氨基脲单克隆抗体制备及其ELISA检测方法的建立

以氨基脲(SEM)和对醛基苯甲酸(CP)为原料合成半抗原(CP-SEM),将半抗原和载体蛋白偶联后免疫Balb/C小鼠,应用杂交瘤技术建立稳定分泌抗氨基脲的杂交瘤细胞株。常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测氨基脲的间接竞争ELISA方法。利用对醛基苯甲酸衍生草鱼肌肉提取的氨基脲,用建立的ELISA方法进行检测,并计算回收率和最低检测限。经细胞融合、筛选及克隆化,共获得7株稳定分泌抗氨基脲的杂交瘤细胞株,其中4株亲和力较高。建立了间接竞争ELISA检测方法,该方法灵敏度达到0.1ng/mL,平均回收率为98.47%,在草鱼组织中的最低检测限为0.76ng/g,回收率为70.55-100.56%,可用于肌肉中氨基脲残留的检测。

Preparation of Anti-semicarbazide Monoclonal .Antibodies and ICELISA for Detection of semicarbazide Residues

The hapten (CP-SEM) which was derivatived by combining 4-carboxybenzaldehyde(CP) with semicarbazide (SEM) was conjugated with carrierprotein to form complete antigen and then was used to immunize Balb/C mice.Hybridoma cells secrecting mAb against semicarbazide were developed.Ascites from the immunized mice were purified by caprylic acid-ammonium sulfate precipitation.The specificity of the purified antibodies was detected and the suitable antibodies were used for establishing indirected ELISA. Seven hybridoma cells stably secrecting mAb were prepared through cell fusion,screening and cloning,and four of these purified mAb had relative high affinity.The indirected ELISA for quantitative analysis of semicarbazide was established with a sensitivity of 0.1ng/mL.The average recovery rate was 98.47% ,Recoveries of SEM were in the range of 70.55-100.56% at 25 ng/kg,12.5 ng/kg and 5 ng/kg. The lowest detection limits of 0.76 ng/mL for SEM in animal tissues were achieved by ELISA.The method was applied for quantitative analysis of Semicarbazide.