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目的制备重金属镉离子(Cd2+)单克隆抗体,并建立Cd2+残留阻断ELISA检测方法。方法用异硫氰酸苄基乙二胺四乙酸(ITCBE)螯合Cd2+合成Cd-ITCBE半抗原,异硫氰酯法制备免疫抗原Cd-ITCBE-BSA和检测抗原Cd-ITCBE-OVA,并进行鉴定;用Cd-ITCBE-BSA免疫BALB/c小鼠,经细胞融合后,间接ELISA和阻断ELISA筛选阳性杂交瘤细胞株,体内诱生腹水法制备Cd2+单抗,并进行鉴定;建立检测Cd2+残留的阻断ELISA法,并进行验证。结果 Cd-ITCBE-BSA和Cd-ITCBE-OVA中BSA与OVA含量分别为7.1和6.7 mg/ml,Cd2+含量分别为191.7和130.1μg/ml;共筛选出1A3、2B7、2E10、4F3 4株杂交瘤细胞,其中2E10株分泌的Cd2+单抗效价最高,细胞培养上清和腹水的间接ELISA效价分别达1∶4.32×102和1∶5.12×105,亲和常数(K)a也最高,为7.58×108 L/mol,对Cd2+的半数抑制浓度(IC50)为16.3μg/L;建立的阻断ELISA法的标准曲线呈典型的S型,符合4参数logit曲线拟合;最低检测限为5μg/L;饲料样、猪尿样的平均添加回收率分别为92.9%和88%,平均变异系数分别为12.35%和12.45%;与Hg2+的交叉反应率为96.5%,与其他化合物未见交叉反应;试剂可在4℃保存180 d。结论已成功制备了高亲和力的Cd2+单抗,并建立了快速、简便、敏感、特异的Cd2+残留阻断ELISA检测方法。
Objective To prepare monoclonal antibodies (Cd2 +) for detection of cadmium (Cd2 +) and to establish a method for detection of residual Cd2 + by ELISA. Methods The Cd-ITCBE haptens were synthesized by the chelation of Cd2 + with ITCBE and the antigens Cd-ITCBE-BSA and Cd-ITCBE-OVA by isothiocyanate method. BALB / c mice were immunized with Cd-ITCBE-BSA. The positive hybridoma cells were screened by indirect ELISA and blocking ELISA after being fused with Cd-ITCBE-BSA. Cd2 + Residual blocking ELISA method and verify. Results The BSA and OVA contents in Cd-ITCBE-BSA and Cd-ITCBE-OVA were 7.1 and 6.7 mg / ml respectively, and the Cd2 + contents were 191.7 and 130.1 μg / ml respectively. A total of 4 strains of 1A3, 2B7, 2E10 and 4F3 Among them, the 2E10 strain secreted the highest titer of Cd2 + monoclonal antibody, the indirect ELISA titer of cell culture supernatant and ascites was 1: 4.32 × 102 and 1: 5.12 × 105, respectively, and the affinity constant (K) a was the highest 7.58 × 108 L / mol and the IC50 of Cd2 + was 16.3 μg / L. The established standard curve of blocking ELISA showed a typical S-curve, which fit the 4-parameter logit curve fitting. The minimum detection limit was 5μg / L. The average recoveries of feed samples and swine urine samples were 92.9% and 88%, respectively. The average coefficient of variation was 12.35% and 12.45% respectively. The cross-reactivity with Hg2 + was 96.5% and no cross reaction with other compounds Reagents can be stored at 4 ° C for 180 days. Conclusion A high affinity Cd2 + monoclonal antibody has been successfully prepared and a rapid, simple, sensitive and specific method for detection of Cd2 + residue blocking ELISA has been established.