Prepration And Application Of Monoclonal Antibody Against Astragaloside ?

Astragalus is come from the dry root of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao or Astraga Lus membranaceus(Fisch.)Bge,a traditional Chinese medicinal.It is necessary to reinforce Qi in traditional Chinese medicine,and is well known as one of the most commonly used health functional food materials.Astragaloside ?(for shot AST)is one of the main active substances of the Chinese medicinal herb Astragalus membranaceus(Fisch.)Bge and the target components for quality identification of it and its products.Studies indicate that AST has many biological activities,such as regulating the immunity of the body,protecting the tissues and organs,reducing blood sugar,anti apoptosis,anti inflammation and anti inflammation.At present,the content of AST in Astragalus and its products is determined by high performance liquid chromatography with evaporative light scattering(HPLC-ELSD).Although this method has high specificity and high sensitivity,the sample pretreatment process is complicated,the cost is high,and it is not suitable for the screening of large quantities of samples.To establish a fast,sensitive and economic method for the determination of AST can be used to supplement the deficiency of this method,and to complement each other.In this study,the artificial antigen of AST was synthesized,monoclonal antibody against AST was prepared,the indirect competitive ELISA method was established,we developed successfully AST specific ELISA detection kit for detecting the AST of Astragalus and its products,which is high sensitivity and strong specificity,simple pretreatment,easy operation,fast response,low cost,suitable for the analysis of the scene detection and large quantities of samples.The main research content and innovative research results are as follows:1.Synthesis and Evaluation of Artificial Antigens for Astragaloside ?.Immuning antigen(AST-BSA)and coating antigen(AST-OVA)were synthesized by sodium periodate.Discussed the synthesis mechanism according to the structural characteristics,and verify the coupling reaction by the simulation of conjugate space structure with molecular dynamics.The immuning antigen AST-BSA coupling ratio was 13:1 determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI TOF MS).Polyacrylamide gel electrophoresis(SDS-PAGE)and animal immunization showed that the antigen synthesis was successful,and the synthesis efficiency was high.The method was simple and feasible,and the immuning antigen(AST-BSA)and the coating antigen(AST-OVA)could be used for the preparation of monoclonal antibody against AST.2.Prepration and identification of monoclonal antibodies against Astragaloside ?.With AST-BSA as immunogen,Balb/ C mice were immunized five times.We detected the antibody titer in the sera of immunized mice with 1?25600 by indirect ELISA.Taking PEG-4000 as fusion agent.The immunized splenocytes were isolated and fused with myeloma cells,SP2/ 0.After HAT screening and the method of limited dilution cloning,two hybridoma cell lines that secretes MAb were obtained,named 1D5 and 3D11 cells.The titer and specificity of 3D11 are better than that of 1D5.The expanded 3D11 hybridoma cells(1 x 106 /m L,1m L)were injected into the mice to induce ascites.The ascites titer detected by indirect ELISA was 1?27000.The indirect competitive ELISA method was used to detect the antibody specificity,and the competitive inhibition rate of IC50 minimum dose is 50 ng/m L.The ascites was collected,then obtained higher purity monoclonal antibodies agaist AST.3.Prepared an immunoaffinity chromatography column with AST and purify monoclonal antibodies against Astragaloside ?.The AST was coupled with the epoxy activated agarose gel(EAS6B),and the conjugates were identified by infrared spectrophotometry(IR).Coupling reaction conditions were optimized by two factor range analysis: temperature 45?,p H 10.Prepared an immunoaffinity chromatography column with AST as the ligand and purified monoclonal antibodies against Astragaloside ?.In order to verify the effect purification of immunoaffinity we compare d the results with Protein G column.The equal amount of ascites,the amount of monoclonal antibody agaist ASTpurified by Protein G column is slightly higher than the AST column;and the titer of monoclonal antibody agaist AST purified by AST column up to 1?25600,higher than the titer of monoclonal antibody agaist AST purified by Protein G column.And the competitive inhibition rate of IC50 minimum dose is 5 ng/m L for monoclonal antibody agaist AST purified by AST column,then 50 ng/m L purified by Protein G column.The results showed that the effect of the self-made AST immunoaffinity column for the purification of monoclonal antibody was significantly better than that of Protein G.4.Establish an indirect enzyme linked immunosorbent assay with monoclonal antibody against Astragaloside ?.The concentration of monoclonal antibody agaist AST is 1?3200 that we selected by indirect competitive ELISA method.The optimal concentration of coating antigen(AST-OVA)was 1.25 ?g/m L that determined by the square(board)titration.The best dilution of enzyme labeled antibody was 6000.AST standard quality concentration of the logarithm of lg C for the horizontal coordinates(X),its corresponding to the competitive inhibition rate of IC for the vertical coordinates(Y),drawing the standard curve,the regression equation is obtained: Y= 0.4192X-0.5677,correlation coefficient R2 was 0.9923,the detection linear range of this method is 39~1830 ng/m L.Study methodology,parallel holes between the precision RSD was 0.44% ~ 1.4%,RSD ELISA plate between the precision is 0.74% ~ 3.2%,recovery of high to low sample rate were 107.5%,104.3%,98.5%,the average recovery rate was 103.4%.In order to verify the feasibility of the method,the content of AST in the same astragalus extract was determined by the method and HPLC-ELSD respectively.The result of the former is 1.3528 mg/m L and the latter is 1.3200 mg/m L.The relative error between the two methods was 2.42%,which indicated that the results obtained by the two methods were basically consistent.It is proved that the method founded can be used for the analysis and determination of AST.5.Preparation of ELISA detection kit for Astragaloside ?.According to the assembly process and the conditions of idc ELISA detecting AST,3 batches of AST idc ELISA Kit have been assembled,and the kit standard repeatability,kit sample repeatability,kit accuracy,specificity and stability of the kit were tested.The results of tests,the 10 standard precision measurement in the range of 4.46% ~ 9.18%,the coefficient of variation of less than 10%,the inter batch coefficient of variation of less than 15%.The recoveries of the two standard concentrations were 103.2% and 106.4%.The cross reaction rates of AST and ginsenoside Re,ginsenoside Rb1,ginsenoside a,ginsenoside D,and so on were <1%.The test results show that the reagent box can be stored at least 6 months or more at 4 degrees.Applicating the researched ELISA kit,examined the content of AST in solid samples such as Radix Astragali,Wujibaifeng pills,Yuping Feng capsule and Buzhongyiqi pills,etc and liquid(or semi solids)samples such as blood Kang oral liquid,aguiyanxue syrup and fitness Changchun paste samples,the results are basically the same.It is showed that the detection kit of AST ELISA can be used for the content determination of AST in Radix Astragali and its products.