Study On Colloidal Gold Immunochromatography Technique For Rapid Detection Of Chloramphenicol Residues In Food

Objective:Chloramphenicol is broad-spectrum antibiotic. It has been applied to treatment bactericidal disease widely. But chloramphenicol has strong side-effect,and has been forbidden to applying in animal food production. But traditional detecting is based on such methods as biochemistry,instrumental analysis,etc.And the operation is complicated and time consuming.So it is very important to detect to chloramphenicol residue rapidly and accurately. Immunochromatography colloidal gold strip is sensitive analytical method for detection of antigens and antibodies. It is a fast,sensitive and selective method compared with conventional analysis procedures. It fits for on-site screening of large numbers of samples.In this dissertation,the main purpose is to develop immunochromatography colloidal gold strip for rapid detection of chloramphenicol in animal food.Methods: The hybridoma cell was expanded cultured and inoculated into mice abdominal cavity to produce ascites. The ascites of the mice was collected and puried to obtain the monoclonal antibody. The ascites were purified with caprylic acid-ammonium sulfate precipitation and affinity column chromatography to obtain the monoclonal antibody. The McAb was determined with coating antigen including concentration,valency as well as cross-reactive rates. Colloidal gold was obtained by reducing the gold chloride with sodium citrate and labels CAP-McAb. The gold conjugate pad was oven-dried after soaked specific immunogold to glass fiber of BT50. The AE98Fast of Whatman corporation were used to prepare the test strip,in which the sheep anti-mouse IgG coated as control line,the concentration of sheep anti-mouse IgG dilution in 1:20,the CAP antigen coated at 0.225mg/mL as the test line. Sample pad, gold conjugating pad,nitrocellulose membrane and absorbing pad were assembled to immunochromatographic test strip. Results: The antibody 4D10 was selected to prepare the colloidal gold- labeled probe.The results showed that the concentration of the McAb was 2.89mg/mL,the titer of the McAb was 1:1024000. ELISA showed that the cross-reactivity with chloramphenicol succinate sodium salt had existence and the cross-reactivity with penicillin,streptomycin, sulfamethazine, thiamphenicol were less than 0.01%. It can be used for sensitive and selective immunochromatography colloidal gold test strip of chloramphenicol. The optimum pH for labelling is about 8.2 and the amount of McAb is 48μg/mL according to the exosyndrome of color,UV-scanning curve and scanning electron microscope.The sensitivity of strips was 100ng/mL.The positive samples were detected with 100% positive result within sensitivity range. The result could be read clearly in 5 minutes.Conclusion: It shows that the strip is time saving,sensitive,easy to be justified and suitable for the on-site detection. The confirmatory test for prepared gold immunochromatographic strip showed that the strip to CAP has strong specificity,simple and fast operation. It lays a foundation for detecting chloramphenicol on-site and batch- exemplar analysis preliminary.