Preparation Of Anti-APOC3 Monoclonal Antibody And Establishment Of Double-antibody Sandwich ELISA Assay

Apolipoprotein C3(apolipoprotein,APOC3)is a class of rich lipoprotein transporter,which plays an important role in the regulation of intrahepatic lipoprotein metabolism.The increasement of APOC3 content will have a direct impact on lipid metabolism,causing hypertriglyceridemia,atherosclerosis,coronary heart disease,metabolic syndrome and other diseases.Recently,studies have shown that APOC3 can be used as an independent predictor of CVD risk compared with TG,which is a more favorable predictor,and APOC3 as the research target in the treatment of CVD has entered clinical trials.Therefore,monitoring of APOC3 levels in real time and making the APOC3 as a clinical risk factor for CVD new biomarkers,will make a favorable contribution to the prevention and treatment of cardiovascular disease.The current method for the detection of blood lipids is more complex.APOC3 as a new risk factor for CVD,it is urgent to set up a rapid,accurate,simple method for detection of APOC3.Double antibody sandwich ELISA has the characteristics of high sensitivity,specificity,simple operation,and high throughput detection.In order to establish APOC3 double antibody sandwich ELISA method,we first prepared APOC3 monoclonal antibody by hybridoma technique,and then established APOC3 double anti-sandwich ELISA method,as follows.1.The preparation of APOC3 antigenMonoclonal antibody is the key to determine the specificity and sensitivity of double antibody sandwich ELISA.In order to prepare monoclonal antibodies with high affinity and specificity,we first used the pre-constructed APOC3 prokaryotic expression system to induce GST-APOC3 fusion protein under optimized conditions,and identified by Western blot.After analyzing the solubility of GST-APOC3 fusion protein,GST-APOC3 protein was expressed in large quantities and then isolated and purified by GSTrap FF affinity chromatography.The purified fusion protein was quantified by Bradford method.2.Preparation of APOC3 monoclonal antibody by hybridoma techniqueBALB / C mice were immunized with purified GST-APOC3 protein.After three immunizations,we detected the titer of the serum.The mice with the highest titer were immunized and we carried out cell fusion on the 3rd day after immunization.At the same time,we optimized the indirect ELISA assay system,and then screened the positive hybridoma cells by indirect ELISA and subcloned.Finally,we obtained six hybridoma cellssecreting APOC3 monoclonal antibodies,and named 1B7,2G8,3G10,3E9,3B7 and 3D9,respectively.3.Preparation,purification and identification of APOC3 monoclonal antibodyUsing hybridoma cells to prepare the mouse ascites by induction method in vivo,and tested the titer of APOC3 monoclonal antibody.The results showed that 6 antibody titers were greater than 1.28 × 105,of which 3B7 and 3D9 antibody titer up to 3.125×107 or more.Then we purified the antibody by ascites ammonium sulfate precipitation method and obtained the anti-APOC3 monoclonal antibody.The type of monoclonal antibody was then analyzed using an indirect ELISA method.The results showed that 1B7 heavy chain was IgG2 a,2G8 and 3B7 heavy chain as IgM,3E9,3G10 and 3D9 heavy chain as IgG1,6antibody light chain were Kappa.Finally,we identified the specificity of anti-APOC3 monoclonal antibody by indirect ELISA and Western blot.It was confirmed that the prepared monoclonal antibody could react specifically with the APOC3 fusion protein.4.Establishment of APOC3 double antibody sandwich ELISA assayThe APOC3 double sandwich ELISA method was established by using the prepared monoclonal antibody and polyclonal antibody,and the detection system was optimized.The samples were tested with the constructed system and compared with the results of the commercialization kit,and the double anti-sandwich ELISA assay system can detect prokaryotic expression of APOC3 protein,but can not detect the natural APOC3 protein..This study provides a useful reference for the establishment of a method for the detection of APOC3 content,which lays the material foundation for the improvement of CVD lipoprotein analysis system with APOC3 as the goal.