| Objective:(1)Synthesis of artificial antigens of saikosaponin c(SSc)to prepare monoclonal antibodies against saikosaponin c.(2)On the basis of the anti-saikosaponin c monoclonal antibody,the corresponding immunoassay method was established,and the method was investigated by a study on methodology.(3)The method of immunoassay of saikosaponin c was used to detect the content of saikosaponin c in Chinese herbal compound,which provided new technical means for Chinese medicine compound research.Methods:(1)SSc-OVA and SSc-BSA were prepared by sodium periodate oxidation method.SSc-BSA was identified by UV spectroscopy and MALDI-TOF-MS.(2)The 6-week-old female BALB/c mice were immunized by subcutaneous injection.The titer and sensitivity of the antibody were measured by ELSIA after four immunizations.Select the enclosed fluid and filter the original working concentration of SSc-OVA.The spleen cells of immunized mice and SP2/0 cells were fused by polypropylene glycol(PEG)method,and the positive monoclonal cell lines were screened by limited dilution method.(3)The expansion of antibody production was carried out by inducing ascites method,and using octanoic acid-ammonium sulfate method for antibody purification.(4)Establish a competitive ELISA method for detecting SSc,based on anti-SSc monoclonal antibody.The specificity,sensitivity,stability and recovery of the method were investigated.(5)The method of SSc immunoassay was used to detect the content of SSc in Chinese herbal compound prepared by Chinese herbal medicine and the content of SSc in the same Chinese herbal compound prepared by Chinese herbal granule.Results:(1)In this study,SSc-BSA and SSc-OVA were successfully prepared by using sodium periodate.SSc-BSA was identified by UV scanning and MALDI-TOF-MS.SSc-BSA was conjugated Successful with coupled ratio of 17.(2)The working concentration of SSc-OVA is 1:4000.Screening blocked liquid species found that 5%skim milk powder closed more complete.titers of anti-SSc antibody induced by SSc-BSA is 1:16000 or more.The determination of serum sensitivity of mice showed that the seropositive competition in number two mice was better,so the spleen of number 2 mice was selected to be fused with SP2/0.(3)This experiment successfully merged SP2/0 cells and number 2 mice spleen cells with fusion rate is 37.76%.The monoclonal cell lines with good positive competition were selected to be monoclonal,and the monoclonal cell lines with stable nature were successfully obtained by repeated passage,cryopreservation and resuscitation.The mice were successfully induced to produce ascites and ascites was purified by octanoic acid-ammonium sulfate method.The ELSIA method was used to detect the titer of antibody before and after purification.(4)On the basis of anti-saikosaponin c monoclonal antibody,the method of enzyme-linked immunosorbent assay(ELISA)was established successfully.The coating concentration of SSc-OVA was 1:10000.The purified antibody was diluted 2000 times as the working fluid concentration.The linearity range is 156.25-2500 ng/mL,the sensitivity is 625 ng/mL,the intra-assay coefficient of variation is less than 7.5%,the inter-assay coefficient of variation is less than 10%,the average recovery rate is 101.7%,and correlation of the ELSIA method and HPLC is better.(5)In this study,SSc immunoassay was used to detect da-chai-hu-tang,xiao-chai-hu-tang and some five Chinese herbal compounds,witch prepared by the traditional Chinese herbal medicine and traditional Chinese medicine granules.The contents of saikosaponinc in chai-hu-gui-zhi-tang,da-chai-hu-tang,xiao-chai-hu-tang,bu-zhong-yi-qi-tang were 10.91mg/g,16.17mg/g,20.45mg/g and 15.06mg/g,respectively,witch prepared by traditional Chinese herbal medicine.The contents of saikosaponin c in chai-hu-gui-zhi-tang,da-chai-hu-tang,xiao-chai-hu-tang,bu-zhong-yi-qi-tang were 10.46mg/g,13.41mg/g,17.70mg/g and 18.46mg/g,respectively,witch prepared by traditional Chinese medicine granules.There is no SSc in gan-cao-xie-xin-tang.So the method is not detected SSc in gan-cao-xie-xin-tang,no matter the traditional Chinese compound was prepared by traditional Chinese herbal medicine and traditional Chinese medicine granules.Conclusion:In this study,we use the sodium periodate oxidation method to synthesize saikosaponin c artificial antigen,and successfully prepared monoclonal antibody of saikosaponin cat the first time.Based on the preparation of monoclonal antibody against saikosaponin c,the enzyme-linked immunosorbent assay(ELISA)was established.The method has the advantages of good stability and high recovery rate,and it can be used for the determination of saikosaponin c.The method has been applied to the detection of traditional Chinese compounds. |
