摘要
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 the monoclonal antibody（mAb） and develop a blocking ELISA for cadmium ion（Cd2＋）.Methods Artificial hapten Cd-ITCBE was synthesized by chelating Cd2＋ with isotrhiocyanobenzyl-EDTA（ITCBE）.The antigens Cd-ITCBE-BSA for immunization and Cd-ITCBE-OVA for detection were prepared by isothiocyanate method.BALB / c mice were immunized with Cd-ITCBE-BSA and,after cell fusion,positive hybridoma cell strains were screened by indirect ELISA and blocking ELISA.The McAb against Cd2＋ was prepared by induction of ascites in vivo and identified,based on which a blocking ELISA for residual Cd2＋ was developed and verified.Results The BSA-OVA contents in Cd-ITCBE-BSA and Cd-ITCBE-OVA were 7.1 and 6.7 mg / ml,while the Cd2＋ contents were 191.7 and 130.1 μg/ml,respectively.Four hybridoma cell strains,i.e.1A3,2B7,2E10 and 4F3,were screened,of which 2E10 secreted mAb with the highest titer and affinity constant（Ka）.The titers of mAb secreted by 2E10 strain were 1 ∶ 4.32 × 102 and 1 ∶ 5.12 × 105 in cell culture supernatant and in ascites respectively as proved by indirect ELISA,while the Ka was 7.58 × 108 L / mol,and the IC50 to Cd2＋ was 16.3 μg / L.The standard curve of developed blocking ELISA was in typical S shape,which was fitted to the four parameter logistic equation,with a detection limit of 5 μg / L.The mean recovery rates of Cd2＋ spiked in forage and in swine urine were 92.9% and 88%,while the mean variation coefficients were 12.35% and 12.45%,respectively.The developed blocking ELISA showed a cross reaction rate of 96.5% to Hg2＋,while showed no cross reaction with other compounds.The validity period of the developed ELISA kit stored at 4℃ was 180 d.Conclusion The high affinity mAb against Cd2＋ was successfully prepared,and a rapid,simple,sensitive and specific blocking ELISA for residual Cd2＋ was developed.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第2期208-212,共5页
Chinese Journal of Biologicals
基金
河南省高校科技创新人才支持计划资助项目(2010HASTIT026)
