| Glycyrrhizic acid(GA) is one of the most important bioactive components in Licorice.Modern research has indicated that GA has extensive pharmaceutical properties such as anti-tumor activity,anti-inflammatory,anti-viral activity, stimulating immune function.It usually serves as a quantification index for Licorice and related pharmaceuticals.Therefore,the quantification of GA has great significance in the study of Traditional Chinese Medicines(TCM).In this study,we successfully synthesized the artificial antigents of GA and carrier protein conjugates.Then,we prepared the monoclonal antibody(MAb) against GA through hybridoma technology.Based on the purified anti-GA MAb,we established an enzyme linked immunosorbent assay(ELISA) method to quantify GA extracted from Licorice.The main results are as following:(1) Artificial antigents for immunization(GA-BSA) and coating(GA-HSA) were synthesized through sodium periodate oxidization.The hapten number in the conjugates determined by matrix-assisted laser desorption/ionization tof mass spectrometry(MALDI-TOF-MS) was eight and five respectively.(2) Three mice were immunized with the artificial antigen.After the fourth boost immunization,the antibody titers in the serum were about 16 000:1,64 000:1 and 32 000:1,respectively.(3) The spleen cells from the mouse with the highest antibody titer value were fused with myeloma cells.The positive wells rate was 27.8%.After three times of limiting dilution,we obtained nine hybridoma cell lines(GA1C3G6,GA1C4D4, GA1D4C10,GA5A1F4,GA6B3C10,GA7A6C5,GA8C5B10,GA10C4F9, GA10A5G5) which can secrete MAb against GA.Their antibody titers in culture solution were determined by indirect ELISA.GA1C4D4 and GA10A5G5 which had the highest antibody titer were injected into mice's abdominal cavity to induce the production of MAbs.(4) The MAbs in the ascites collections were purified by protein G.The MAb concentration in the ascites is more than 2 mg/mL. (5) The antibody titers in the ascites were above 512 000:1;The MAbs had low cross-reactivity with similar compounds except glycyrrhetinic acid;The affinity constant and sensitivity of the MAbs were about 1×10-7 mol/L and 0.1μg/mL, respectively.(6) With this purified anti-GA MAbs,A competitive ELISA method was established to directly quantify GA in Licorice.The content determined by ELISA Kept high agreement with that determined by high performance liquid chromatography(HPLC).The results indicated that the anti-GA MAbs prepared are qualified in the establishment of sensitive and reliable ELISA method which shows a broader application in TCM studies.
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