莱克多巴胺单克隆抗体的制备与免疫学检测方法的建立

发布时间：2018-01-15 12:28

  本文关键词：莱克多巴胺单克隆抗体的制备与免疫学检测方法的建立　出处：《中国人民解放军军事医学科学院》2007年硕士论文　论文类型：学位论文

  更多相关文章： 莱克多巴胺 单克隆抗体 药物残留 酶联免疫吸附试验

【摘要】： 本研究采用混合酸酐法将莱克多巴胺(ractopamine, RCT)分别与钥孔血蓝蛋白(KLH)和卵清蛋白(OVA)偶联,制备免疫抗原(RCT-KLH)和包被抗原(RCT-OVA)。用免疫抗原免疫8周龄雌性Balb/c系小鼠,通过四次免疫后,将小鼠断尾采血,以包被抗原包被酶标板,用间接ELISA法选出血清具有抗RCT的高阳性鼠,取这些小鼠的脾细胞与SP2/0细胞融合,通过检测、克隆、筛选等步骤生产抗RCT单抗。 通过以上步骤得到3株具有稳定、特异分泌抗RCT的单克隆抗体,分别命名为2B6(A株)、3D3(B株)、2F7(C株),通过鉴定3株单抗的效价分别可达到1:8.0×104、1:3.2×105、1:4.0×104。应用间接抑制ELISA将3株单抗与其它结构类似物进行竞争抑制,实验表明,抗RCT单抗对RCT的IC50为40.5ng/ml,对克伦特罗、肾上腺素、去甲肾上腺素、异丙肾上腺素、麻黄碱和氨茶碱的IC50均≥3906ng/ml,这表明抗RCT单抗与异丙肾上腺素的交叉反应为1.02%,与其它一些结构类似物的交叉反应均小于1.02%。 应用3株抗RCT单抗,基于间接抑制ELISA方法,建立RCT免疫学检测方法。通过对此方法中的相关条件进行优化,可以使本检测体系对RCT的检测范围达到1.25～1280ng/ml,表明基于此原理建立的检测方法可以用于动物性食品RCT残留的普查。
[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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