Preparation And Immunoassay Of Monoclonal Antibodies Against Bioactive Paeoniflorin Of Chinese Materia Medica

Immunoassay systems using monoclonal antibodies(MAbs) against naturally occurring bioactive compounds having lower molecular weights have become one of important tools for the studies on receptor binding analysis,enzyme assay,and qualitative and/or quantitative analysis techniques in herbal medicines owing to their specific affinity.However,there are not many reports and references about the applications of MAbs in the tranditional Chinese medicine(TCM) reserches. Therefore,it is necessarily urgent to introduce the immunoassay techniques in the studies of TCM exploitation and applications.This work aimed at the preparation of the anti-PF MAbs and the establishment of an enzyme-linked immunosorbent assay(ELISA) to detemine the concentration of paeoniflorin(PF),which is a major bioactive constituents in Paeoniae Radix.This immunochemical approach can be used to determine the PF in crude drug and/or prescription samples.This work mainly includes the procedures:Synthesis of antigen conjugates and determination of hapten number in PF-carrier protein conjugates,Immunization and hybridization,Screening and purification of MAbs,Characterization of the MAbs against PF,Establishment of an ELISA for PF by using anti-PF MAbs and its validation.PF-carrier protein conjugates(PF-HSA / PF-OVA) were artificially synthesized by the method using NaIO₄,and the hapten number of the conjugates of PF-HSA was determined to be nine by MALDI-TOF-MS and UV scanning technology.This hapten number was estimated to be enough for the successive animal immunization. Splenocytes yielded from PF-HSA-immunized Balb/c mice were fused with hypoxanthine-aminopterin-thymidine(HAT)-sensitive mouse myeloma cell SP2/0 by PEG 1450.The fusion rate was 87.19%.Following fusion,the cells were selected by HAT medium.Then hybridomas were cloned by the limiting dilution method and screened by indirect ELSA.Two hybridomas producing MAbs reactive to PF were obtained.They were named P1E6 and P5H10.Large-scale antibody production was performed by the culture of established hybridomas in serum-free eRDF medium and by the generation of ascites in mice. Each MAb was purified using a Protein G column.The purity of the obtained MAbs was estimated by the SDS-PAGE and the MALDI-TOF MS to be high enough for the ELISA test.The characteristics of the MAbs were also tested by ELISA.The titre of ascites containing MAbs was 1:256000.The sensitivity were 0.0797μg/ml and 0.124μg/ml respectively.The affinity constant of MAbs P1E6 And P5H10 were 1.819×10^-7 and 2.815×10^-7 mol/L,respectively.They have very high reactivity with PF.while MAbs P1E6 and MAbs P5H10 cross-reacted with albiflorin weakly,0.322%,0.524%. The cross-reactivities of MAbs P1E6 and P5H10 against camphor were 0.018%and 0.078%.The other structural analogs nearly did not cross-react.This showed the high specific activity of PF MAbs against PF.Base on the above-mentioned characteristics of purified anti-PF MAbs to determine the concentration of the extraction.A series of Paeoniae Radix samples have been determined by the competitive ELISA using anti-PF MAbs,and the results showed good agreement with that determined by traditional high-performance liquid chromatography(HPLC),whereas the established competitive ELISA was much sensitive than the HPLC.Some obvious advantages of the newly established ELISA are its rapidity, sensitivity,simplicity and good reproducibility in comparison with HPLC.It's will also useful especially when large number of small and impure samples are to be analyzed.Not only the ELISA can be used to determine the total concentration of PF in Paeoniae Radix and/or compound prescription of TCM study,quality control and standardization of pharmacological activity of a crude drug and its prescription.