Study On The Enzyme Linked ImmunoSorbent Assays Of Chloramphenicol In Chicken Muscle Tissue

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%.