Study On Enzyme-Linked Immunosorbent Assay And ELISA Kit For Chloramphenicol

Two haptens (CAP-HS1 and CAP-HS2) of chloramphenicol (CAP) were synthesized by chemical reaction between CAP and succinic anhydride, and identified by ESI-MS. Then, two haptens were covalently coupled onto bovine serum albumin (BSA) with the modified active ester method (CAP-HS1-BSA and CAP-HS2-BSA), the ratios of conjunction haptens and BSA were determined by UV spectrometer. The anti-CAP-HS1 antibody (Ab1) and anti-CAP-HS2 antibody (Aba) were obtained by immunising the New Zealand white rabbits with CAP-HSi-BSA and CAP-HS2-BSA. Titers of Ab, and Ab2 were 8.39 × 10~6 and 6.55 × 10~4, respectively. In terms of the affinity of two antibodies by ELISA, Ab1 was better than Ab2.Compared with different methods of coupling horseradish peroxidase (HRP), the modified method of sodium periodate was proved to be more effectively to tag HRP on Abi.The effects of various factors, such as immuno-plate, coating buffer, coating temperature, coating time, solvent, ionic intensity and non-specificity adsorption were studied. The optimum condition for ELISA was given as following: NUNC Immuno plate, coated with the coating antigen(1μg/mL,100 μ L per well) in 0.05M carbonate-bicarbonate buffer (pH9.6), and then incubated at 37°C for 2 hours. Some other materials such as 2% defatted milk powder, 10% fetal calf serum ,0.1% BSA and 1% glutin in 0.01M pH7.4 phosphate-biphosphate buffer could improve the sensitivity of the ELISA method.Adopting the optimized analytic condition and HRP tagged Ab1, the affinity and specifity of Ab1 were evaluated by direct competed-ELISA method. Results indicated that Ab1 had high affinity and specifity to CAP. The linear regression equation for the method was y = 10.94Ln(x) + 30.394, r= 0.999. The limit of detection (IC20) was 0.386ng/mL. The percentages of cross reactions with thiamphenicol and florfenicol were 0.025% and 0.166%, respectively, and those for the other relative antibiotics were less than 2 × 10-5, according with the literature.The ELISA kit was preparated to detect the CAP in different matrices, such as shrimp, pork, chicken and egg. Recovery of methods to detect CAP were ranged from 71.6 to 117.6% with coefficient of variations of less than 13.9% at spiking levels of 0.5, 5, 50ng/mL。 It was suggested that the ELISA Kit could be applied to detect the residue of CAP in shrimp, pork, chicken and egg.