Preparation Of Monoclonal Antibody Against Arctiin And Establishment Of ELISA Immunoassay Method

Objective:Monoclonal antibody(MAb)against arctiin(ARC)was prepared which is the active constituent and biomarker of Fructus Arctii.An icELISA method was established base on the prepared ARC-MAb and then the methodological investigation of this icELISA method was carried out.This study provides a new alternative analysis technique for pharmacodynamics,quality control,metabolism and distribution study of ARC as well as Chinese medical prescription containing ARC.Methods:The artificial antigen of ARC including ARC-BSA and ARC-OVA are prepared by sodium periodate oxidization method with some modification.The structure of artificial antigen is identified by thin layer chromatography(TLC)and matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).BALB/c mice were immunized with the prepared ARC-BSA conjugate bi-weekly.The tilter and specificity of the anti-serum was detected by ELISA and indirect competive ELISA(icELISA)respectively.The sprenocytes immunized with ARC-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.Then based on the prepared ARC-MAb,an icELISA method for ARC was developed and investigated,and the recovery,sensitivity,specificity,accuracy and variation of this method was studied.Results:(1)Result of TLC and MALDI-TOF-MS indicate that ARC-BSA and ARC-OVA conjugate were synthesized successfully.And MALDI-TOF-MS results showed that the coupling ratio of ARC-BSA is 6:1.BALB/c mice were immunized with the ARC-BSA,after the forth 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 1:16000.(2)The spleenocytes from the mouse were fused with myeloma cells SP2/0.After three times of limiting dilution,we obtained hybridoma cell line ARC-MAb-C9 which can secrete Mab against GRe.It was injected into mice's abdominal cavity to induce the production of MAbs.(3)An icELISA using the Mabs to detect ARC was established.Under optimum conditions,a linear relationship between optical density and doses in the range of 125-3981ng/mL could be achieved with a half-maximal inhibitory concentration of 1466.0ng/mL.The standard curve equation was y =-0.2031n(x)+ 2.1102,R2=0.9969.ARC recoveries were 97.5?107.4%(mean,102.2%).The intra-assay CV were<4.62%,while the inter-assay CV were<8.67%.The cross reaction rate of ARC-MAb with other active ingredients was tested by icELISA.The results showed that the cross reaction rate of ARC-MAb with other components was less than 0.10%,which demonstrate the high specificity of this anti-ARC MAb.Conclusion:In this experiment,the artificial antigen of ARC was successfully synthesized,which was the basis for the screening and preparation of ARC monoclonal antibodies and the establishment of corresponding immune methods.Monoclonal antibodies against ARC were first prepared.Then an icELISA method was established for the first time by using the anti-ARC MAb.