本文選題：雌酮硫酸鈉 + 膠體金免疫層析法��； 參考：《新疆醫(yī)科大學(xué)》2010年碩士論文

【摘要】：目的：利用抗原和抗體的特異性結(jié)合反應(yīng)對(duì)微量抗原或抗體進(jìn)行測(cè)定的方法,研究制備雌酮硫酸鈉(Sodium estrone sulfate,ESS)多克隆抗體(polyclonal antibody,PcAb)及建立簡(jiǎn)單快速、靈敏、經(jīng)濟(jì)的膠體金免疫層析試紙條和酶聯(lián)免疫試劑盒分析雌酮硫酸鈉的方法。方法：(1)以雌酮硫酸鈉和牛血清蛋白(Bovine serum albumin,BSA)為主要原料,將ESS進(jìn)行結(jié)構(gòu)修飾,經(jīng)各種譜圖鑒定確證結(jié)構(gòu)后,再用混合酸酐法制備完全抗原,并以紫外掃描和凝膠電泳法分析完全抗原的偶聯(lián)比率,運(yùn)用合成的免疫原(Hapten-BSA,Hap-BSA)免疫家兔,采集血清,純化獲得雌酮硫酸鈉多克隆抗體。(2)用檸檬酸三鈉還原氯金酸制備的適宜粒徑的膠體金進(jìn)行多克隆抗體標(biāo)記,膠體金標(biāo)記抗體經(jīng)純化后,噴涂于玻璃纖維膜上制成膠體金結(jié)合墊。將雌酮硫酸鈉的多克隆抗體(檢測(cè)線)和羊抗兔IgG(質(zhì)控線)分別噴涂在硝酸纖維素(Nitrocellulose,NC)膜上。干燥后依次將NC膜、膠金結(jié)合墊、樣品墊、吸水墊粘貼在膠板上,使用切刀切成3mm×5.5cm的試紙條,并通過(guò)檢測(cè)孕馬尿中雌酮硫酸鈉來(lái)確證它的特異性和靈敏度。(3)用過(guò)碘酸鈉改良法制備辣根過(guò)氧化物酶-雌酮硫酸鈉多克隆抗體蛋白結(jié)合物(Horseradish peroidase conjugated anti-ESS IgG, HRP-anti-ESS-IG),方陣滴定法確定包被最適工作濃度及]IRP-anti-ESS-IgG最適稀釋倍數(shù)后,用雙抗夾心法制備用于檢測(cè)孕馬尿中雌酮硫酸鈉的酶聯(lián)免疫檢測(cè)試劑盒。結(jié)果：(1)衍生后產(chǎn)物經(jīng)各種譜圖鑒定為雌酮硫酸鈉-17-肟。半抗原衍生物與載體蛋白BSA成功偶聯(lián),其偶聯(lián)比為25.74。采用間接ELISA測(cè)定產(chǎn)生的抗血清稀釋度可以達(dá)到1.28×105；經(jīng)親和層析法純化后蛋白濃度為3.179mg/mL。(2)采用檸檬酸三鈉法還原法制備膠體金,金顆粒直徑在10-20nm之間,滿足試紙條的要求,經(jīng)比較試驗(yàn)選用Millipore公司產(chǎn)品型號(hào)為HF135的硝酸纖維膜作為層析試紙條材料；質(zhì)控線羊抗兔IgG包被濃度為2mg/mL,每條1.5μl；檢測(cè)線ESS-PAcb的包被濃度為1.6mg/mL,每條1.5μl；樣品墊、結(jié)合墊均采用Millipore公司的專用材料。檢測(cè)ESS標(biāo)準(zhǔn)品靈敏度為30ng/mL。(3)雙抗夾心ELISA的檢測(cè)程序?yàn)椋河脻舛葹?0μg/mL抗體蛋白包被酶標(biāo)板,包被液為CB,包被條件為4℃包被過(guò)夜；封閉液為1%牛血清白蛋白,封閉條件為室溫(23℃-25℃)孵育2h；加孕馬尿樣本室溫(23℃-25℃)孵育1h；酶標(biāo)二抗室溫(23℃-25℃)下作用時(shí)間60min;底物作用時(shí)間為30min。結(jié)論：本課題首先建立了半抗原衍生化的方法,即通過(guò)化學(xué)合成的方法將甾體激素雌酮先合成為雌酮-17-肟,再將雌酮-17-肟合成為雌酮硫酸鈉-17-肟。在此基礎(chǔ)上建立了制備雌酮硫酸鈉多克隆抗體的方法,并初步建立了簡(jiǎn)單、快速、方便的膠體金免疫層析試紙法和雙抗夾心ELISA法檢測(cè)孕馬尿中混合雌激素。
[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，

本文編號(hào)：1940719