2,4-D人工抗原合成及2,4-D类农药特异性多克隆抗体的研制  

Synthesis of the Antigens for 2,4-D Type and Development of Its Specific Polyclonal Antibody

作　　者：王升吉[1] 尚佑芬[1] 赵玖华[1] 杨崇良[1] 路兴波[1] 孙红炜[1] 

机构地区：[1]山东省农业科学院植物保护研究所,山东济南250100

出　　处：《农业环境科学学报》2008年第5期2052-2057,共6页Journal of Agro-Environment Science

基　　金：山东省自然科学基金项目(Y2006D32)

摘　　要：通过改良EDC法,将2,4-DB、2,4-D分别与载体蛋白(BSA、OVA)偶联合成了2,4-D的免疫原2,4-DB-BSA、2,4-D-BSA和包被原2,4-DB-OVA、2,4-D-OVA。免疫抗原测定结果表明,所得紫外吸收光谱,具有载体蛋白BSA和半抗原2,4-DB或2,4-D的特性,最大吸收峰为282nm。合成2,4-DB-BSA、2,4-D-BSA免疫抗原的结合比分别为1∶24、1∶20,合成的2,4-DB-OVA、2,4-D-OVA包被原的结合比分别为1∶10、1∶8。上述两种免疫原分别免疫新西兰白雄兔,获得2,4-D类农药多克隆抗体。其中免疫2,4-DB-BSA的两只兔PBD1和PBD2的效价较高,分别是1∶102400和1∶51200。选择效价较高的PBD1多克隆抗体进一步优化筛选出抗原抗体的工作浓度,2,4-D-OVA包被原3μg·mL-1,PBD1多克隆抗体1∶12800。经测定,该多克隆抗体对目标检测物2,4-D、2,4-Dbutylester敏感度较高,抑制中浓度(I50)、检测限(I20)分别为,2,4-D:179.8ng·mL-1,1.09ng·mL-1;2,4-Dbutylester:3010.0ng·mL-1,1.53ng·mL-1。对2,4-D识别的特异性也较强。另外测定了制备2,4-D多克隆抗体对几种2,4-D类似结构化合物交叉特异性反应,2,4-DB最强,2,4-D次之,之后依次为2,4-D丁酯、2,4-D异丙酯,交叉反应性较差的有2甲4氯、2,4,5-涕、2,4-滴丙酸。2,4-DB, 2,4-D,as two haptens, were conjugated to bovine serum albumin （BSA）and ovalbumin （OVA）with improved EDC method, two immunogens of 2,4-DB-BSA, 2,4-D-BSA and two coating antigens of 2,4-DB-OVA, 2,4-D-OVA were obtained. The characteristics in ultraviolet absorption spectrum of immunogen possessed those of both carrier protein and haptens, the location of the absorption peak was at 282 nm, same as that of happten, 2,4-D. The reaction ratios of 2,4-DB-BSA and 2,4-D-BSA were 1：24 and 1：20 respectively. Those of 2,4-D-OVA and 2,4-DB-OVA, were 1：10 and 1：8. The specific polyclonal antibody for 2,4-D were obtained by immunizing New Zealand male white rabbits with the two antigens. Two rabbits PBD1 and PBD2 were immunized with 2,4-DB-BSA, produced high titers of polyclonal antibody （antiserums）as follows, 1：102 400 and 1：51 200 respectively. The PBD 1 polyclonal antibody was selected to next study. Work concentration of antigens was 3 μg· mL^-1, the PBD1 polyclonal antibody was 1：12 800. The polyclonal antibody to 2,4-D, 2,4-D butyl ester had high sensibility. The midmost inhibitory concentration （I50）and the detection limit （I20）of 2,4-D were 179.8 ng·mL^-1 and 1.09 ng·mL^-1, and those of 2,4-D butyl ester were 3 010.0 ng·mL^-1 and 1.53 ng·mL^-1. The peculiar cross-reactivity of the polyclonal antibody against 2,4-D analogous compounds were also tested, the sensibility to 2,4-DB was highest, then 2,4-D, 2,4-D butyl ester, and 2,4-D isopropyl ester, but the sensibility to MCPA, 2,4,5-T, Dichiorprop were lower.

关 键 词：2 4-D 半抗原 免疫抗原 酶联免疫吸附测定(ELISA) 抗体 

分 类 号：X592[环境科学与工程—环境工程]