Development Of Simultaneous Determination For 9 Kinds Of Veterinary Drugs In Animal Food By Membrane-based Dot Immunoassay

Veterinary drug residues means that veterinary drugs, chemicals, metabolic product andimpurities areaccumulated in cells, tissues and organs of loadanimals because of abuse. Excessive contents of veterinary drug residues not only are harmful to human health, but also could lead to the enhancement of microorganism drugs resistance and toxicity of the ecological environment. Meanwhile, the frequent events related to food safetycaused by veterinary drug residue make it a non-tariffbarrier in international trade, which draw great attention to the international community and governments. Subsequently, measures are taken to strictly monitor veterinary drug residue. Therefore, iturgently needs todeveloping of a rapid, simple and easy, high sensitive, high throughput technology to detection veterinary drug residues.In this study, membrane-based immunoassay was developed to simultaneously detect three categories(9 kinds) of common veterinary drug including GM, SAs,FQNs in milk and pork liver samples. This high throughput method was of screening,easy, rapid and accurate. The results of this method were consistent with the results of HPLC and commercial ELISA. In the field of small molecules material residues with poison, this method showed a good application potential. The main research content was as follows:1. The chemical synthesis of the detecting antigen GM- OVA and CPFX- BSAGM was activated by 1-ethyl-3-(3-dimethy-lamiopropyl) carbodiimide hydrochloride(EDC), and conjugated to ovalbumin(OVA) as the GM detecting antigen(GM-OVA). CPFX was activated by glutaraldehyde, and conjugated to bovine serum albumin(BSA) as the CPFX detecting antigen(CPFX-BSA). The concentration of GM-OVA and CPFX-BSA antigen were measured using Nanodrop ultramicro spectrophotometer. It was concluded that the GM-OVA and CPFX-BSA antigen concentration was 1.0 and1.05 mg/m L, respectively.2. Research on the Ic-ELISA to detecting SAs, GM and FQNs in milk and pork liver, respectively2.1 Research on Ic-ELISA to detecting SAs in milk and pork liverAccording to the checkerboard titration, the optimal working concentration of SAs-BSA was 6 g/m L and anti SDZ monoclonal antibody was diluted to 1/8000,IC50 was 0.063 g/L, and the linear working concentration ranged 0.016 0.202 g/L.The recovery rate was between 63.0% and 117.9%. The cross reactivity to SMR, SM2 and SMDZ was 56.3%, 50.0% and 52.1%, respectively, and no cross reaction was found with other common antibacterial drugs.2.2 Research on Ic-ELISA to detecting GM in milk and pork liverAccording to checkerboard titration, the optimal working concentration of SAs-BSA was 1 g/m L and IC50 of anti GM monoclonal antibody after diluted to1/8000 was 0.996 g/L, and the linear working concentration ranged 0.287 2.473g/L. The recovery rate was between 70.9% and 94.4 %, and no cross reaction was found with other common antibacterial drugs.2.3 Research on Ic-ELISA to detecting FQNs in milk and pork liverAccording to checkerboard titration, the optimal working concentration of CPFX-BSA was 4 g/m L and IC50 of anti CPFX monoclonal antibody was diluted to1/8000 was 0.346 g/L. The linear working concentration range was 0.099 0.831g/L. The recovery rate varied from 60.5% to 120.4%. The cross reactivity to NFLX,ENLX, DFLX were 432%, 20% and 15.5%, respectively, and no cross reaction was found apparently with other common antibacterial drugs.3. Research on simultaneous detection of three catalogues(9 kinds) by membrane immune-chipAccording to the theory of Ic–ELISA, the detected antigen was fixed on the PVDF membrane. A membrane-based dot immunoassay was to detect the residues level of GM, SAs and FQNs(9 kinds) in milk and pork liver samples. Using this method, the cut-off level of the sum of SAs, GM and FQNs were 50, 50 and 75 g/kg in milk and pork liver, respectively. These results suggest that this method could be a useful on-site screening tool for the rapid detection within 30 min. The concentration of GM, SAs and FQNs were determined in 20 milk and 8 pork liver samples using both the PVDF membrane-based immunoassay and Ic-ELISA. Meanwhile, the results showed consistent with the detected method of HPLC or the ELISA kit.