| The hapten of norfloxacin was synthesized by chemical paths. After purification,the structure of hapten was confirmed by infrared spectroscopy,mass spectroscopy and nuclear magnetic resonance spectroscopy.The hapten was covalently conjugated to carrier proteins BSA and OVA to synthesize to prepare immunogen and coating antigen by the method of modified active ester. After the conjugating was verifixed by UV spectrometry, the NFLX-Hp-BSA was used as immunogensto immunize New Zealand white rabbits to prepare the specific antiserum to the related antigen can be obtained. The middle titers for anti-NFLX-Hp-BSA serum was 6.4×104 determined by the indirect ELISA, and 1/64 measured by the method of agar double immunodiffusion. The polyclone antibodies separated from antiserum by salting out method with 35% saturated ammonium sulfate (S.A.S) and freeze-dried in a high vacuum. The NFLX-Hp was conjugated to HRP .The HRP tagged hapten was purified by dialysis and Sephadex chromatography. The NFLX-Hp-HRP maintained both immunological activity and enzyme activity.The effects of pH value, ionic intension and organic solvent(methanol,acetonitrile and acetone) on the (enzyme-tagged-hapten, E-H) direct competitive ELISA were evaluated. The results showed that the optimal pH for antibody coating and norfloxacin -antibody reaction is 6.2. In a certain range the affinity of norfloxacin-antibody could be improved if the ionic intension in buffer was increased. Acetone,methanol and acetonitrile had an obvious influence on E-H direct competitive ELISA if their concentration is more higher 5% in reaction medium.The direct competitive ELISA of norfloxacin has been developed successfully, and the ELISA conditions were selected by phalanx test, 10μg/mL of antibody was used to coat microplate and the NFLX-Hp-HRP was diluted 16000 times. Under optimized conditions, the standard ELISA inhibition curve for the detection of norfloxacin was established and the regression equation was I = 16.17LogC + 67.57 ( r 2= 0.978),the linear concentrations of detection is ranged from 100μg/mL to 0.01μg/mL, the half-maximal inhibition(IC50)was 0.082μg/mL and IC20 was 1.14ng/mL with the relative standard deviation (RSD) of 8.33(n=5).The cross reactions (CR%) of hapten to antibody was 38139.535 which was highest than other. The levofloxacin,ciprofloxacin and lomefloxacin's cross reactions to antibody were 8.884,3.306 and 0.869.The standard norfloxacin was spiked in chicken serum at the levels of 0.5mg NFLX/kg or 5mg NFLX/kg. The sample was extracted by 1.0mol/mL hydrochloric acid, and determined by ELISA. The recovery rate was 86.34%～107.40%, the average was 97.285% and the RSD(n=4) was 6.71 at the spiked level of 0.5 mg NFLX/kg. The recovery was 81.11%～101.91%, the average was 93.035% and the RSD(n=4) was 9.53 at the spiked level of 5 mg NFLX/kg. It reached the requirements of NFLX residue analysis.
|
PDF Full Text Request