Study On The Technology Of The Immunoassay Method Rapidly Determination Of Residue Streptomycin

Streptomycin was common antibiotic drug to treat tubercle bacillus, and found as a major residue in food, especially in milk.The Immunological analyticak technique, which has many merits of sensitiveness, specificity, rapidity and convenience, have been generally used in determination of drug residues in animal food. This study aimed at immunological rapid determination of SM residues in animal food, including the immunogencity of SM, sythesis of artifivial antigen, preparation and special properties of SM polyclonal antibody, SM monoclonal antibodies and regent kits, the preparation and quality of immunological gold-labled strip by LC-MS.1. Immunogen BSA-EDC-SM and coating antigen OVA-EDC-SM were synthesised by using EDC one-step after inleted carboxy group by O-carboxymethyl-hydroxylamine, BSA-GA-SM and BSA-di-SM were synthesized by using glutaraldehyde and 1,4-butanediol diglycidyl ether. Corresponding identification was made by SDS-PAGE and UltraViolet scan. Bind score molecule of BSA and SM of BSA-EDC-SM,BSA-GA-SM,BSA-di-SM were 1∶23,1∶15,1∶10 respectively. The high-titer, sensitive and specific SM antiserum had been produced by immunized 6W Balb/C mice.IC50 of pAb of BSA-EDC-SM immunity class miceⅠ-1# anti-SM was 45.8μg/mL, cross reaction rate to DHSM was 90.6%, and no cross reaction to other antibiotics. BSA-di-SM,BSA-GA-SM immunity class mice were no cross reaction to DHSM and other antibiotics.2. Six new Zealand white rabbits were immunized with BSA-EDC-SM by different dosage and different intermission, and SM pAb had produced. The results were 1# rabit inhibiting effection was better, the half maximal inhibition concentration (IC50) was 5.81μg/L.Mice were immunized with BSA-EDC-SM to prepare mAb. The lymphocytes from Balb/c mouse spleen were fused with NS0 myeloma cells. The hybridoma cell lines of F139-3A9-C4 was selected by indirect ELISA. The hybridomas secret high affinity monoclonal antidodies (McAb) and ascites antibodies with the titers of 1∶320 and 1∶512000, Ka was 7.5×1010 L/moL. The immunoglobulin isotypes of the McAb were identified with the Immunoglobulin Isotype Kit as IgG2a/κ. The chromosome number of this hybridomas was 96~102, the average number was 98.6. The IC50 of ascites antibodies to SM was 8.99μg/L, the cross reaction rate to DHSM was 93.6%, no cross reaction to other antibiotics.3. Based on the anti-SM McAb and the enzyme-linked immunosorbent assay, and were developed and applied to the screening for SM and DHSM. The an direct competitive ELISA-kit was better than indirect competitive ELISA- kit.The standard curve regression equations of the direct ELISA-kit was"S"type. IC50 was 6.22μg/L,the range of detection was 1.0~256.0μg/L,sensitivity was 0.52μg/L. The recovery rate in milk and urine was 83.2% and 86.89%, the average CV of intra-batch of milk and urine were less than 15%. The CR to DHSM was 82.71%, and no cross reaction to other antibiotics. The matrix had not effect sensitive of this kit.4. Based on the anti-SM McAb and the gold immunochromatography assay (GICA), SM-Strip was developed and applied to the screening for SM and DHSM.The diameter colloidal bead by transimission electron microscope were 25±1.0nm. The proper density was 12.5μg/mL, the most proper was 13.75μg/mL. The standard curve regression equations of the SM-Strip was"S"type. The machine reading sensitivity was 0.42μg/L, and eye-measurement was 4.14μg/L. The cross reaction to DHSM was 4.0μg/L by eye-measurement, and no cross reaction to other antibiotics.5. The sensitivity, specificity, simplicity and reliability of ELISA-kit and SM-Strip were confirmed by procedure utilizing LC-MS for the identification and quantification of SM in pig urine and milk. The result of refence-analyse indicated that ELISA-kit and LC-MS had more better dependability, the coincidence rate was 100%.