固相微球双抗体夹心ELISA法检测NDV抗原

Microball Double Antibody Sandwich Elisa for the Detection of Newcastle Disease Virus Antigen in Chickens

用抗新城疫病毒单克隆抗体FN_(29)和FN_(29)—HRP,分别作为捕捉抗体和示踪抗体,建立了MDS—ELISA法检测NDV抗原.FN_(29)为400μg—IgM时,可检出3μg/ml NDV蛋白.MDS—ELISA敏感性高于DFA,DFA阳性的标本,MDS—ELISA亦阳性,MSD—ELISA阳性的部分标本,DFA为阴性.两者符合率92%.人工感染病鸡的组织中NDV抗原检出率为:盲肠扁桃体、肺和肾混合液100%,肾100%,肺90%,肝、脾和盲肠扁桃体均为80%.鸡接种Ⅰ系弱毒疫苗后6天内,部分鸡肺组织中可检出NDV抗原.鸡接种Ⅱ系弱毒疫苗后,在组织中未能检出NDV抗原.该方法特异性强,灵敏度高,快速、经济、简便,克服了DFA需要特殊仪器的限制,易于基层推广应用.

A mieroball double antibody sandwich ELISA (MDS-ELISA) was developed to detect Newcastle disease virus (NDV) antigen in the tissues of chickens infected with NDV. In the assay , a monoclonal antibody (specific for NDV.IgM isotype), and its conjugate of horse radish peroxidase (FN-HRP) were served as the capture antibody and the indicator antibody, respectively. When FN was at 400g/ml, the detectable antigen amount was as low as 3g NDV protein per millilitre. The results of detection of NDV antigen from NDV -infected chickens by the MDS-ELISA and the direct immunof luorescent- antibody test (DFA) showed that the sensitivity of the MDS-ELISA was higher than of the DFA. All samples that were positive inthe DFA were also positive in the MDS-ELlSA. However, the MDS -ELISA indicated that a small number of samples were NDV antigen positive which were negative by the DFA. The concordance rate between tese two methods was 92%. The MDS- ELISA was evaluated on detection of the samples from chickens inoculated with various NDV strains. The experiment demonstrated that: 1 ) the positive rates in the various organs from the chickens infected with F48 strain of NDV were 100% in a mixture of lung, kidney and ileocecal tonsil, and in kidney. 90 % in lung, 80% in ileo-cecal tonsil, liver and spleen, respectively; 2 ) only some positive samples were found in Mukteswar-inoculated chickens in 6 days post-injection; 3 ) no positive samples had been found in Bl strain infected chickens. The MDS- ELISA was proved to be a most sensitive, specific, rapid and convenient method. It would be most useful for diagnosis of ND in poultry farms.