【作者】 杨智洪；

【导师】 王川庆；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本研究利用碳化二亚胺（EDC）法分别合成了链霉素（SM）与载体蛋白BSA（牛血清白蛋白）、HSA（人血清白蛋白）、OVA（卵清白蛋白）和KLH（血蓝蛋白）的连接物,制备了四种人工抗原,并免疫小鼠,获得针对链霉素的抗体。用紫外分光光度法进行了鉴定,并计算连接比率,结果分别是22:1（SM-BSA）、26:1（SM-HSA）、29:1（SM-OVA）和495:1（SM-KLH）。间接ELISA检测结果显示,SM-BSA、SM-HSA、SM-OVA三种连接物在三免后抗体效价均达到1:3200以上,SM-KLH免疫后抗体水平参差不齐,高者可达1:12800,低者只有1:400。 成功建立了检测抗链霉素抗体的间接ELISA方法,其主要条件如下:pH9.6、0.005mol·L^-1的碳酸盐缓冲溶液（CBS）为包被液,0.5μg·mL^-1的连接抗原SM-HSA（链霉素—人血清白蛋白）为包被抗原,0.5%明胶为封闭液,1:1000稀释的HRP酶标记的羊抗小鼠抗体作为二抗,TMB（3,3,5,5-四甲基联苯胺）作为底物。试验证明本法特异性强,有较好的重复性。用该法对SM免疫小鼠的血清抗体及其杂交瘤细胞单抗上清进行检测,获得满意的效果。 通过细胞融合,获得一株能稳定分泌抗链霉素特异性单克隆抗体的杂交瘤细胞株,命名为1B6。其细胞培养上清效价达1:12800,腹水效价为1:819200,单克隆抗体亚型为IgG₁/κ。该单抗与其他抗生素的交叉反应率均低于0.01%。该杂交瘤细胞株连续传代26代,6个月内冻存、复苏3次,仍能稳定分泌抗SM的单抗。 利用1B6杂交瘤细胞株的小鼠腹水单抗建立了检测SM残留的竞争ELISA方法。其关键步骤的最佳试验条件为:最佳包被抗原浓度为0.5μg·mL^-1,腹水的最佳稀释比例是1:5×10⁴。最适检测范围为10μg·mL^-1-10ng·mL^-1,检测限量为10ng·mL^-1,得到的回归方程是y=-0.2162x+0.2304,R²=0.9599。该方法有待进一步研究、改进,并结合金标免疫层析做成试纸条,以用于生产实践中链霉素残留的快速检测。更多还原

【Abstract】 Streptomycin(SM) was conjugated with bovine serum albumin(BSA), human serum albumin(HSA), ovalbumin(OVA) and keyhole limpet hemocyanin(KLH) separatedly with EDC method to form artificial antigens. Mice inoculated with different immunogens above produced antibodies against streptomycin. The conjugants were identified by UV spectrum method. The rate of conjugation were 22:1 (SM-BSA), 26:1(SM-HSA), 29:1(SM-OVA) and 495:1(SM-KLH). The results of the indirect Enzyme-linked Immunosorbent Assay(ELISA) showed the titers of the first three conjugatants(SM-BSA,SM-HSA and SM-OVA) were above 1:3200. The titers of SM-KLH lineared from 1:400 to 1:12800.Indirect Enzyme-linked Immunosorbent Assay(ELISA) method was developed to detect antibodies against streptomycin. The optimum condition of the ELISA are set as follows: 0.5ug mL~-1 of SM-HSA(streptomycin-human serum albumin) conjugation used as a coated antigen dissolved in carbonate buffer solution(CBS,pH 9.6,0.05mol L~-1), 0.5 % glutin in PBS(pH 7.4,0.0 lmol L~-1) as a blocking agent post-coating, horse radish heroxidase-labeled sheep anti-mouse IgG(HRP-anti IgG) diluted by PBS(pH 7.4,0.lmol L~-1) in 1:1000 as a secondary antibody, and TMB (3,3’,5,5’-tetramethyl benzidine dihydrochloride) as a sbustrate. The specificity and repetitiveness of the ELISA was primely proved by the blocking test. The ELISA was conducted to detect the antisera of mouse and presented good results.One clone which can stably secrete the McAbs against streptomycin was obtained after fuse and named cell strain 1B6. The ELISA titers of the McAbs in cell supernatant and ascite were 1:12800 and 1:819200, respectively. The McAb was categorized into IgGl/ k. subtype and the rate of cross reactivity with other antibiotics were below 0.01%. The cell strain 1B6 also can stably secrete the McAbs after twenty-six setial passages and freezing ang recicfing three times in six months.更多还原