重金属镉特异性抗体的制备及icELISA检测方法的建立

为检测食品中的重金属镉残留问题,本研究建立了基于重金属镉抗体的酶联免疫分析方法。利用异硫氰酸苄基乙二胺四乙酸(Isothiocyanobenzyl-EDTA,ITCBE)螯合Cd~(2+)并偶联牛血清白蛋白(BSA)和卵清白蛋白(OVA),合成了针对Cd~(2+)的免疫原Cd-ITCBE-BSA和包被原Cd-ITCBE-OVA,通过紫外扫描鉴定说明偶联成功;以Cd-ITCBE-BSA免疫Balb/c小鼠,制备出Cd~(2+)抗血清并对其免疫学特性进行鉴定,进一步说明了抗原合成成功,在此基础上进行了条件优化并建立了针对镉的icELISA检测方法。方法的检测限(IC10)为0.18 ng/mL,IC50为1.15 ng/mL,线性检测范围(IC20～IC80)为0.24～5.54 ng/mL。除了与Hg~(2+)有2.16%的交叉反应率,该方法对Pb~(2+)、Fe~(3+)、Cu~(2+)和Cr3+交叉反应率均小于0.3%,板内变异系数和板间变异系数都低于10%。此抗体特异性强灵敏度高,建立的icELISA方法可应用于食品中重金属镉的快速检测。

Preparation of Specific Antibody to Cadmium and Establishment of icELISA Detection Method

An enzyme-linked immunosorbent assay (ELISA) for the determination of heavy metal Cadmium ion (Cd2+) residues in food was established. ITCBE was coupled with Cd2+ and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to obtain Cd-ITCBE-BSA as immunogen and Cd-ITCBE-OVA as coating antigen, which were identified by ultraviolet spectrum (UV). Cd-ITCBE-BSA was injected to Balb/c mice to produce antiserum. The immunological characteristics of the antiserum were identified, which further proved the success of synthesis. On this basis, an ic-ELISA was established after optimizing the conditions. The limit of detection (IC10) was 0.18 ng/mL, the 50% inhibitory concentration (IC50) was 1.15 ng/mL, and the linear range (IC20~IC80) was 0.24~5.54 ng/mL. Except for a cross-reactivity of 2.16% with Hg2+, the cross-reactivity with Pb2+, Fe3+, Cu2+ and Cr3+ were all less than 0.3%. The coefficients of variation within and between plates were both less than 10%. This antibody has strong specificity and high sensitivity, and the established ELISA method can be applied to the rapid detection of heavy metal cadmium in food.