Synthesis Of Saikosaponin D Artificial Antigen And Preparation Of Monoclonal Antibodies

Objective:Saikosaponin is one of the most widely used and important drugs in th Febrile and Miscellaneous Diseases.To obtain monoclonal antibodies of saikosaponin D(SSD),and establish the enzyme linked immunosorbent assays based on the monoclonal antibodies.The artificial antigen and artificial package of SSD were synthesized.The method of enzyme-linked immunosorbent assay(ELISA)was established by using SSD monoclonal antibody to detect the content of SSD in Chinese herbal medicine containing Saikosaponin,and the new method was established for the quality control of traditional Chinese medicine compound.The mechanism and mechanism of Saikosaponin Lay the foundation.Methods:(1)The hapten of SSD is prepared by sodium periodate oxidization.Then the hapten was coupled respectively to BSA and OVA which give the antigenicity to SSD to prepare the artificial antigen.(2)BALB/c mice were immunized with the prepared SSD-BSA conjugate biweekly.The specificity of the anti-serum was detected by ic-ELISA respectively.The splenocytes immunized with SSD-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)The peg of mouse spleen cells and mouse bone marrow cells were fused with peg method,and the monoclonal antibody was produced by ascites assay.(4)Using monoclonal antibody to establish ELISA method,the specificity,sensitivity,cross reaction rate,recovery rate and accuracy of the method were tested.(5)Using SSD ELISA method to detect the content of SSD in compound traditional Chinese medicine and compound quality control with SSD.Results:(1)The results of TLC identification showed that SSD-BSA and SSD-OVA was successfully synthesized;The mouse serum titer is above 1:10 000,the standard competitive inhibition curve shows that the antibodies in serum can be specifically combined with SSD.The mouse spleen cells were fused with the spleen cells and the limited dilution method was used to screen the monoclonal cell lines secreting SSD antibody.Based on the monoclonal antibody of SSD,the corresponding ELISA was established.(2)The statistical analysis showed that in the linear range 156.25?5000 ng/ml,the inhibition rate and the linear relationship curve of SSD monoclonal antibody was obtained under the condition of 1:4000 dilution.The regression equation was:y =-0.158In(x)+1.7693,R2=0.9856.The IC50 was 425.81 ng/ml.The recovery rate was 98.15%?104.37%%(mean,101.19%),the difference between plates<8.16%,difference between plates<13.78%.The cross reaction rate of reaction with other traditional Chinese medicine was tested by ELISA.(3)The results showed that the cross reaction rate of SSD and other components was less than 0.10%.It shows that the monoclonal antibody of saikoside has good specificity.Conclusion:In this experiment it is the first time the artificial antigen of SSD was successfully synthesized,which laid the foundation for the screening and preparation of SSD monoclonal antibody and the establishment of corresponding immunization methods.The monoclonal strain of SSD was first screened;The monoclonal antibodies of SSD were prepared for the first time.The enzyme-linked immunosorbent assay(ELISA)was established for the first time using the monoclonal antibodies of the production of SSD.These studies can provide a new method for the quality control of Saikosaponin in the traditional Chinese medicine compound,and lay the foundation for the study of the separation of the active ingredients of the drug,the study of the effective substance base of the traditional Chinese medicine compound,the metabolism of the body and the mechanism of action.