Using The Immunological Affinity Chromatography Of Monoclonal Antibody To Specifically Knock Out The Chemical Constituents Of Gegen Qinlian Decoction

Gegen Qinlian Decoction (GQD) was reported originally in treatise on Febrile and Miscellaneous Diseases both written by Zhang Zhongjing in the last years of the Han dynasty in China. The decoction is prepared by boiling Radix Puerariae, Rhizoma Coptidis, Liquorice and Radix Scutellariae. In the clinical practice of TCM science, GQD has been used to treat bacillary dysentery, typhoid fever and other diseases, and it has anti-bacterial, anti-pyretic, spasmo lysis, anti-virus, and anti-arrhythmic pharmacological effects. Baicalin and Puerarin are the most important active ingredients in GQD.We use anti-Baicalin monoclonal antibody (anti-BAL MAb) and anti-Puerarin monoclonal antibody (anti-Pue MAb) as a tool to establish the specific immunoaffinity knockout technology methods. The efficacies of anti-bacteria, fever-reducing and anti-diarrhea of GQD water extract were compared before and after knockout, by this way we can clarify the influence of the content of the BAL and Pue on the effects of GQD.This study will offering a new approach, technology and methods for the research of pharmacology efficacy studies of Chinese herb medicine.Objective:To more clearly clarify the influence of the knocking out compounds to GQD, we prepared immunoaffinity chromatography column (IAC) for knocking out GQD and identified the compounds.Methods:1 We prepared the anti-BAL MAb and anti-Pue MAb by the ascites induced method, then we purified MAbs by the caprylic acid-ammonium sulfate method, and determinated the titer, competition and cross reaction rate.2 We prepared the IAC, inspected the elution process parameters, examined gel antibody coupling ratio, column capacity and recovery.3 We prepared the GQD extract, determinated BAL and Pue in GQD by HPLC and ic-ELISA, and verified the accuracy of the ic-ELISA.4 Specific knockout technology provides a powerful tool for confirming the role of the target compounds in plant or its priscriptions, its principle as gene knockout. In the present study, we generated the IAC conjugated with anti-BAL MAb, then loaded the GQD extract washed with the washing solvent, followed by the elution solvent. We established the HPLC fingerprint of GQD, prepared the knockout samples, and we identified the knockout compounds by LC-MS.5 In the present study, we generated the IAC conjugated with anti-Pue MAb, then loaded the GQD extract washed with the washing solvent, followed by the elution solvent. We established the HPLC fingerprint of GQD, prepared the knockout samples, and we identified the knockout compounds by MS.Results:1 The anti-BAL MAb titers in the ascites were above 128000:1, the MAb was highly specific for BAL with<0.01% cross-reactivity with structurally related chemicals, except for Baicalein(30.41%), Wogonin(31.98%); the anti-Pue MAb titers in the ascites were above 128000:1, the MAb was highly specific for Pue with<0.01% cross-reactivity with structurally related chemicals, except for Daidzin(72.3%), Baicalein(58.1%).2 To specifically knock out BAL from GQD, an IAC was prepared. The column was made by covalently coupling the anti-BAL MAb to CNBr-activated Sepharose 4B and used for knocking out BAL from GQD. The knockout and elution conditions were optimized and the knockout buffer was preferred distilled water, elution buffer was preferred HCl(pH 3), the MAb-coupled was 97.9%, the gel coupling was 2.5 mg/mL, and the column capacity was 771 μg. To specifically knock out Pue from GQD, an IAC was prepared. The column was made by covalently coupling the anti-Pue MAb to CNBr-activated Sepharose 4B and used for knocking out Pue from GQD. The knockout and elution conditions were optimized and the knockout buffer was preferred distilled water, elution buffer was preferred HCl(pH 3), the MAb-coupled was 99.3%, the gel coupling was 3.1 mg/mL, and the column capacity was 1000 μg.3 An ic-ELISA was established to directly quanlify BAL and Pue in GQD. The content determined by ic-ELISA kept high agreement with that determined by HPLC.4 The results of MS showed that the IAC using anti-BAL MAb can specifically knock out BAL,Oroxylin A-7-O-glucuronide, Wogonoside, Baicalein, Wogonin from GQD;The IAC can knock out BAL,Galengin-7-O-glucuronide and Baicalein from scutellaria baicalensis; The IAC can knock out Quercetin from pagodatree flower bud. In conclusion, a reliable and one-step method for specifically knocking out BAL has been established.5 The results of MS showed that the IAC using anti-Pue MAb can specifically knock out 3’-hydroxy puerarin, Pue,3’-methoxy puerarin, Daidzin, BAL from GQD; The IAC can knock out 3’-hydroxy puerarin, Pue, Puerarin-7-xyloside, Daidzin from Radix Puerariae.In conclusion, a reliable and one-step method for specifically knocking out Pue has been established.Conclusion:An IAC using anti-BAL MAb was successfully prepared for the first time, and the IAC using anti-Pue MAb was also successfully prepared.The IAC can not only specifically knock out the antigen but also knock out chemical structures very similar to antigen.