【作者】 魏书林；

【导师】 沈建忠；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 本研究采用活化酯法制备氯霉素(Chloranmphenicol,CAP)免疫抗原，分别免疫家兔和Balb／c小鼠获得抗CAP抗血清，并用间接酶联免疫吸附检测法(ELISA)对抗血清效价进行了检测，分别建立了检测CAP残留的间接竞争ELISA方法，可用于检测鸡肌肉组织中CAP的残留量。 CAP免疫抗原(CAP-BSA)的偶联比率为8∶1，包被抗原(CAP-OA)的偶联比率为2∶1。 在以多克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为1.7μg／ml，抗血清最高效价为1∶640000，抗血清纯化后最适工作浓度为1∶20000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～36.45ng／ml，检测限为0.05ng／ml。以0.5、1、2.5、5ng／g的浓度添加时，鸡肌肉组织中的回收率为42.00％～92.80％，变异系数为4.76％～26.29％。 在以单克隆抗体建立的ELISA方法中，用方阵滴定法确定包被抗原最适包被浓度为0.85μg／ml，腹水最高效价为1∶10~8，纯化后最适工作浓度为1∶10000。采用间接竞争ELISA法建立检测CAP的标准曲线，线性检测范围为0.1ng／ml～25ng／ml，检测限为0.1ng／g。以0.5、1、2．5、5ng／g的浓度添加时，鸡肌肉组织中回收率为71.01％～127.86％，变异系数为2.68％～16.16％。 上述2种ELISA方法快速、灵敏、方便，满足了氯霉素残留检测的要求，适用于残留大规模的快速筛选检测。更多还原

【Abstract】 In this study, one kind of immunogen was prepared: chloramphenicol(CAP) was coupled to carrier protein (BSA) using the N-hydroxysuccinimide activated ester method. Then the anti-CAP serum were raised in rabbits and in Balb/c mice respectively. After monitoring the titer of anti-serum by the indirect Enzyme Linked ImmunoSorbent Assay (ELIS A), the indirect competivive ELISA (ciELISA) detecting residues of CAP in chicken muscle tissues was developed.The coupling ratio of chloramphenicol to BSA was 8:1 and chloramphenicol to OVA was 2:1.In the ciELISA of polyclonal antibody(PcAb), the optimal concentration of coating antigen (CAP-OVA) was 1.7 u g /ml and the titer of anti-serum was 1:640000. The optimal working dilution of the purified PcAb was 1:20000. The linear range of calibration curves to detect CAP was 0.1ng/ml~36.45ng/ml and the limit of detection was 0.05ng/ml. The recoveries ranged from 42% to 92.80% for detecting CAP residues in chicken muscle tissues and the coefficients of variation was 4.76%~26.29%.In this study, two hybridomas against CAP were obtained and designated ID10 and 5E6. The monoclonal antibody (McAb) was obtained by the production of ascite. Some characteristics of the McAb were detected, the results were as following: the subclass was IgG1, the titer of supernatant was 1:512, the titer of ascitic fluid was 1 :1×108, the molecular weight was 150KD and 156 KD, the chromosomal numbers were 94-105, the affinity constant was 1.26×1010L/M. Except the chloramphenicol succinate salt, no other CAP’s analogues and the two carrier proteins had cross reactivity with the antibody. The antibody had excellent stability in this study. The ciELISA of McAb was developed after the purification of the ascite fluid. In the ciELISA the optimal concentration of coating antigen (CAP-OVA) was 0.85 u g /ml , the optimal working dilution of the McAb was 1:10000, the limit of detection was 0.1ng/ml, the linear range of calibration curves to detect CAP was 0.1ng/ml~25 ng/ml, the recoveries ranged from 71.01% to 127.86% for detecting CAP residues in chicken muscle tissues, and the coefficients of variation was 2.68%~16.16%.更多还原