【作者】 孔晶；

【导师】 吴艳涛； 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2004， 硕士

【摘要】 禽流感（Avian Influenza，AI）是由A型流感病毒感染引起的一种禽类传染病，有重要的公共卫生意义。本研究的目的是以高致病性禽流感病毒A／Chicken／Shanghai／1／2000（H5N1）作为免疫原研制单克隆抗体（McAbs），并将研制的McAbs初步应用于AIV检测。尿囊液用甲醛灭活，差速离心法提纯，三次免疫BALB／c小鼠，取免疫小鼠的脾细胞与骨髓瘤细胞SP2／0-Ag-14融合。用间接ELISA和血凝抑制（HI）试验筛选阳性细胞株。将获得的阳性细胞株用有限稀释法进行多次亚克隆，筛选稳定分泌McAbs细胞株。 本研究一共获得14株能够稳定分泌特异性McAbs的杂交瘤细胞株，分别命名为2B6、3A5、4B3、5A5、5C2、F2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4，其中2B6和3A5的免疫球蛋白亚类为IgM，4B3、F2、A6、1D3、4A6和6C4的免疫球蛋白亚类为IgG_2a，其余6株McAbs均为IgG₁。McAb A6具有HI特性，并可以与重组鸡痘病毒表达的H5亚型AIV HA蛋白反应，能与64株H5亚型AIV中的2株病毒发生HI反应。用间接ELISA试验测定McAbs与125株AIV分离株的反应性，结果显示有11株McAbs即2B6、3A5、5C2、A6、1A2、1A4、1D3、4A6、5A2、6B5和6C4能与H5亚型AIV反应，反应的比例均低于56％（36／64）；3株McAbs（5A5、4B3与F2）能与H5亚型和H9亚型AIV发生反应，其中5A5与H5亚型AIV反应的比例为48％（31／64）、与H9亚型AIV反应的比例为49％（30／61）；4B3与H5亚型AIV反应的比例为89％（57／64）、与H9亚型AIV反应的比例为83％（51／61）；F2与H5亚型AIV反应的比例为89％（57／64）、与H9亚型AIV反应的比例为90％（55／61）。用McAbs（4B3和F2）进行交叉反应实验，确定了McAbs可用于双抗体夹心ELISA的最佳包被和酶标方式，以McAb 4B3为基础建立的双抗体夹心ELISA对64株H5亚型AIV、61株H9亚型AIV、36株H3亚型AIV、6株H1亚型AIV、2株H4亚型AIV、2株H6亚型AIV和2株H11亚型AIV进行检测，阳性检出率分别为98％（63／64）、90％（55／61）、97％（35／36）、扬州大学硕士学位论文100%（2/2）、100%（2/2）、100%（2/2）和100%（2/2）;与30株新城疫病毒和l株传染性支气管炎病毒、1株传染性法氏囊病毒和1株产蛋下降综合征病毒进行交叉反应，结果均呈阴性。以McAb 4B3为基础建立的双抗体夹心EUSA对AIV的最小检测限为0.4卜g/ml。更多还原

【Abstract】 Avian influenza has raised great concerns for its economic importance to poultry industry and potential threat to the public health. In this representation, a highly pathogenic avian influenza virus, A/Chicken/Shanghai/2000(H5Nl), was chosen to immunize BALB/c mice in order to product 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 and limited dilution method was performed to subclone positive clones.Fourteen of McAbs specifically against avian influenza virus (AIV) were obtained, designated as 2B6, 3A5, 4B3, 5A5, 5C2, F2, A6, 1A2, 1A4, ID3, 4A6, 5A2, 6B5 and 6C4 respectively. Two of these 14 McAbs, 2B6 and 5A5, were of IgM subtype. Four McAbs, 4B3, F2, A6 and 6C4, were of IgG2a and the others were of IgG1 subtype. One of these McAbs, A6, had the ability of HI, and could be detected in two samples of 64 H5 subtype AIV tested. Furthermore, A6 could react with the rFPV-H5-HA in the infected chicken embryo fibroblast cell (CEF). It was verified that 11 McAbs could react with H5 subtype AIV and all the rates of reaction were lower than 56% (36/64) in indirect ELISA. Three McAbs, 5A5, 4B3 and F2, could react with H5 and H9 subtype AIV and the rates reacting with H5 subtype AIV were 48% (31/64), 89% (57/64) and 89% (57/64) respectively. And that of H9 subtype AIV were 49% (30/61), 83% (51/61) and 90% (55/61) respectively. Two McAbs, 4B3 and F2, were coated and conjugated with horseradish peroxides (HRP). The specificity of the coated/HRP-conjugated McAbs was detected respectively by sandwich ELISA and direct ELISA respectively.Sandwich ELISA was established with the optimal McAbs to be coated and HRP-conjugated. By using the selected 4B3 for both coating and conjugating of HRP, the allantoic fluids of 64 strains of H5 subtype AIV, 61 strains of H9 subtype AIV, 36 strains of H3 subtype AIV, 6 strains of H1 subtype AIV, 2 strains of H4 subtype AIV, 2 strains of H6 subtype AIV, 2 strains of H1 1 subtype AIV were tested, the positive testing rates were 98(63/64), 90% (55/61), 97% (35/36), 100% (2/2), 100% (2/2), 100% (2/2) and 100% (2/2) respectively. No cross-reaction was observed in the samples of NDV, IBV, IBDV and EDSV, additionally, the lowest detection concentration of AIV was 0.4g/ml.更多还原