Preparation Of Monoclonal Antibody Against Heart Failure Marker ST2 And Study On Electrochemical Biosensor For Determing ST2

Heart failure（Heart Failure,HF）is a serious and terminal stage of various heart diseases with high incidence.It is one of the most important cardiovascular diseases today.In recent years,clinically,it is advocated to detect the changes of myocardial injury markers to reflect arrhythmia,so as to judge the type of disease.At present,the commonly used markers of heart failure are B-type natriuretic peptide（BNP）and N-terminal brain natriuretic peptide（NT-pro BNP）.In recent years,the growth stimulating gene 2 protein（ST2）of interleukin-1（IL-1）receptor family has been widely studied as a new marker of heart failure,which has far-reaching clinical significance in pre-screening,treatment guidance and post-treatment monitoring of heart failure.Heart failure marker ST2 can help diagnose acute myocardial infarction and heart failure-related diseases and predict readmission events and mortality in patients with heart failure.The purpose of this paper is to prepare ST2 antigens and antibodies with strong specificity and high activity,and use them as reagent materials to develop a high sensitive electrochemical immunosensor based on MXene modified screen printed electrode for the detection of ST2 and to provide a new detection technique for the diagnosis of heart failure.The research contents of this paper are as follows:（1）According to the ST2 gene sequence published in Genbank（entry number:D12763.1）,the whole ST2 gene was synthesized chemically and cloned into p ET28a（+）vector.The recombinant plasmid p ET28a（+）-ST2 was constructed and introduced into BL21（DE3）receptive cells.The temperature,time and concentration of inducer were optimized,and ST2 protein was expressed in large quantities according to the optimal expression conditions obtained by screening.The Ni column（His-tag）in the protein purification system was used to separate and purify the ST2recombinant protein.The purified ST2 recombinant protein was analyzed by SDS-PAGE gel electrophoresis,Western-blot specificity analysis,and concentration（2）determination by BCA method.The active ST2 recombinant protein was obtained by dialysis.（3）The ST2 recombinant protein was emulsified with adjuvant as immunogen,and BALB/c mice were immunized by long-term immunization.The spleen of mice after four times of immunization and one step of booster immunization was taken and the spleen cell suspension was prepared and mixed with SP2/0 myeloma cells to induce cell fusion with PEG1500.The supernatants of fusion cells and subcloned cells were repeatedly screened by indirect ELISA.The obtained monoclonal cell lines were expanded and cultured,and then injected into the abdominal cavity of BALB/c mice to prepare ascites.The ascites was collected and the antibody was purified by protein A column,and the biological characteristics（purity,concentration,specificity,titer）of the purified antibody were identified.（4）Construction of a highly sensitive electrochemical biosensor based on MXene modified screen printed electrode for the detection of ST2.A highly sensitive electrochemical biosensor based on MXene modified screen-printed electrode was constructed for the detection of ST2.The preparation of MXene was characterized by X-ray diffraction pattern（XRD）,field emission electron microscopy（SEM）and SEM-Energy dispersive X-ray energy spectroscopy（EDX）.Cyclic voltammetry（CV）and differential voltammetry（DPV）were used to monitor the synthesis of the sensor.The linear measurement range of ST2 electrochemical biosensors was determined,and then the specificity,reproducibility and clinical performance of the sensors were evaluated.Result:（1）After double enzyme digestion of recombinant plasmid p ET28a（+）-ST2,the target band appeared at 1014bp,which was completely consistent with the corresponding known gene sequence after sequencing,indicating that the recombinant plasmid was successfully constructed.The plasmid was transformed into Escherichia coli BL21（DE3）receptive cells for optimal expression conditions.The optimal expression conditions were 30℃,0.2mmol/L,10h.The results of ST2 electrophoresis after purification by Ni column affinity chromatography showed that there was a（2）single band around 38KD,which was consistent with the target band.Western-blot specificity analysis showed that there was an obvious band at 38KD,indicating that ST2 protein was successfully expressed.（3）The cell fusion rate reached 100%and the positive rate was 99.8%.Six monoclonal hybridoma cell lines of ST2 were obtained and named as C-4D-6G,B-9E-12C,B-11G-11F,C-4D-4C,B-11G-11A,B-11G-11E.The results of subtype identification showed that C-4D-6G was Ig G2b,and the rest were Ig G1 subtype.Cell lines C-4D-6G and B-9E-12C were selected to prepare and purify ascites monoclonal antibodies,and then biological characteristics were identified.SDS-PAGE results showed obvious bands at 25KD and50KD.The concentration of monoclonal antibody to ST2 was 2.0485mg/m L by BCA method.Western-blot results showed that there were obvious specific bands at 38KD,indicating that the prepared monoclonal antibody to ST2 had good specificity.The titers of C-4D-6G and B-9E-12C monoclonal cell lines could reach more than 1:1024000 by ELISA.（4）MXene was successfully prepared,and a high-sensitivity electrochemical biosensor based on MXene modified screen-printed electrode was established.The linear detection range(1ng·m L^-1to 400ng·m L^-1)of the immunosensor was determined,and the detection limit（LOD）was 0.171ng·m L^-1.It had good repeatability（RSDS were 3.46%）.The specificity of the immunosensor was verified by detecting clinical samples of degamma-abnormal prothrombin（DCP）,anti-Muller hormone（AMH）,α-fetoprotein（AFP）and ST2.The results showed that the change of peak current value of the reaction between the sensor and ST2 was greater than that of DCP,AMH and AFP.Through the detection of 3 positive clinical samples and 3negative clinical samples of ST2,the results show that the change of peak current value of the positive clinical samples is higher than that of the negative samples.Through statistical analysis,the sensor has a significant difference in the detection of the positive samples and the negative samples.Conclusion:In this paper,the prokaryotic expression system was used to prepare highly active ST2 protein,and the monoclonal antibody of ST2 with high efficiency and high specificity was prepared.The ST2 electrochemical immunosensor based on MXene modified screen printing electrode was constructed by using the prepared ST2antigen antibody,which provides a new detection technology for the diagnosis of heart failure and has important clinical application value.