| Objective:To establishIcELISA for dectecting daidzin and IAC-HPLCfor knocking out puerarin and daidzinbased on anti-puerarin monoclonal antibody (MAb).Methods:1 The hybridoma cells were injected to mice abdominal cavityand anti-Pue MAbwas produced.Then the titerof ascites was verifiedand standard curve of inhibition by DA using MAb was established by icELIS A.2 TheMAbs were purified by caprylic acid precipitation, and IAC based on anti-Pue MAbs was prepared.3 Establish IAC-HPLC for knocking out puerarin based on aBio-RadDuoFlow low pressureliquidchromatographysystemand study the parameters of IAC (washing buffer (double distilled water and PBS), elution buffer (glycine-HCl and HCl), incubating time (0,10,20 min), loading speed (0.4,0.8,1.6 mL. min-1), loading volume (0.33,0.5,1 mL), elution speed (0.5,1.5,2.5 mL·min-1), ionic concentration of elution buffer (0.5,1,1.5 M), and pH of elution buffer (2,3,4)).Precision and accuracywere assessed.4 Synthetizedaidzein-ovalbuminusing sodium periodate and identify artificial antigen by ultraviolet spectrophotometry.Establish an indirect competitive enzyme linked immunosorbent assay to determine daidzin and knockout soybeans by IAC-HPLC.Results:1 Comparing the results, the titers of MAb before and after purification were 1:64000 and 1:32000, respectively. There is no significant difference between MAb purified or not, indicating that purification was successful.2 The immunoaffinity chromatography was prepared successfully with the results that coupling ratio was calculated to be 8.3:1 and column capacity was 16.8 μg per mL medium gel.3 The method was successfully established with the parameters verified, which showed that the double distilled water and glycine-HCL were chosen to be wash buffer and elution buffer, respectively. The results also showed that incubating time, loading speed and volume, ionic concentration of elution buffer have little effect on knocking out, while conditions of streaking occurred at a 0.5 mL·min-1 elution speed and effect decline of elution with pH 4 of elution buffer. Precision and accuracy, recovery and stability were confirmed to reach the requirement. In addition, the Kudzu root extract from knocked out was determined.4 While the peak absorption for DA-OVA was located at 297 nm, the shape of the DA-OVA curve exhibited the curve characteristics of both DA and OVA, which indicated that there might generate new substance, and that the synthesis might be successful. Indirect competitive enzyme linked immunosorbent assay for detecting daidzin was established with a liner range of 10 ng·mL-1 to 10 μg ·mL-1, the detected sensitivity of 823 ng·mL-1 and a linear equation Y=-0.0971nX+1.1512 (R2= 0.9914). Precision and accuracy were detected with intra-assay (<4.1%) and inter-assay (<6.3%). Average recovery was detected to be 105.4%, which indicating that the assay could be applied to determination of daidzin.We developed a rapid one-step chromatography protocol for separating DA from its original medicinal plants using an immunoaffinity column.Conclusion:A novel method, IAC-HPLC, was developed for isolation of Pue and DA. An icELISA method based on anti-Pue MAb was developed for determining DA, which provided a simpler, more efficient, and sensitive approach to determine the DA content. Meanwhile, this study provides a new ideology and method for the extraction and concentration of minor compounds from complex matrices. |
