Based on the preparation of an anti-diethylstilbestrol(DES) monoclonal antibody,a simple and convenient indirect competitive enzyme-linked immunosorbent assay(ELISA) method for DES detection has been developed.The monoclonal antibody demonstrated high sensitivity to DES with an IC50 value of 275 pg mL-1 and detection limit(LOD) of 90 pg mL-1.The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol(<7%),estrone(<0.1%),estriol(<0.1%),and diethylstilbestrol benzoate(<0.1%).Chicken,fish,shrimp,urine and bile spiked with different concentration of DES were detected by the developed method,and the recovery rates were greater than 79.5%.Intra-and inter-assay variations were about 6%.This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples.The LODs in fish/shrimp,liver,feed and urine spiked with DES were 600,600,4800 and 600 pg mL-1,respectively.These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals.
To detect gatifloxacin(GAT)residue in swine urine,an electrochemical immunoassay was established.An indirect competitive immunoassay was developed,in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay(ELISA)plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody.Horseradish peroxidase(HRP)conjugated to goat anti-rabbit IgG was used as the enzymatic label.A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator.The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration(IC50)of 8.9 ng/ml was obtained.Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin(3.0%),ciprofloxacin(3.0%),and ofloxacin(1.9%)among commonly used(fluoro)quinolones.In conclusion,the immunoassay system developed in this research can be used as a rapid,powerful and on-site analytical tool to detect GAT residue in foods and food products.
Jian YIMeng MENGZhong-qiu LIUJin-fang ZHIYuan-yang ZHANGJing XUYa-bin WANGJin-ting LIURi-mo XI
