摘要
接种H5N1亚型禽流感病毒 (AIV)鸡胚尿囊液,经甲醛灭活后 ,差速离心提纯病毒 ,三次免疫BALB/c小鼠 ,取免疫小鼠脾细胞与骨髓细胞SP2/0-Ag-14融合。用间接ELISA和血凝抑制 (HI)试验筛选阳性杂交瘤细胞株。本研究共获14株能够稳定分泌特异性单克隆抗体 (McAbs)的杂交瘤细胞株 ,其中McAbA6具有HI特性 ,并可以与重组鸡痘病毒表达的H5亚型AIVHA蛋白反应。用间接ELISA试验测定McAbs与125株AIV分离株的反应性。用反应谱广的McAbs(4B3和F2)进行交叉反应实验 ,确定了McAbs可用于夹心ELISA的最佳包被和酶标方式。以McAb4B3为基础建立的夹心ELISA对175株各亚型的AIV的阳性检出率在90
H5N1 subtype avian influenza virus was chosen to immunize BALB/c mice in order to produce the monoclonal antibodies(McAbs).The allantoic fluid was inactivated by 0.1% formaldehyde,purified and concentrated by differential centrifugation.The spleen cells of immunized mice were fused with Sp2/0-Ag-14 myeloma cells.Indirect enzyme linked immunosorbent assay(ELISA) and hemagglutinatin inhibition(HI) test were used to screen hybridoma cells.Fourteen McAbs specifically against avian influenza virus(AIV) were obtained.One of these McAbs,A6,had the ability of HI,and could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell(CEF).Two McAbs,4B3 and F2,were coated and conjugated with horseradish peroxides(HRP).Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated.By using the selected 4B3 for both coating and coniugating of HRP,the allantoic fluids of 175 strains of AIV were tested,the positive rates were above 90%.No cross-reaction was observed in the samples of NDV,IBV,IBDV and EDSV.
出处
《中国动物检疫》
CAS
2004年第10期20-22,共3页
China Animal Health Inspection
基金
国家十五攻关科技项目
