Development Of Immunoassay Based On Carbon Nanotubes For The Detction Of Gentamicin

Gentamicin is a kind of aminoglycoside antibiotics and widely used in clinical treatment and animal husbandry. Antibiotic abuse will do harm to the environment and human. In China, antibiotic production and use are huge, which means a large amount of antibiotic residues may occur in the ecological environment. High sensitivity analysis method is demanded because of the trace gentamicin in environmental samples. Enzyme-likned immunosorbent assay(ELISA) as a rapid,special and sensitive biological method, is gradually applied to detect pollutants in the environment. This research establish a novel method of immunoassay based on carbon nanotubes with preparation of gentamicin monoclonal antibodies.GEN-BSA and GEN-OVA were successfully synthesized by direct chemical crosslinking method. The BALB/C mice were immunized subcutaneously with GEN-BSA conjugates. The spleen cells of the immunized mouse which has the highest inhibition rate was fused with SP2/0 cells. Adopted indirect ELISA method,utilized the clone technology of limit dilution to clone and screen 3 times. Six kind of hybridization tumor cell 2G5D12, 2G5F8, 2G5E7, 2G5D7, 2G5F5G7, 2G5F5H7 were obtained. The immunoglobulin subtypes of the six MAbs were identified as IgG2 b,Kappa light chain. Mice were injected with 2G5D12 hybridoma for production of ascites in large quantity. Ascites were purified by caprylic acid ammonium sulfate precipitation method.The experimental conditions were optimized, and competition standard curve with optimal working concentration was developed. According to the fitting curve, the detection limit(IC90) was 0.026 ng/mL, the half inhibition(IC50) was 0.095 ng/mL,and the linear range(IC80~IC20) was 0.042~0.2438 ng/mL. The cross reactivity of2G5D12 MAbs with tobramycin was 1.73%,and cross reactivity with streptomycin,neomycin, kanamycin, ribostamycin and amikacin were below 0.01%. The rate of recovery was 74.3~126.8%, and the coefficient of variation was less than 13.2% and the method has a good stability. Gentamicin content in environmental samples collected from Jiangsu University was analyzed. The concentration of gentamicin range was 0.04~0.1 ng/mL in water samples, and 0.115~0.167 ng/g in soil sample.Gentamicin concentration was 0.832~1.075ng/mL in milk samples, and1.946~3.577ng/g in animal tissue, which were lower than the standards for maximum limited residue.GEN-HRP was synthesized and carbon nanotubes conjugated with GAM weredeveloped using two kinds of methods. Nonspecific binding of carbon nanotube composite was discussed. After optimizing the experimental conditions, 2% BSA as the diluent, and MWCNTs-PEG-GAM was selected as the carrier to establish a new method based on carbon nanotubes. Gentamicin competition standard curve was obtained. According to the fitting curve calculation of detection limit(IC90) for 0.048ng/mL, half inhibition(IC50) for 0.195 ng/mL, the linear range is 0.080~0.512 ng/mL(IC80~IC20). Four kinds of sample recovery rate and repetition rate were measured, the recovery rate is in the range of 60.1~126.8%, the coefficient of variation is less than18.7 %. The method has a good stability. Qualitative method was developed by filter separation with 0.22 μm membrane. The color becomes shallow at 0.1 ng/mL and disappear at 1ng/mL. GEN concentration in environmental samples was analysed. The concentration of Jiangsu University range was 0.07~0.146 ng/mL in water samples,and 0.198~0.38ng/g in soil sample. Using qualitative method, water samples(NO. 6, 7,10) was negative, and soil samples(NO. 9, 11, 13) were positive.