克百威的ELISA试剂盒研制及多克隆抗体解毒功能研究
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摘要
本研究以2，3-二氢-2，2-二甲基-7-苯并呋喃氯甲酸酯为基础合成了克百威的两种半抗原，6—[[(2，3-二氢-2，2-二甲基-7-苯并呋喃基氧)羰基]氨基]已酸(BFNH)和4—[[(2，3-二氢-2，2-二甲基-7-苯并呋喃基氧)羰基]氨基]丁酸(BFNB)。同时将这两种半抗原与载体蛋白牛血清蛋白(BSA)和卵清蛋白(OVA)偶联合成克百威免疫原和包被抗原，并用紫外光谱法测定克百威与载体蛋白结合物结合比。
用合成的克百威免疫原免疫兔子后获得高效价、高特异性的抗克百威多克隆抗血清，用间接ELISA测定效价为1：16000～1：128000，纯化抗体效价为1：500000～1：4000000。所得克百威多抗血清与丁硫克百威的交叉反应率6.19％，与呋喃酚的交叉反应率为4.00％，与供试的其它5种氨基甲酸酯类农药的交叉反应率均小于0.44％，对于三种有机磷农药的交叉反应率均低于0.12％。并选择由BFNB-BSA免疫兔子所得的一种纯化抗体与辣根过氧化物酶偶联制备成酶标抗体，同时了测定了酶标抗体的活性，效价为1：4000。
在优化免疫学反应条件后，建立了克百威间接竞争ELISA法和直接竞争ELISA法，检测限分别为0.005mg／L和0.01mg／L，检测线性范围分别为0.005～10mg／L和0.01～10mg／L，并在此基础上研制了两种用于检测水、土壤、蔬菜、中毒样品的酶联免疫试剂盒，其试剂盒的重现性好，批内批间及整体变异系数均低于8.0％，试剂盒检测的准确度高，除呕吐物样品外，其余样品的回收率均在89.62％以上。
同时，根据抗体抗原的特异性结合反应的原理，进一步研究了抗克百威抗体对小鼠克百威中毒后的解毒作用，试验结果表明，抗体可以较好地缓解克百威对乙酰胆碱酯酶抑制作用的症状，使中毒小鼠逐渐恢复正常。
Two kinds of haptens 6-{[(2,3-Dihydro-2,2-Dimethyl-7-benzofuranyloxy)carbonyl] -amino}hexanoicAcid(BFNH)and4-{[(2,3-Dihydro-2,2-Dimethyl-7-benzofuranyloxy) carbonyl]-amino}butanoic Acid(BFNB) has been synthesized by chemical way. Then they were covalently conjugated to bovine serum albumin (BSA) or ovalbumin(OVA) to prepare antigens(immunogens) and coating antigen The conjugates were identified and their molecular ration of hapten to carrier protein were determined by UV spectrophotometry. Rabbit were immunized with synthetic antigens and then two kinds of antisera specific to carbofuran were obtained by using BFNH-BSA, BFNB-BSA as immunogens. The titers of the antisera were ranged froml: 16,000 to 1:128,000 and the purified antibody's titers were ranged from 1:500,000 to 1:4,000,000, respectively. The cross reactives of anti-BFNB-BSA serum to carbofuran-phenol, carbosulfan were 4.00% and 6.19%, other carbamates were less than 0.44%, three organophosphorus pesticide were less 0.12%.
The enzyme tagged antibody(E-Ab) was prepared by coupling horseradish peroxidase(HRP). The enzyme conjugate maintained both immunological activity and enzyme activity and its liter was 1:4000.
Two ELISA kits(Enzyme-linked Immunosorbent Assay kit) including indirect ELISA and E-Ab direct ELISA, have been established to detect the residue of carbofuran in specimens of water > soiK vegetable and food poisoning samples. The in-ELISA kit's linear detection was ranged from 0.005 to 10.00 mg/L, and the E-Ab ELISA kit's linear detection was ranged from 0.01 to 10.00 mg/L. The detection limits were 0.005mg/L and O.Olmg/L for two kinds of kits, respectively. The mean within-assay > between-assay and total-assay variability were less than 8.0%. The average recoveries of carbofuran added to sample were above 89.62% except for vomitus sample. The validity of kits was six months or more.
