An Enzyme-linked Immunosorbent Assay For The Rapid Detection Of Chloramphenicol In Animal Food

Chloramphenicol(CAP) is a broad-spectrum antibiotic to Gram-positive and negative bacterial.And its pharmacodynamic action is stable in the animal feeds and drugs.As a result of abusing drug,the residue problem appeared seriously. Chloramphenicol has been forbidden to applying in animal food production in some developed countries.As traditional detections are based on the methods such as biochemistry and instrumental analytical which are complicated and time consuming,it is very important to establish a rapidly and accurate method.Therefore,the study on chloramphenicol ELISA kit plays a significant social and economic role for protecting consumers' interests,food safety testing as well as international trade.This study focused on synthesizing the artificial antigen,purifying Immunoglobulin G and establishing ELISA method.The active ester reaction and diazotization reaction were used respectively to couple the hapten chloramphenicol with carrier protein BSA and OVA.Two immunogens were obtained and proved by ultraviolet scan,infrared spectrogram scan and SDS-PAGE electropheresis.The anti-CAP antibody was obtained by immuning the New Zealand white rabbits with HAP-CAP.Titer of Ab was 1:1000000 by ELISA.A primary purification was conducted according to the method of the ammonium sulphate precipitations.Using bis(3-aminopropg) amine as a spacer between the gel and the CAP ligand,a separation material was synthesized for affinity chromatography to purify the specific anti-CAP IgG. The results showed that highly purified rabbit anti-chloramphenicol antibody could be obtained by the self prepared immunoaffinity chromatography column.Compared with the different methods of coupling HPR,the modified method of mixed anhydride was proved to be more effectively to tag HRP,and the activity of the conjugate was proved by ELISA.The effects of various factors,such as coating buffer, washing times,coating temperature,coating time,ionic intensity and non-specificity adsorption were studied.The optimum condition was given as following:coated with the coating antibody in 0.05mol/L pH9.6 CBS,and then incubated at 37℃for 2h.The PBS buffer with 1.0%OVA could improve the sensitivity of the ELISA methods.Adopting the optimized analytic condition and HRP tagged antigen,the affinity and specificity of Ab were evaluated by direct competed-ELISA method.The results indicated that Ab had high affinity and specificity to CAP.And the results of square matrix titration showed that anti-CAP IgG were 28.30μg/mL,HRP tagged antigen were 5.36μg/ml.The sensitive range to detect CAP varied from 0.2ng/mL to 64ng/mL.The linear regression equation from the method was y=-14.8431ogx+56.692,R²=0.9965,with the detection limit(IC₁₀) 0.451ng/mL,and the sensitivity IC₅₀ was 2.82ng/mL.The cross-reactivity with related analytes was all bellow 0.01%.The ELISA kit was prepared to detect CAP in different matrices,such as chicken, milk and shrimp.Recovery of methods to detect CAP was ranged from 72.6%to 113.6%, and the coefficient of variations was ranged from 4.26%to 17.75%.The results of performance measurement demonstrated that the ELISA test kit showed good repeatability and accuracy,and its preserving duration could be 4 months at least.The ELISA test kit could be used for the screening analysis of CAP in a batch of samples such as shrimp,chicken and milk within a short time at low cost.