Preparation And Establishment Of ELISA Method Of Ginsenosides Re Monoclonal Antibodies

Objective: To obtain monoclonal antibodies of GRe, and establish the enzyme linked immunosorbent assays based on the monoclonal antibodies. To provide a new method for quality control and metabolism study.Methods:(1) The hapten of GRe is prepared by sodium periodate oxidization. Then the hapten was coupled respectively to BSA and OVA which give the antigenicity to ginsneoside Re to prepare the artificial antigen. The structure of artificial antigen is identified by thin layer chromatography(TLC) and ultraviolet spectrophotometry(UV).(2) BALB/c mice were immunized with the prepared GRe-BSA conjugate bi-weekly. The tilter and specificity of the anti-serum was detected by indirect ELISA and indirect competive ELISA respectively. The sprenocytes immunized with GRe-BSA was fused with a mouse myeloma cell line SP2/0using PEG method. Monoclonal cells were selected and cultivated using limited dilution method. The ascites was induced with monoclonal cells by intraperitoneal injection.(3) Then GRe ELISA method was developed and investigated, and all the assay recovery, sensitivity, specificity, accuracy and variation meet the requirements of the biological detection. GRe in15Traditional Chinese Medicine prescriptons and human saliva was detected by GRe ELISA method.(4) GRe immune affinity chromatography was prepared by agarose gel activation, antibody-coupled, closed, wash miscellaneous balance, column packing and preparation. The capacity of the column was detected by HPLC and ELISA method after knockout of ginseng sample.Results:(1) The TLC and UV results had showed that GRe-BSA and GRe-OVA was successfully synthesized. BALB/c mice were immunized with the GRe-BSA, after the third immunization, the BALB/c mice were selected by indirect ELISA. The results showed that the anibody titers in all of the mice serum were over1:60000.(2) The spleenocytes from the mouse were fused with myeloma cells SP2/0. After three times of limiting dilution, we obtained hybridoma cell line anti-GRe Mab2F4-3H10which can secrete Mab against GRe. It was injected into mice’s abdominal cavity to induce the production of MAbs.(3) An indirect competitive ELISA (ic-ELISA) with the MAbs to detect GRe was established. Under optimum conditions, a linear relationship between optical density and doses in the range of7.8～500ng/mL could be achieved with a detection limit of0.39ng and a half-maximal inhibitory concentration of64.4ng/mL, the standard curve equation was y=0.2261og2(x)+1.4384, R2=0.998. GRe recoveries were92.7～107%(mean,99.32%), the intra-assay RSDs were<5.77%, while the inter-assay RSDs were<8.76%. The anti-GRe MAb cross-reacted with GRg1and GRd at100%,25.5%respectively and reacted with GRhi, GRg2, and GRg3at low rates but had no reactivity with other flavonoids.(4) GRe concentration is the highest in Ban-xia-xie-xin-tang and the lowest in Zhi-gan-cao-tang.(5) The GRe immunoaffinity chromatography was successfully prepared and GRe can be successfully separated.(6)The results showed that salivary GRe peaked at100min after the oral administration of0.01g/kg of ginseng, with an average Cmax of258.83ng/mL, AUCo-t of72,594(min)·(ng/mL), and MRT of384.978min.Conclusion:The GRe ELISA method can be used for trace analysis more quickly and accurately than conventional HPLC methods. Immune affinity chromatography can be used in traditional Chinese medicine extraction or the compound material knockout.