抗阿维菌素单克隆抗体的制备与鉴定-临床兽医学专业毕业论文.docxVIP

摘要试验采用杂交瘤技术制备了两株抗阿维菌素(AVM)的单克隆抗体的的杂交瘤细胞株 摘要 试验采用杂交瘤技术制备了两株抗阿维菌素(AVM)的单克隆抗体的的杂交瘤细胞株 2F2、2IqIo’对获得的单抗进行了初步的纯化和鉴定。并建立了检测AVM的间接竞争ELISA (Ci-ELISA)方法。研究结果如下： 用合成抗原AVM-BSA免疫BALB／e小鼠，四次免疫后，采集血清，建立间接非竞争 ELISA方法，并测定血清效价。结果表明：包被抗原(AVM-OVA)最适浓度为6．75p．g／ml， 抗体最适稀释度为1：200。 BALB／c小鼠经AVM-BSA五次免疫后第三天，取脾细胞与SP2／0骨髓瘤细胞进行细胞 融台，选择PEGl450作为融合剂，两种细胞比例为10：1，融合率88％，阳性率为3。16％。采 用有限稀释法经过5次亚克隆。得到两株特异性分泌抗AVM的单克隆抗体的杂交瘤细胞株， 分别命名为2F2、2Hloo体内诱生腹水法制各单克隆抗体，收集腹水并进行单克隆抗体特性 的鉴定。经抗体亚类试剂盒测定，ELISA检测，染色体计数和SDS．PAGE电泳检测，表明该 单克隆抗体的免疫球蛋白亚类均为IgM，上清滴度为1：800，腹水滴度达1：6 400。分子质量 为925KD，染色体数目介于89--,96之间，稳定性良好。 绘制了单克隆抗体2Hio的标准曲线，绘制曲线在10-,10 000rig之间线性关系良好，检测 限为10ng，IC5q=101ng／ml，单克隆抗体与伊维菌素、多拉菌素、红霉素、自霉素的交叉反应 率均小于0．01％。 关键词阿维菌素；单克隆抗体；Ci．ELISA 东北农业大学农学硕士学位论文Abst：ract 东北农业大学农学硕士学位论文 Abst：ract Two hybfidoma cells(2F2，2H10)against AVM were prepared by hybridoma technique， purified and dentified in this study．Ci-ELISA to detect AVM was established．The results were∞ foIlows： BALI]／c mouse were immunized with artificial antigen(AVM-OVA)．Atier the fourth immunity,the serum was separated and tested the titer through the indirect ELlSA．The result showed that the optimal concentration ofcoating antigen(AVM-OVA、was 6．75pg／m1．The optimal dilution ofantibody was 1：200． The fusion between splenic lymphocytes and SP2／o cells(10：1)was performed at the third day aider the fifth immunity with AVM-OVA，PEGl450∞fusogen．The fusion rate was 88％，the positive Was 3．16％．Two hybfidoma cells producing anti—AVM monoclonal antibodies were obtained by limited dilution after 5 times subclone，named 2F2，2Hio respectively．Large amounts of McAb were prepared by production ofascite．All the ascite was collected and used to identification of antibody specificity．The characterization of antibodies were detected by antibody subtype kit， ELISA．chromosomal counting and SDS，PAGE．ne results indicated that the antibody subtype was IgM，the titer of supematant Was】：800，the titer of ascite was l：6 400，the chromosomal numbers were 89-96 and the molecular weight was 925KD