| Olaquindox (OLA) is a kind of an artificial quinoxaline medicine, having wide spectrum of antibacterial. As feed additives, OLA can significantly promote animal growth, and prevention and treatment of bacterial infections. However, the study found Olaquindox has cumulative toxicity when unconscionable used, which causes distortion of animals, cancer and even death. More importantly, OLA presents a risk to consumers as the drug residues in the animal tissues. A variety of methods, such as chromatography and ELISA, have been utilized to determine OLA.These methods, however, are instrument dependent, time consuming and inconvenient to perform on the spot. With the rapid development of detection technologies, the gold immune chromatographic assay (GICA) with a lot of adventages including convenient, quick, low cost etc., has been used to monitor pesticide and veterinary drug residues in foods. Therefore, the aim of this study was to develop a rapid and quantitative detection to detect OLA in animals’ tissues in the based of GICA.Trial 1 was conducted to prepare the polyclonal antibody of olaquindox. Olaquindox hemisuccinate (OLA-HS), the hapten was prepared using succinic anhydride method, and then immunogen (OLA-HS-BSA) and coatingen (OLA-HS-OVA) were prepared using esterification method by coupling the hapten and the carrier protein(BSA and OVA). Ultraviolet method was used to measure the concentration and scan for identification of immunogen and coatingen. Six-week-old BALB/c mice were immunized by immunogen, and then ELISA was used to determine antiserum titer for the identification of the sensitivity and specificity. After determination, the conjugation ratio of OLA-HS with BSA and OVA were 16:1 and 6:1 respectively. Antiserum titer reached 1:51200. The antibody was used for the detection of IC50 and IC20 value, and cross-reactivity. The IC50 and IC20 value were 17.4 ng/mL and 3.86 ng/mL respectively, and the cross rate of carbadox was 0.53%. There was no cross-reactivity detected with mequindox and 3-methyl-quinoxaline-2-hydroxy acids. These results suggested that the prepared polyclonal antibody of olaquindox in the present study had high titer, sensitivity and specificity.Trial 2 was aimed to develop a new GICA test strips for rapid detection of olaquindox residues in animal origin food. Combining the prepared and purified polyclonal antibody of olaquindox with colloidal gold and sprayed on glass fibre mat. Coatingen (OLA-OVA) and sheep anti-mouse Ig were fixed on nitrocellulose membrane, and the immunochromatography testing paper strip was then manufactured, to detect its sensitivity, specificity, accuracy and stability. We also used this test strip for detection of olaquindox residues in pork and pig livers. The results showed that the test strip could be accomplished qualitative detection of olaquindox in 5 min, and the limit of detection of the strips was 0.05 μg/mL in both pork and pig livers. There was very little cross-reactivity detected with carbadox, mequindox and 3-methyl-quinoxaline-2-hydroxy acids. The false positive rate was less than 5%, and the false negative rate was 0. In addition, no effectiveness losing occurred during 6 months of storage at room temperature under dry conditions was observed. These results suggested that the prepared GICA test strips can be applied in rapid detection and on-site screening of olaquindox residues in animal tissues.Tiral 3 was carried out to assess the prepared GICA test strips for rapid and quantitative detection of OLA residues in animal tissues and market applications. Based in GICA to establish method on the T/C ratio to detect OLA residue, and compared results with ELISA and high performance liquid chromatography (HPLC) detection. When the animal tissues has OLA concentration at a range of 1 ng/mL to 200 ng/mL, the test strips showed a higher linear relationship, and the limit of dection (LOD) of immunochromatographic strip for OLA was 6.83 ng/mL. For negative liver samples, and their recovery rates were 86.9%～105.0% at 25,50 and 100 ng/mL. By ELISA and HPLC test, the results are basically the same.In summary, the test strips of high titer, sensitivity and specificity.had been used to detect OLA residues in animal tissue samples, and the results compared with ELISA and HPLC test are basically the same, so the strips can be used to rapid and quantitative detection of OLA residues in animal tissues... |
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