雌酮硫酸钠多克隆抗体的制备与免疫分析法的研究

发布时间：2018-05-27 05:45

  本文选题：雌酮硫酸钠 + 胶体金免疫层析法　； 参考：《新疆医科大学》2010年硕士论文

【摘要】：目的：利用抗原和抗体的特异性结合反应对微量抗原或抗体进行测定的方法,研究制备雌酮硫酸钠(Sodium estrone sulfate,ESS)多克隆抗体(polyclonal antibody,PcAb)及建立简单快速、灵敏、经济的胶体金免疫层析试纸条和酶联免疫试剂盒分析雌酮硫酸钠的方法。方法：(1)以雌酮硫酸钠和牛血清蛋白(Bovine serum albumin,BSA)为主要原料,将ESS进行结构修饰,经各种谱图鉴定确证结构后,再用混合酸酐法制备完全抗原,并以紫外扫描和凝胶电泳法分析完全抗原的偶联比率,运用合成的免疫原(Hapten-BSA,Hap-BSA)免疫家兔,采集血清,纯化获得雌酮硫酸钠多克隆抗体。(2)用柠檬酸三钠还原氯金酸制备的适宜粒径的胶体金进行多克隆抗体标记,胶体金标记抗体经纯化后,喷涂于玻璃纤维膜上制成胶体金结合垫。将雌酮硫酸钠的多克隆抗体(检测线)和羊抗兔IgG(质控线)分别喷涂在硝酸纤维素(Nitrocellulose,NC)膜上。干燥后依次将NC膜、胶金结合垫、样品垫、吸水垫粘贴在胶板上,使用切刀切成3mm×5.5cm的试纸条,并通过检测孕马尿中雌酮硫酸钠来确证它的特异性和灵敏度。(3)用过碘酸钠改良法制备辣根过氧化物酶-雌酮硫酸钠多克隆抗体蛋白结合物(Horseradish peroidase conjugated anti-ESS IgG, HRP-anti-ESS-IG),方阵滴定法确定包被最适工作浓度及]IRP-anti-ESS-IgG最适稀释倍数后,用双抗夹心法制备用于检测孕马尿中雌酮硫酸钠的酶联免疫检测试剂盒。结果：(1)衍生后产物经各种谱图鉴定为雌酮硫酸钠-17-肟。半抗原衍生物与载体蛋白BSA成功偶联,其偶联比为25.74。采用间接ELISA测定产生的抗血清稀释度可以达到1.28×105；经亲和层析法纯化后蛋白浓度为3.179mg/mL。(2)采用柠檬酸三钠法还原法制备胶体金,金颗粒直径在10-20nm之间,满足试纸条的要求,经比较试验选用Millipore公司产品型号为HF135的硝酸纤维膜作为层析试纸条材料；质控线羊抗兔IgG包被浓度为2mg/mL,每条1.5μl；检测线ESS-PAcb的包被浓度为1.6mg/mL,每条1.5μl；样品垫、结合垫均采用Millipore公司的专用材料。检测ESS标准品灵敏度为30ng/mL。(3)双抗夹心ELISA的检测程序为：用浓度为40μg/mL抗体蛋白包被酶标板,包被液为CB,包被条件为4℃包被过夜；封闭液为1%牛血清白蛋白,封闭条件为室温(23℃-25℃)孵育2h；加孕马尿样本室温(23℃-25℃)孵育1h；酶标二抗室温(23℃-25℃)下作用时间60min;底物作用时间为30min。结论：本课题首先建立了半抗原衍生化的方法,即通过化学合成的方法将甾体激素雌酮先合成为雌酮-17-肟,再将雌酮-17-肟合成为雌酮硫酸钠-17-肟。在此基础上建立了制备雌酮硫酸钠多克隆抗体的方法,并初步建立了简单、快速、方便的胶体金免疫层析试纸法和双抗夹心ELISA法检测孕马尿中混合雌激素。
[Abstract]:Objective: to study the preparation of polyclonal body polyclonal antibody (PcAb) of sodium estrone sulfate ESSs by the specific binding reaction of antigens and antibodies, and to establish a simple, rapid and sensitive method for the determination of trace antigens or antibodies. Economic colloidal gold immunochromatographic strip and enzyme linked immunosorbent kit for the analysis of sodium estrone sulfate. Methods ESS was modified with sodium estrone sulfate and bovine serum albumin (BSA) as the main raw materials. The structure was confirmed by various spectra, and then the complete antigen was prepared by mixed anhydride method. The coupling ratio of complete antigen was analyzed by ultraviolet scanning and gel electrophoresis. The rabbits were immunized with the synthetic immunogen Hapten-BSAA and the serum was collected. The polyclonal antibody of sodium estradione sulfate was purified. The polyclonal antibody was labeled with colloidal gold of suitable size prepared by trisodium citrate reduction of chlorauric acid. The colloidal gold binding pad was prepared by spraying the colloidal gold labeled antibody on glass fiber membrane after purification. The polyclonal antibody (detection line) and goat anti-rabbit IgG (quality control line) of sodium estrone sulfate were sprayed on the nitrocelluloseine (NCC) film respectively. After drying, NC film, colloidal gold binding pad, sample pad, water absorbent pad were pasted on the rubber plate in turn, and the test paper strip of 3mm 脳 5.5cm was cut by cutting knife. The specificity and sensitivity of sodium estradione sulfate in pregnant horse urine were confirmed by the method of sodium periodate.) horseradish peroidase conjugated anti-ESS IgG, HRP-anti-ESS-IGG, square titration were prepared by modified method of sodium periodate and estradiol sodium sulfate polyclonal antibody protein conjugate of horseradish peroxidase (HRP-anti-ESS-IGG). After determining the optimal working concentration of the coating and] the optimal dilution multiple of IRP-anti-ESS-IgG, Enzyme linked immunosorbent assay (Elisa) kit for the determination of estradiol sulfate in pregnant horse urine was prepared by double antibody sandwich method. Results the derivative product was identified as estrone sodium sulfate-17-oxime by various spectra. Hapten derivatives were successfully coupled with carrier protein BSA with a coupling ratio of 25.74. The dilution of antiserum determined by indirect ELISA can reach 1.28 脳 10 ~ 5 and the concentration of protein purified by affinity chromatography is 3.179 mg / m 路L ~ (-2). Colloidal gold is prepared by citric acid trisodium reduction method. The diameter of gold particles is between 10-20nm and meets the requirements of test strip. Millipore's nitric acid fiber membrane (HF135) was used as the material for chromatographic test strip. The coating concentration of sheep anti-rabbit IgG was 2 mg / mL, 1.5 渭 l per piece, and the coating concentration of ESS-PAcb was 1.6 mg / mL, 1.5 渭 l per piece. Combined with the pad are used Millipore company's special materials. The detection procedure of double antibody sandwich ELISA was as follows: the concentration of 40 渭 g/mL antibody protein was coated with enzyme labeled plate, the coating solution was CBB, the coating condition was 4 鈩，

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