| There often exists many imperfect issues in enzyme-linked immunosorbent assay (ELISA), such as sensitivity, stability, specificity and other issues and so on. As one kind of green reaction medium, imidazole ionic liquids(ILs) can improve and enhance the activity of enzyme reaction, stability, and stereoselective, but the impact of the law is not clear now . Horseradish peroxidase (HRP) is the most commonly used in enzyme-linked immunosorbent assay(ELISA). In the subject, filtered a series of structure and the nature of the symmetry of regularly graded imidazole ionic liquids, applied them to horseradish oxide-catalyzed reaction, and then selected one kind of friendly ILs against HRP. And then used it in the deoxynivalenol (DON) enzyme-linked immunosorbent assay media system. Compared with the traditional immune system that the the this ionic liquid was not involved in, sensitivity, reproducibility and other characteristics was improved significantly.Firstly, the response characteristics of horseradish peroxidase (HRP) in such systems containing containing 1-butyl -3-methylimidazolium tetrafluoroborate, 1-isobutyl-3- methylimidazolium tetrafluoroborate ([i-Bmim]BF4) and 1-butyl-3-methylimidazolium was studied. The results showed that, the catalytic activity and stability of HRP is better than other systems, when it contained appropriate [i-Bmim]BF4 (0.0035g/ml to 0.017g/ml) in the system. And the law of temperature and pH influencing on HRP is similar to the system that [i-Bmim]BF4 was not contained. And compared with non-ionic liquid reaction medium, the Vmax and Km of HRP were increased.Secondly, the conjugation of DON and bovine serum albumin BSA were produced according to the method of EDC catalyze. And the initial molar ratio of DON and BSA was 70:1, compared to the molar ratio of 11.8:1 in the complete antigen. Through infrared spectroscopy and the ELISA test, verified the coupling of DON-BSA is successful. Using the The rabbits were immunized by injection of DON-BSA., the most effective antibody was about 51,200. Through indirect competitive immunoassay method, the best work concentration of antigen-antibody and antigen was 1:6400 and1:100 respectively, and its IC50 value was about 549.5ng/ml.Finally, [i-Bmim]BF4 ionic liquids was used in DON ELISA. The results showed that when the concentration of this ionic liquid was 0.014g/ml in the system, the IC50 value of was about 331.1ng/ml, whose sensitivity was increased about 39.7% than that the liquid was not contained. Determination of the reproducibility of experiments, when content of ionic liquid in the system were 0.014g/ml and 0g/ml, their coefficients of variation were 1.14% and 3.99%. It indicated that the involvement of ionic liquids improved the reproducibility of the reaction. In addition, samples of beer standard addition recovery experiment was made. And the results showed that the sample recovery was 93.1% ~ 98.6% when the concentration of [i-Bmim] BF4 was 0.014g/ml the system, and the average coefficient of variation of 5.0%, which was increased by about 10 percentage points than this tionic liquids was not contained.
|
PDF Full Text Request