Development Of Colloidal Gold Immunochromatographic Assay For Rapid Detection Of Streptomycin Residue In Food And Milk

Streptomycin(SM) is an Aminoglycosides antibiotics.It is effective to treat cow mastitis, infectious coryza and endometritis, and is frequently added into feeds for the prevention or treatments of animal diseases. However, illegal administration will lead to residues in edible tissues, and finally do latent harm to human health, resulting in hearing toxicity and kidney toxicity. Many countries and international organizations have established the maximum residue limit of Streptomycin in animal tissues for human consumption.To find and establish the best way for detecting SM residues, this research synthesized the antigen, developed the antibodies and colloidal gold to establish the GICA method for detecting SM by the technology of monoclonal antibody and GICA, and finally prepared the strip.In this study, hybridoma 5C10 producing anti-streptomycin monoclonal antibody (McAb) was used to inoculate into Balb/c mice abdominal cavity. The ascites of the mice was collected and purificated by caprylicacid-ammonium sulfate precipitation (CA-AS) to obtain the monoclonal antibody. The McAb was determined with coating antigen and the results showed that the concentration of the McAb was 4mg/mL, the titer of the McAb was 1?1.6×105 and the 50% inhibition concentration was 102.28ng/mL. The specificity of the McAb was well.Tri-sodium citrate with aqueous gold chloride were warmed up and mixed to make colloidal gold were labeled with monoclonal antibody against SM and were coated onto the glass fiber after the mixure veered purified. Str-OVA and the sheep anti-mouse immunoglobulins were coated onto a nitrocellulose membrane which the distance. Nitrocellulose membrane, gold conjugate pad, sample application pad, filter paper andabsorbent pad were assembled onto a sheet and cut into individual strips. The optimal diameter of colloidal gold particles was about 18nm, colloidal gold labeled antibodies was 15μg/mL. The glass fibre of Ahlstrom 8964 and nitrocellulose (NC) of Millipore 135 were used as chromatographic materialsThe various factors and conditions of the immunochromatographic lateral-flow assay were explored, and the optimal reaction conditions of the assay were ascertained. A GICA for rapid detection of Streptomycin residues in food derived from animals was successfully established. The GICA device had a cut-off value of 100ng/ml for detecting the SM dissolved in the phosphate buffer sodium. The cross-reactive rates of GICA to other seven antibiotics were negligible.The research has established the way to detect SM residues in milk, desiccated milk, honey and so on. The results detected by the CiELISA method and the strip show that the strip can meet the testing requirements.The major advantages of the strip were that results could be obtained within 10 minutes and that all needed reagents were included in the strip. The strip could be used to detect the Streptomycin residue in food derived from animals. The method of producing the one step strip for SM could be used for reference in the development of strips for the detection of other veterinary drugs.