| With the social development and the rising of the living standard, people pay more attention to food quality, especially to the possible adverse effects of the residues of veterinary drugs in foodstuffs on human health. Recently, β-agonists, one kind of the veterinary drugs, have been widely illegal used in livestock due to the function of fat distribution. The residues of β-agonist in animal origin production can accumulate in human body and pose a grave threat to the comsumers. The β-agonist food safety incidents had been happened frequently, thus it is urgent to establish convenience and fast methods for the detection of β-agonists. The purpose of this work is to produce the monoclonal antibodies (mAb) against β-agonists (herein phenylethanolamine A and salbutamol) and develop corresponding ELISA for detecting phenylethanolamine A and salbutamol in food and tissue samples.Phenylethanolamine A (PA), an emerging new P-agonist, has been illegally used as growth promoter in livestock in China. Herein, the mAb against PA was produced and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) with highly sensitivity and specificity was developed for the detection of PA in tissue samples. The synthesized PA derivative with an amino group was coupled to carrier proteins (BSA or OVA) to form coating antigen and immunogen. The most sensitive mAb against PA was successfully prepared through hybridoma technology with PA-BSA conjugate as an immunogen. And corresponding ic-ELISA was established and characterized in terms of sensitivity, specificity, precision and accuracy under optimal experimental conditions. The half inhibition concentration (IC50) and limit of detection (LOD) values of the ic-ELISA using homogeneous combination of coating antigen/antibody for PA detection were 0.16 ng/mL and 0.011 ng/mL, respectively, which are 1.3 to 17 times lower than those obtained by other ELISAs. The cross-reactive (CR) values of the assay with other fourteen β-agonists were lower than 0.59%. Pork and swine liver spiked with different content of PA were analyzed by the ic-ELISA. The recovery rates and intra-assay coefficients of variation of the samples were 91.40-105.51% and 1.56-9.92%(n=3), respectively. The ic-ELISA for spiked tissue samples showed high correlation coefficients with liquid chromatography tandem mass spectrometry (LC-MS/MS). Therefore, the proposed ic-ELISA should be a simple and feasible quantitative/screening method for PA analysis in tissue samples with high sensitivity and specificity.Salbutamol is the most commonly abused β-agonist except for clenbuterol. In this study, the synthesized salbutamol derivative with a carboxylic spacer by succinic anhydride was coupled to carrier proteins to form immunogen and coating antigen. The antisera obtained from four immunized mice displayed high titer (104). Corresponding ic-ELISA based on the antisera was established and displayed high sensitivity with IC50 and LOD of 6.91 ng/mL and 0.19 ng/mL, respectively. The process to get stable antibody-producing clones is still going on. |
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