Preparation Of Monoclonal Antibody Against Icariin And Establishment Of ELISA Method And Preliminary Study Of FLISA Method

Objective:To obtain monoclonal antibodies of Icariin (ICA), and establish the enzyme linked immunosorbent assays based on the monoclonal antibodies. To provide a new method for quality control and metabolism study. Try to develop a fluorescent immunoassay approach to detect Icariin (ICA) using a fluorescently labelled monoclonal antibody. The ic-FLISA method can be successfully applied for not only the detection and quantification of ICA in medicines and biological samples, but visualization research on TCM.Methods:(1) The hapten of ICA is prepared by sodium periodate oxidization. Then the hapten was coupled respectively to BSA and OVA which give the antigenicity to Icariin to prepare the artificial antigen. The structure of artificial antigen is identified by TLC and UV.(2) BALB/c mice were immunized with the prepared ICA-BSA conjugate biweekly. The specificity of the anti-serum was detected by ic-ELISA respectively. The splenocytes immunized with ICA-BSA was fused with a mouse myeloma cell line SP2/0 using PEG method. Monoclonal cells were selected and cultivated using limited dilution method. The ascites was induced with monoclonal cells by intraperitoneal injection.(3) Then ICA ELISA method was developed and investigated, and all the assay recovery, sensitivity, specificity, accuracy and variation meet the requirements of the biological detection. ICA in 5 Traditional Chinese Medicine prescriptions was detected by ICA ELISA method and HPLC.(4) Develop a fluorescent immunoassay approach to detect ICA using a fluorescently labelled monoclonal antibody. The ICA-specific antibody was purified by the caprylic acid-ammonium sulfate method and then labelled with Fluoresce Inisothiocyanate (FITC). The FITC-labelled monoclonal antibody was then used to develop indirect competitive fluorescence-linked immunosorbent assay (ic-FLISA).Results:(1) The TLC and UV results had showed that ICA-BSA and ICA-OVA was successfully synthesized. BALB/c mice were immunized with the ICA-BSA, after the fourth immunization, the BALB/c mice were selected by indirect ELISA. The results showed that the anibody titers in all of the mice serum were over 1:8000.(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-ICA Mab which can secrete Mabs against ICA. It was injected into mice’s abdominal cavity to induce the production of MAbs.(3) Ic-ELISA with the MAbs to detect ICA was established. Under optimum conditions, a linear relationship in the range of 7.81～1000 ng/mL could be achieved with a half-maximal inhibitory concentration of 156 ng/mL, the standard curve equation was y=-0.9913Lg(x)+0.9799, R2=0.9828. ICA recoveries were 93.80～118.80%(mean,105.00%), the intra-assay CVs were<3.76%, while the inter-assay CVs were <9.84%. The anti-ICA MAbs had no reactivity with other flavonoids.(4) We also observed good correlation between ELISA and HPLC analyses of total ICA in a crude extract. We successfully developed a reliable ELISA method which will be a useful tool for further exploration into the role of ICA in formulated Chinese medicines.(5) Ic-FLISA with the MAbs to detect ICA was established, a linear relationship between optical density and doses in the range of 1pg～100 μg/mL could be achieved with a half-maximal inhibitory concentration of 531.79 ng/mL, but it remains to be further optimized.