【摘要】： 睪丸酮叢毛單胞菌含有降解甾核的關(guān)鍵酶3α-羥類固醇脫氫酶(3α-HSD),通過(guò)包含許多酶的氧化甾核的復(fù)雜代謝途徑,可以分解類固醇類物質(zhì)。 本研究采用單克隆抗體技術(shù)制備抗3α-HSD單克隆抗體,獲得了高表達(dá)抗3α-HSD的單克隆抗體的雜交瘤細(xì)胞株。主要方法是:將3α-HSD純化后免疫雌性Balb/c小鼠,取其脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行融合,經(jīng)克隆和間接ELISA篩選,獲得3株穩(wěn)定分泌抗3α-HSD單克隆抗體的雜交瘤細(xì)胞株。經(jīng)過(guò)鑒定:其腹水效價(jià)分別為1×10~6、1×10~5、1×10~4。 采用G蛋白親和層析柱對(duì)腹水進(jìn)行純化,將純化后的腹水單抗分別用辣根過(guò)氧化物酶(HRP)和異硫氰酸熒光素(FITC)標(biāo)記,制備酶標(biāo)抗體和熒光抗體,并對(duì)該酶標(biāo)抗體和熒光抗體進(jìn)行鑒定。制備所得酶標(biāo)抗體和熒光抗體具有特異性強(qiáng)、敏感性高的特點(diǎn)。我們應(yīng)用本研究制備的酶標(biāo)單克隆抗體建立了雙抗體夾心ELISA方法,對(duì)采集的水源樣品進(jìn)行檢測(cè)。結(jié)果表明,該單克隆抗體顯示了對(duì)類固醇類物質(zhì)的高特異性,為建立科學(xué)完善的環(huán)境激素檢測(cè)方法奠定了基礎(chǔ)。
[Abstract]:The steroid degradation key enzyme 3 偽 -hydroxysteroid dehydrogenase (3 偽 -HSD) is found in Trichotoma tumega, which can decompose steroids through the complex metabolic pathway of oxidative steroid nucleus containing many enzymes. In this study, monoclonal antibodies against 3 偽 -HSD were prepared by monoclonal antibody technique, and hybridoma cell lines with high expression of anti 3 偽 -HSD monoclonal antibody were obtained. The main methods were as follows: female Balb/c mice were immunized with 3 偽 -HSD, and the spleen cells were fused with SP2/0 myeloma cells. By cloning and indirect ELISA screening, three hybridoma cell lines secreting monoclonal antibodies against 3 偽 -HSD were obtained. The ascites titers were 1 脳 10 ~ (6), 1 脳 10 ~ (5) and 1 脳 10 ~ (4), respectively. Ascites were purified by G protein affinity chromatography. The purified ascites McAbs were labeled with horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC), respectively. The enzyme labeled antibody and fluorescent antibody were identified. The enzyme labeled antibody and fluorescent antibody were prepared with strong specificity and high sensitivity. A double antibody sandwich ELISA method was developed for the detection of water source samples. The results showed that the monoclonal antibody showed a high specificity to steroids, which laid a foundation for the establishment of a scientific and perfect method for the detection of environmental hormones.
【學(xué)位授予單位】：長(zhǎng)春理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王雅文;陳華林;王維;馬帥;王一東;冀偉;;抗3α-HSD單克隆抗體制備方法的初步研究[J];長(zhǎng)春理工大學(xué)學(xué)報(bào)(自然科學(xué)版);2013年05期

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本文編號(hào)：2139646