孔雀石绿多克隆抗体的制备与筛选

Preparation and Selection of Polyclonal Antibodies against Malachite Green

设计并成功合成了三种孔雀石绿半抗原,所有半抗原均采用活化酯法分别与血匙兰蛋白(KLH)偶联制备成免疫抗原,与卵清蛋白(OVA)偶联制备成包被抗原。利用所制备的三种免疫原免疫新西兰大耳白兔都获得了高效价的抗孔雀石绿抗体,并将每一种抗体都与三种包被抗原进行组合配对,通过间接竞争酶联免疫吸附检测(ELISA)方法筛选出最佳的抗体-包被抗原组合,筛选出的抗体采用Sepharose FF-ProteinA亲和层析柱纯化,采用间接ELISA法测定效价及鉴定特异性。经测定筛选出的抗体效价达到1:12000,IC50值为0.65 ng/mL,交叉反应表明该抗体有较好的特异性,与结构类似物结晶紫的交叉反应率为18.73%,与隐性孔雀石绿及隐性结晶紫的交叉反应率低于10%。本研究为建立快速检测食品中孔雀石绿残留的免疫分析方法奠定了基础。

Three haptens for malachite green were designed and synthesized. Then the haptens were separately coupled to keyhole limpet hemocyanin （KLH） as immunogens and coupled to ovalbumin （OVA） as coating proteins by using active ester method. New Zealand white rabbits were immunized with the three immunogens, and all produced high titer antisera antibodies angainst malachite green. All the antibodies and the coating proteins were screened and then the optimal antibody-coated antigen combination was obtained. The selected polyclonal antiserum was purified by Sepharose FF-Proteins A affinity chromatography. The titer and characteristic were determined by indirect ELISA. The results showed that the antibody titer and IC50 reached 1：12000, and 0.65 ng/mL, respectively. And the antibody had good specificity. The cross-reactivity was less than 10% for leucomalachite green and leucocrystal violet but the antibody exhibited significant cross reactivity with crystal violet （18.73%）. The study provided basis for rapid detecting malachite green residues in food by immune analysis.