high VN tiers(103) even though the HI tiers was 0.1C6 and 4D5 were used as coating and peroxidase-labelling McAbs respectively by antibody pairing test. Based on them, we developed and optimized the sandwich ELISA. The results shown that: (1)the plate which irradiated by X-ray could adsorb more coating protein; (2)1C6 and 4D5-HRP should diluted by 800 and 1600 times respectively; (3) 0.05mol/L pH9.6 Na2CO3-NaHCO3, l%BSA and 4%NCS-PBS were suitable to work as coating buffer, blocking buffer and peroxidase-labelling antibody diluting buffer respectively; (4)the reacting time of 4D5-HRP and OPD was 30min and 15min respectively.The sandwich ELISA based on McAbs was experimented with specificity, sensitivity and repetition. The result demonstrated that: (1)NDV-specific antibodies could stop the reaction between 4D5-HRP and NDV; (2)the minimum detectable concentration by this assay was 2.5ng/mL; (3)CV of intra-assay was smaller than 5%(n=6).
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