氯霉素单克隆抗体的制备及ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.09.029

Abstract

Abstract:

This study was aimed to establish an indirect competition ELISA (ci-ELISA) method for determination of chloramphenicol (CAP) residue in animal-derived foods.An active carboxyl group (-COOH) was introduced to the hydroxy (-OH) locus of phenyl of CAP to form CAP-HS.Via the method of mixed anhydride,CAP-HS was conjugated with BSA and OVA to obtain artificial immunogen (CAP-HS-BSA) and the coating antigen (CAP-HS-OVA) was obtained in the same way.CAP-HS-BSA was used to immunize BALB/c mice.Cell fusion spare mice were screened by indirect ELISA and ci-ELISA.CAP mAb were established using hybridoma technology.ci-ELISA based on CAP mAb and CAP-HS-OVA for CAP was developed.The result showed that one hybridoma cell line (2C4) was isolated,which produced mAb that could bind CAP,the titer of mAb was 4.8×10^-5.The optimal conditions for ci-ELISA based on 2C4 for CAP were as follows:0.4 μg/mL CAP-HS-OVA coating at 37℃ for 2 h;5% pig serum blocking at 37℃ for 1 h; 1:6.4×10⁴ CAP mAb incubating at 37℃ for 15 min;1:1 000 goat anti-mouse enzyme-conjugated secondary antibody (GaMIgG-HRP) incubating at 37℃ for 30 min;TMB was used for color development at room temperature for 9 min.The calibration curve of ci-ELISA CAP showed typical sigmoid and fitted to the four parameters logistic equation.The IC₅₀ in ci-ELISA was 0.53 ng/mL.The recoveries of CAP in negative carp meat and pork were 93.3% to 96.6% and 93.7% to 96.8%,respectively;The coefficient variation of the variability of intra-assay were 2.3% to 5.0% and 2.2% to 4.6%,respectively;The coefficient variation of the variability of inter-assay were 2.7% to 4.1% and 2.3% to 3.6%, respectively.HPLC comparative test results showed no significant difference between ci-ELISA and HPLC.The ELISA method for residue detection of CAP was successfully established in this experiment.This method had high sensitivity,accuracy and precision,and could meet the requirements for CAP residue detection in animal-derived foods.

Key words: chloramphenicol; residue detection; monoclonal antibody (mAb); ci-ELISA

CLC Number:

S859.84

FAN Suju, YANG Xingdong, GAO Run. Preparation of Chloramphenicol Monoclonal Antibody and Establishment of ELISA Detection Method[J]. , 2018, 45(9): 2566-2574.

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Preparation of Chloramphenicol Monoclonal Antibody and Establishment of ELISA Detection Method

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