Development Of Visual Gel-based Immunoaffinity Test Column For The Detection Of Veterinary Drugs And Illegal Additives

In this paper, visual gel-based immunoaffinity test column had been developed to detect quinolones, furaltadone metabolite AMOZ, chloramphenicol and melamine in food of animal origin as a rapid detection technology.The norfloxacin monoclonal antibody from mouse ascites was purified by octanoic acid-ammonium sulfate method. The enzyme-labelled antigen (NOR-HRP) was prepared by the active ester method. Agarose gel activated by CNBr was respectively conjugated with norfloxacin antibody and HRP antibody to prepare antibody gel and HRP antibody gel.By optimizing the working conditions, the dilution times of NOR-HRP, norfloxacin antibody gel and HRP antibody gel were respectively 1:500000, 1:7 and 1:12 (v/v). The PBS solution (pH 7.4) was choosed as sample dilution. With the optimal conditions, the detection limit of gel-based immunoaffinity test column analysis method was 5 μg/L (in terms of norfloxacin). The results of specificity test showed that the visual gel-based immunoaffinity tset column has cross-reactivity with quinolones such as ciprofloxacin,enrofloxacin and ofloxacin and so on, which indicated that the test column could be used to detect these quinolones at the same time. The detection limit of norfloxacin in food of animal origin was 25～75 μg/kg (μg/L).Furaltadone metabolite AMOZ polyclonal antibody from rabbit serum was purified by protein A-Sepharose 4B immunoaffinity chromatography method. The enzyme-labelled antigen (CPAMOZ-HRP) was prepared by the active ester method. By optimizing the working conditions,the dilution times of CPAMOZ-HRP,AMOZ antibody gel and HRP antibody gel were respectively 1:70000, 1:39 and 1:39 (v/v). The PBS solution (pH 7.4)was choosed as sample dilution. With the optimal conditions, the detection limit of AMOZ gel-based immunoaffinity test column analysis method was 20 μg/L (in terms of 2-NPAMOZ). The detection limit of AMOZ in food of animal origin was 3～6 μg/kg (μg/L).The method of purifying the chloramphenicol polyclonal antibody is the same as AMOZ polyclonal antibody. The enzyme-labelled antigen (CAP-HRP) was prepared by the active ester method. By optimizing the working conditions, the dilution times of CAP-HRP,chloramphenicol antibody gel and HRP antibody gel were respectively 1:100000, 1:9 and 1:19 (v/v). The PBST solution (pH 8.5) was choosed as sample dilution. With the optimal conditions, the detection limit of chloramphenicol gel-based immunoaffinity test column analysis method was 3 μg/L (in terms of chloramphenicol). The detection limit of chloramphenicol in food of animal origin was 3-15 μg/kg (μg /L).The method of purifying the melamine polyclonal antibody is the same as AMOZ polyclonal antibody. The enzyme-labelled antigen(MEL-HRP)was prepared by the active ester method. By optimizing the working conditions, the dilution times of MEL-HRP,melamine antibody gel and HRP antibody gel were respectively 1:10000, 1:4 and 1:149(v/v). The PBST solution(pH 5.7) was choosed as sample dilution. With the optimal conditions, the detection limit of melamine gel-based immunoaffinity test column analysis method was 200 μg/L (in terms of melamine). The detection limit of melamine in dairy products was 1000 μg/kg.