| Florfenicol(FF) is a synthetic and broad-spectrum antibiotic and developed for veterinary use. Owning to its misusing, the residues in foods of animal origin happened frequently and may cause potential risks to human health. Many countries including China, USA and member states of Europen Union have strictly control its clinical applications. For example, florfenicol’s maximum residue limit(MRL) in musle is about 100 ug/kg. Therefore, it’s of great importance and urgency to develop rapid and sensitive methods for surveillance the florfenicol residues in animal tissues.1. Preparation and identification of polyclonal antibody against florfenicol. This study focused on preparating the specific polycolonal antibody against florfenicol. Two artifical antigens of florfenicol (FFA-BSA, FFA-OVA) were prepared by glutaraldehyde method and examined by ultraviolet spectrophotometry. BALB/C mice were immunized with FFA-BSA. Polycolonal antibody was generated from antiserum and detected by indirect ELISA. The results of UV spectrometric scanning showed that the artificial antigen FFA-BSA and FFA-OVA were synthesized successfully and their conjugation ratios were 12:1 and 11:1 respectively. The titer of the polyclonal antibody was over 1:32000. The florfenicol inhibition concentration (IC50) was 36.9 ng/mL. The polyclonal antibody against florfenicol in this experiment had high sensitivity and specificity, which made it possible to establish immunoassay of florfenicol residues.2. Preparation of colloidal gold immunochromatography strip for detection of florfenicol residues. Colloidal gold particles (mean diameter,25 nm) were prepared according to the Sodium citrate reduction method. Using the labeling antibody with colloidal gold particles. The Rapid Test Dispenser HM3030 is used for quantitative lines coating one strip membrane. Subsequently, the absorption pad, the nitrocellulose membrane, the relased antibody-gold conjugation pad and the sample application pad were pasted onto a PVC backing card with adhesive orderly. With the prepared colloidal gold immunochromatography test strip to determine the standard FF solution, the results demonstrated a detection limit of approximately 1.0 μg/mL. Cross-reaction indicated that the strip had a high specificity to FF. The strips were stable for at least 6 months at 4℃. Therefore, the immunochromatography detection method is an ideal and rapid determination method for monitoring of florfenicol and it’s of great potential for the preliminary screening of florfenicol in food of animal origin and feed samples.3. Comparative applications of colloidal gold immunochromatographic assay (GICA) and liquid chromatography-mass spectrograph (LC-MS) for the determination of florfenicol residues in food of animal origin. Colloidal gold immunochromatographic assay and LC-MS was separately used for the analysis of florfenicol residues in creatural products. Florfenicol residues at concent rations above 1.0 μg/mL could be detected by GICA. The average recenicol overy rates of LC-MS were 86.5%,93.7% and 90.9% respectively with relative standard deviation (RSD) of 2.1%～5.0% and the detection limit was 1μg/kg. The results of 80 animal samples tests confirmed that 0 sample was positive by LC-MS. Therefore, GICA is an ideal and rapid detrmination method for monitoring of florfenicol, and has increased application in food safety field especially in developing countries, while LC-MS is sensitive and suitable to be used for quantification of positive samples.In summary, the test strips of high titer, sensitivity and specificity had been used to detect florfenicol residues in animal tissue samples, and the results compared with LC-MS are basically the same, so the strips could be used as a convenient qualitative tool for rapid screening of florfenicol residues in foods of animal origin. |
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