Since specific antibodies could be generated to target antigens, the experiment study was carried out on application of antibody in medical way to prevent inhibition in mice roisonned by carbofuran. The result indicated that the toxic effect of carbofuran on mice acetylcholine esterase was significantly reduced by the antibody, and the mice were recovered as normal.
用合成的克百威免疫原免疫兔子后获得高效价、高特异性的抗克百威多克隆抗血清，用间接ELISA测定效价为1：16000～1：128000，纯化抗体效价为1：500000～1：4000000。所得克百威多抗血清与丁硫克百威的交叉反应率6.19％，与呋喃酚的交叉反应率为4.00％，与供试的其它5种氨基甲酸酯类农药的交叉反应率均小于0.44％，对于三种有机磷农药的交叉反应率均低于0.12％。并选择由BFNB-BSA免疫兔子所得的一种纯化抗体与辣根过氧化物酶偶联制备成酶标抗体，同时了测定了酶标抗体的活性，效价为1：4000。
在优化免疫学反应条件后，建立了克百威间接竞争ELISA法和直接竞争ELISA法，检测限分别为0.005mg／L和0.01mg／L，检测线性范围分别为0.005～10mg／L和0.01～10mg／L，并在此基础上研制了两种用于检测水、土壤、蔬菜、中毒样品的酶联免疫试剂盒，其试剂盒的重现性好，批内批间及整体变异系数均低于8.0％，试剂盒检测的准确度高，除呕吐物样品外，其余样品的回收率均在89.62％以上。
同时，根据抗体抗原的特异性结合反应的原理，进一步研究了抗克百威抗体对小鼠克百威中毒后的解毒作用，试验结果表明，抗体可以较好地缓解克百威对乙酰胆碱酯酶抑制作用的症状，使中毒小鼠逐渐恢复正常。
Two kinds of haptens 6-{[(2,3-Dihydro-2,2-Dimethyl-7-benzofuranyloxy)carbonyl] -amino}hexanoicAcid(BFNH)and4-{[(2,3-Dihydro-2,2-Dimethyl-7-benzofuranyloxy) carbonyl]-amino}butanoic Acid(BFNB) has been synthesized by chemical way. Then they were covalently conjugated to bovine serum albumin (BSA) or ovalbumin(OVA) to prepare antigens(immunogens) and coating antigen The conjugates were identified and their molecular ration of hapten to carrier protein were determined by UV spectrophotometry. Rabbit were immunized with synthetic antigens and then two kinds of antisera specific to carbofuran were obtained by using BFNH-BSA, BFNB-BSA as immunogens. The titers of the antisera were ranged froml: 16,000 to 1:128,000 and the purified antibody's titers were ranged from 1:500,000 to 1:4,000,000, respectively. The cross reactives of anti-BFNB-BSA serum to carbofuran-phenol, carbosulfan were 4.00% and 6.19%, other carbamates were less than 0.44%, three organophosphorus pesticide were less 0.12%.
The enzyme tagged antibody(E-Ab) was prepared by coupling horseradish peroxidase(HRP). The enzyme conjugate maintained both immunological activity and enzyme activity and its liter was 1:4000.
Two ELISA kits(Enzyme-linked Immunosorbent Assay kit) including indirect ELISA and E-Ab direct ELISA, have been established to detect the residue of carbofuran in specimens of water > soiK vegetable and food poisoning samples. The in-ELISA kit's linear detection was ranged from 0.005 to 10.00 mg/L, and the E-Ab ELISA kit's linear detection was ranged from 0.01 to 10.00 mg/L. The detection limits were 0.005mg/L and O.Olmg/L for two kinds of kits, respectively. The mean within-assay > between-assay and total-assay variability were less than 8.0%. The average recoveries of carbofuran added to sample were above 89.62% except for vomitus sample. The validity of kits was six months or more.
Since specific antibodies could be generated to target antigens, the experiment study was carried out on application of antibody in medical way to prevent inhibition in mice roisonned by carbofuran. The result indicated that the toxic effect of carbofuran on mice acetylcholine esterase was significantly reduced by the antibody, and the mice were recovered as normal.
引文
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