重金属铅免疫复合物的制备与鉴定

Preparation and identification of immuno-complex for plumbum

目的制备Pb-DTPA-BSA复合物,作为完全抗原,用以免疫动物制备铅抗体。方法首先用金属螯合剂二乙基三胺五乙酸(Diethylene triamine pentacetate,DTPA)与铅离子(Pb2+)结合,再分别用1-(3-二甲氨基丙基)-3-乙基碳二亚胺(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC)、戊二醛、三乙胺作偶联剂将其与大分子载体蛋白-牛血清白蛋白(Bovine Serum Albumin,BSA)偶联,制备Pb-DTPA-BSA复合物。将复合物做聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate polylamide gel electrophoresis,SDS-PAGE)、基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption Time of Flight mass Spectrometry,MALDI-TOF-MS)鉴定并免疫小鼠,获得抗PbDTPA抗体。结果 SDS-PAGE和MALDI-TOF-MS结果均验证合成了复合物Pb-DTPA-BSA,平均分子量76435;Pb-DTPA与BSA的偶联比为11∶1。免疫小鼠后,小鼠血清中产生了抗Pb-DTPA抗体,抗体最高效价达到1.0×105。结论经分析证明成功合成了免疫复合物Pb-DTPA-BSA,可用于重金属Pb抗体的制备。

Objective To prepare immuno - complex for plumbum （Pb） as the complete antigen to immune animal and produce antibody against Pb. Methods Firstly, Pb2 ＋ was chelated to diethylene triamine pentacetate （DTPA） and then coupled to the carrier protein bovine serum albumin （BSA） by 1 - （ 3 - dimethylaminopropyl） - 3 -ethylcarbodiimide hydrochloride （EDC）, glutaraldehyde and triethylamine. Then the conjugate was assessed by sodium dodecyl sulfate polylamide gel electro- phoresis （SDS -PAGE） and the matrix -assisted laser desorption time of flight mass spectrometry （MALDI -TOF- MS）. Fi- nally the complex was used to immune mice to obtain the anti - Pb - DTPA. Results Under the determination of SDS - PAGE and MALDI - TOF - MS, the conjugate was approved successfully, with average molecular weight of 76435 and coupling ratio of 11 ： 1. The antibody to Pb - DTPA was produced in serum of the immunizing mice with the maximum valence of 1.0 ×105. Conclusion The immunological conjugate Pb - DTPA - BSA has been proved successfully to be prepared, and it could be used for preparation of the Pb - antibody.