| Streptomycin (SM) is an important aminoglycoside antibiotics, with stableproperty, wide antibacterial spectrum, simple production process, good curativeeffect and other characteristics, and powerful antibacterial effect for Mycobacteriumtuberculosis. So it was frequently added into feeds for the prevention or treatments ofanimal diseases. The streptomycin residue in animal food has been attractedwidespread attention at home and abroad because of its serious totoxicity andnephrotoxicity. The development of rapid, accurate and economical detection methodfor streptomycin residue in production and life is of great significance.In this study, the two current widespread application of immunoassay methods,enzyme-linked immunosorbent assay (ELISA), and a novel method which based onaptamer，were developed to detect streptomycin residues in food. The study resultswere obtained as the following.1. SM artificial antigen were prepared and identified. By using1-ethyl-3-(3-dimethylaminopropyl) Carbodilmide (EDC) and glutaraldehyde method(GA), SM was successfully coupled with bovine serum albumin (BSA) andovalbumin (OVA) to prepare the immune antigen (SM-BSA) and the coating antigen(SM-OVA). The conjugants were identified by UV scanning and SDS-PAGE. Thecoupling rates of SM-BSA and SM-OVA were7.6:1and17.7:1, respectively.2. SM polyclonal antibody and monoclonal antibody were prepared andidentified.6New Zealand rabbits and3Balb/C mice were immuned with SM-BSA.The titers and half inhibitary concentration (IC50) of antiserum against SM wereestimated by indirect ELISA and indirect competitive ELISA. The best mouse waschosed to further prepare the monoclonal antibodies against SM according to the titerand median inhibitory concentration.After8immunes, the rabbit polyclonal antibodies (rSM pAbs) against SM wereobtained rSM pAb from the antisera of New Zealand white rabbits. The rSM pAb ofrabbit3was best with the titer was of1:8000, the IC50of3.32ng/mL and thecross-reactivity rate to streptomycin and dihydrostreptomycin of100%and120.78%, no cross-reactivity to another antibiotcs. The antisera (mSM pAbs) of Balb/C micewere estimated after5immunes. According to indirect ELISA, the titer of mouse2antiserum was up to1:80000; IC50is3.44ng/mL. The spleen of Balb/C mouse2was chosed to fuse with sp2/0tumor cell. One sensitive and specific hybridoma cellline1G1-E3secreting monoclonal antibodies against SM was screened and cloned.The hybridoma cell could secret high affinity monoclonal antidodies and ascitesantibodies with the titers of1:1280and1:1x106. The IC50of ascites antibodiesagainst SM was2.91ng/mL, the cross reaction rate to SM and DHSM were100%and116.40%, no cross reaction to other antibiotics. The antibodies prepared areavailable for SM residual detection, but mSM mAb has better performance.3. One direct competitive ELISA kit (dcELISA kit) based on mSM mAb wasdeveloped for rapid detection of streptomycin residue. The standard curve of SMdcELISA kit was showed S shape with the linear detection range of0.5~40.5ng/mL,the IC50of3.60ng/mL. The average recoveries of spiked milk, honey samples were80%~120%. The average intra-assay and interassay variability coefficient were lessthan15%. The effect of different biological matrix on SM-Kit testing was small.SM-Kit could be maintained for more than6months at4℃.4. Two rapid detection schemes were developed based on SM aptamer. Thestandard curves were showed with the IC50of160.83ng/mL and168.16ng/mL forrapid detection of SM. The results were suggested that the RNA aptamer-basedmethod was suitable for rapid detection of SM and laied a good foundation forfurther study. |
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