Research On The Preparation And Application Of Human SST2 Monoclonal Antibody

Heart failure is a serious and terminal stage of cardiovascular disease,which has a high fatality rate.It is one of the cardiovascular diseases that seriously affect human health.In recent years,many studies have found that the expression of soluble growth stimulation expressed gene 2(sST2)is significantly increased in patients with heart failure,and sST2 is directly related to the myocardial fibrosis response,which can reflect and predict the degree of myocardial fibrosis and ventricular remodeling in patients with heart failure,and is not easily influenced by age,sex,renal function and other factors,so it is an ideal indicator to evaluate heart failure and has gradually become a new biomaker for prognosis of heart failure.Therefore,the establishment of a sensitive method to detect the level of sST2 is of great significance not only for the diagnosis and prognosis evaluation of heart failure,but also for studying the transcriptional regulation of the sST2 gene.Because double-antibody sandwich ELISA has the advantages of high sensitivity,high specificity and simple operation,thus this study intends to establish a double-antibody sandwich ELISA for the detection of sST2.Obtaining monoclonal antibodies with high quality is the key to determine the specificity and sensitivity of double-antibody sandwich ELISA.At present,the commonly used method for antibody preparation is to use full-length protein as the immunogen,and the antibodies prepared by this method are difficult to obtain suitable matching antibodies,and it may require a lot of screening work to obtain relatively satisfactory antibody pairs,so we have designed two strategies to prepare monoclonal antibodies to the above problems.First,we used different bioinformatics software to predict the B-cell linear epitopes of sST2,and screened out some potential B-cell linear epitopes that were not interfered with each other in spatial structure.Three sST2 peptides containing two or more epitopes(sST2 F1,F2 and F3)and two B-cell linear epitopes(sST2 EP1 and EP2)were designed.Because three peptides and B-cell epitopes have low molecular weight,it is difficult to be purified and the immunogenicity is weak.Therefore,we use the glutathione sulphur transferase(GST),which is highly expressed and easy to purify,as immune carrier three peptides.Using strong immunogenicity of hepatitis B virus core granule protein HBcAg and adenovirus as immune carrier for B-cell epitopes.Monoclonal antibodies against human sST2 peptide and B-cell epitope were prepared by hybridoma technology,and a double-antibody sandwich ELISA method for detection of sST2 was established by using the obtained antibodies.The specific research content of this topic includes the following aspects.1.Expression and purification of immunogen and screening antigen used for the preparation of monoclonal antibody against human sST2 peptide and B-cell epitopes.1)Prediction B-cell epitope of sST2.The commonly used bioinformatics software was used to predict the B-cell epitope of sST2.Three peptides and two B-cell epitopes of sST2 were designed.2)Expression and purification of immune proteins and screening proteinsused for the preparation of monoclonal antibody against human sST2 peptide and B-cell epitopes.The fusion protein sST2-6His,GST-sST2 F1-6His,GST-sST2 F2-6His,GST-sST2 F3-6His,Grn E-sST2 F1-6His,Grn E-sST2 F2-6His,Grn E-sST2 F3-6His,HBcAg-sST2 EPI-6His,HBcAg-sST2 EP2-6His,Grn E-sST2 EP1-6His,Grn E-sST2 EP2-6His were expressed and purified by the prokaryotic expression system and Ni-NTA affinity chromatography,respectively.3)Preparation and purification of recombinant adenovirus pAd5/El-CMV-HBcAg-sST2 EP1-2/E3-Luciferase-T2A-eGFP/Hexon HVR5-sST2 EP1-2 carrying expression cassette secreting HBcAg-sST2 EP1-2 in E1 region and sST2 EP 1-2 modification in Hexon HVR5.sST2 B-cell epitope was inserted into the adenovirus E1 shuttle vector pShuttle-E1/H1H2-HBcAg and the adenovirus backbone vector pAd5/E3-Luciferase-T2A-eGFP/Hexon-HVR5-LacZ,respectively.Recombinant adenovirus shuttle vector pShuttle-E1/H1H2-HBcAg-sST2 EP1-2 and backbone vector pAd5/E3-Luciferase-T2A-eGFP/Hexon-HVR5-sST2 EP1-2 were obtained by enzyme digestion sequence analysis.Scal linearized pShuttle-E1/H1H2-HBcAg-sST2 EP1-2 and ClaI linearized PAd5/E3-Luciferase-T2A-eGFP/Hexon-HVR5-sST2 EP1-2 were co-transformed into E.eoli BJ5183 by homologous recombination.Recombinant adenovirus plasmids pAd5/E1-CMV-HBcAg-sST2 EP1-2/E3-Luciferase-T2A-eGFP/Hexon HVR5-sST2 EP1-2 were obtained through enzyme digestion.Three recombinant adenovirus vectors were linearized by Pad and then transfected HEK293 cells.Amplified adenovirus was purified by CsC1 density gradient method.4)Expression of sST2 eukaryotic protein.The sST2 gene was cloned into pUC57-kozac-IL-2 signal peptide-MCS vector which used to establish HEK293 Ientiviral cell line expressing sST2 protein.2.Preparation and application of human sST2 monoclonal antibody.1)Monoclonal antibodies against human sST2 peptide and B-cell epitope were prepared by hybridoma technology,and the specificity of the obtained antibodies was identified by Western Blot and Immunoprecipitation2)Establishment of the double-antibody sandwich ELISA method for detection of human sST2.The prepared monoclonal antibody was used for antibody pairing to establish double-antibody sandwich ELISA for detection of human sST2.3)Then the double-antibody sandwich ELISA for detection of sST2 was finally established by the optimization of the concentration of capture antibody,detection antibody and Avidm.HRP,and its standard curve was established.The stability of the double-antibody sandwich ELISA for detection of sST2 was proved by the repeated detection within and between groups.In conclusion,the following results were obtained in this study.(1)Immunogen and screening antigen carrying peptide and B-cell epitope of sST2 were successfully prepared.(2)Six strains of monoclonal antibodies with high potency and specificity against human sST2 were successfully prepared by hybridoma technology,including two strains of monoclonal antibodies against sST2 F2,three strains of monoclonal antibodies against sST2 F3 and one strain of monoclonal antibodies against sST2 EP2.(3)The double-antibody sandwich ELISA for detection of sST2 was successfully established by using.the prepared antibodies and its detection range is 1.524 ng/ml-10 g/ml,its sensitivity is 4.7 ng/ml,its intra-and inter-assay reproducibility is less than 10%.The established double-antibody sandwich ELISA for sST2 has good stability.The results of this study show that the preparation strategy of monoclonal antibody based on B-cell epitope is feasible.This study provides a useful reference for the development of a variety of specific and sensitive methods for the detection of sST2.At the same time,it provides a new idea and method for the establishment of double-antibody sandwich ELISA for other biomarkers and the preparation of neutralizing monoclonal antibodies.