本文關(guān)鍵詞：萊克多巴胺單克隆抗體的制備與免疫學(xué)檢測(cè)方法的建立　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 萊克多巴胺 單克隆抗體 藥物殘留 酶聯(lián)免疫吸附試驗(yàn)

【摘要】： 本研究采用混合酸酐法將萊克多巴胺(ractopamine, RCT)分別與鑰孔血藍(lán)蛋白(KLH)和卵清蛋白(OVA)偶聯(lián),制備免疫抗原(RCT-KLH)和包被抗原(RCT-OVA)。用免疫抗原免疫8周齡雌性Balb/c系小鼠,通過(guò)四次免疫后,將小鼠斷尾采血,以包被抗原包被酶標(biāo)板,用間接ELISA法選出血清具有抗RCT的高陽(yáng)性鼠,取這些小鼠的脾細(xì)胞與SP2/0細(xì)胞融合,通過(guò)檢測(cè)、克隆、篩選等步驟生產(chǎn)抗RCT單抗。 通過(guò)以上步驟得到3株具有穩(wěn)定、特異分泌抗RCT的單克隆抗體,分別命名為2B6(A株)、3D3(B株)、2F7(C株),通過(guò)鑒定3株單抗的效價(jià)分別可達(dá)到1:8.0×104、1:3.2×105、1:4.0×104。應(yīng)用間接抑制ELISA將3株單抗與其它結(jié)構(gòu)類(lèi)似物進(jìn)行競(jìng)爭(zhēng)抑制,實(shí)驗(yàn)表明,抗RCT單抗對(duì)RCT的IC50為40.5ng/ml,對(duì)克倫特羅、腎上腺素、去甲腎上腺素、異丙腎上腺素、麻黃堿和氨茶堿的IC50均≥3906ng/ml,這表明抗RCT單抗與異丙腎上腺素的交叉反應(yīng)為1.02%,與其它一些結(jié)構(gòu)類(lèi)似物的交叉反應(yīng)均小于1.02%。 應(yīng)用3株抗RCT單抗,基于間接抑制ELISA方法,建立RCT免疫學(xué)檢測(cè)方法。通過(guò)對(duì)此方法中的相關(guān)條件進(jìn)行優(yōu)化,可以使本檢測(cè)體系對(duì)RCT的檢測(cè)范圍達(dá)到1.25～1280ng/ml,表明基于此原理建立的檢測(cè)方法可以用于動(dòng)物性食品RCT殘留的普查。
[Abstract]:In this study, ractopamine (RCTs) was coupled with keyhole hemocyanin (KLH) and ovalbumin (OVA) by mixed anhydride method. The immune antigen (RCT-KLH) and the coated antigen (RCT-OVA) were prepared. The 8-week-old female Balb/c mice were immunized with the immune antigen. The high positive mice with anti RCT were selected by indirect ELISA method. The spleen cells of these mice were fused with SP2/0 cells. Screening and other steps to produce anti-RCT monoclonal antibody. Through the above steps, three McAbs with stable and specific secretion of RCT were obtained. The titers of the three McAbs were 1: 8.0 脳 10 ~ (4) ~ (-1): 3.2 脳 10 ~ (5) respectively. 1: 4.0 脳 104. The three McAbs were competitively inhibited with other structural analogs by indirect inhibition of ELISA. The IC50 of anti RCT McAb to RCT was 40.5 ng / ml, to clenbuterol, epinephrine, norepinephrine and isoproterenol. The IC50 of ephedrine and aminophylline were 鈮，

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