Study On Tetracycline Immunogold Fast Detection Technique

Purpose:As a broad-spectrum antibiotic, tetracycline antibiotic includes Tetracycline, Terramycin, Aureomycin and doxycycline, and is widely used in the treatment of various infectious diseases. However, excessive remnant of tetracycline antibiotic in animal tissues, milk and bee products due to non-standard dosing is a potential risk for human health and has aroused great concern with methods for detection developing continuously. The research adopts monoclonal antibody technology and colloidal gold immunochromatography technique to prepare tetracycline immunogold fast detection test paper, with a purpose to rapidly detect the remnant of tetracycline in foods.Method:The study adopts Mannich method to synthesize artificial antigen TC-BSA, TC-OVA and confirm the cross bonding of the hapten TC and the carrier BSA and OVA through UV scanning. The anti-etracycline monoclonal hybridoman cell strain is inoculated to the abdominal cavity of the mice to induce ascites, which is purified and salted out by ammonium sulphate. The concentration of the monoclonal antibody protein is 4.2mg/mL and the IC50 is 1.9ng/mL. Monoclonal antibody is especially specific for tetracycline antibiotic. The colloidal gold is prepared by reduction of Citrate Sodium. The marked monoclonal antibody is then attached to the binding pad of the colloid gold. TC-OVA (test line) and goat anti-mouse IgG (control line) were wrapped on nitrocellulose membrane, which is assembled with the sample pad and absorbent pad to make the test paper. The result may be judged by comparing the difference of the shade of the test line and the control line of the test paper. In this experiment, the most appropriate dosage of antibody protein in stable colloidal gold is 7.5μg/mL. Ahlstorm8964 fiberglass membrane is selected as the binding pad of the colloidal gold, MILLIPORE135s nitrocellulose membrane is used as chromatographic material, SB06 is the material for sample pad and CH37M is for absorbing pad.Result:The test paper indicated tetracycline test line of lOng/ml. The terramycin, aureomycin and doxycycline will have cross reaction with the tetracycline when the concentration is over 15ng/ml, lOng/ml and 20ng/ml respectively, while have no cross reaction with penicillin, streptomycin, gentamicin, chloramphenicol, erythromycin and sulfonamides. The tetracycline test line is 10ng/ml in pork sample and 50ng/ml in milk and honey, meeting the national standard for the products.Conclusion:The tetracycline colloidal gold fast detection test paper prepared in the research is fast and convenient for testing and meets the testing requirements in term of sensitivity, specificity and stability.