2,4-D完全抗原的合成及其免疫性能评价

以2,4-D为半抗原,BSA为载体蛋白,通过EDC的偶联反应条件优化试验,合成了多种结合比的完全抗原,制备了小分子环境污染物的多克隆抗体.结果表明,偶联反应优化步骤是2,4-D、BSA和EDC同时反应,4°C条件下反应18h.偶联反应可以在2,4-D的浓度为10.0～12.0 mg/mL的0.05mol/L的磷酸盐缓冲溶液中进行,反应缓冲体系pH控制在5.4～6.1之间.EDC的加入量低于12 mg时,采用2,4-D和多聚赖氨酸的偶联物为包被抗原.通过balb/c小鼠免疫试验评价结合比分别为6、12、18和25的完全抗原的免疫性能,结果表明结合比为12和18的完全抗原具有很好的免疫原性.其中以结合比为18的完全抗原免疫小鼠获得的抗血清对包被抗原载体的非特异性吸附低于阴性血清,2,4-D特异性血清含量高,适于作为完全抗原免疫小鼠进一步制备单克隆抗体.

Conjugation and Immunogenic Evaluation of Complete Immunogen for the Small Molecular Environmental Pollutants 2,4-D

Conjugation of complete immunogen for 2,4-dichlorophenoxyacetic acid (2,4-D) was studied. 2,4-D was cross-linked to bovine serum albumin (BSA, carrier) by 1-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide hydrochloride (EDC). The conjugation reaction was found to be more effective at 4 degrees C and incubated for 18 hour. 2,4-D was dissolved in 0.05 mol/L phosphate buffer between 10.0 - 12.0 mg/mL concentration, pH was adjusted to be 5.4 - 6.1. When the weight of added EDC was below 12 mg, the more EDC was added the higher substitution degree complete immunogen was synthesized. Complete immunogen of various substitution degree (2,4-D: protein) were applied to immunize balb/c mice. The conjugates of 2,4-D and poly-L-lysine was applied as coating antigen. It was experimentally found that complete immunogen of substitution degree 12 and 18 are more immunogenic than that of substitution degree 6 and 25. None-specific adsorption between antiserum that was produced by complete immunogen of substitution degree 18 and coating antigen was very weak, and the antiserum contained more 2,4-D specific antibody. It could be used as the immunogen for the preparation monoclonal antibody.