Study On The Methods For Purifying Anti-HBcAg Monoclonal Antibody

Hepatitis B is a serious viral infectious disease to hazard human health,which has the the greatest hazard in various viral hepatitis.There are more than 120 million people of hepatitis B surface antigen （HBsAg）-positive and more than 30 million patients with chronic hepatitis B in China,which cause serious burden to society and families and has become a very prominent problem to today’s society.Hepatitis B core antigen（HBcAg） is the core of the hepatitis B virus which is one of important indicators to determine the replication and activity of hepatitis B virus,but the monoclonal antibody for hepatitis B detection mainly relies on foreign imports,which is very expensive.The mouse ascites containing monoclonal antibodies of anti-hepatitis B core antigen（HBcAg） were prepared and the effects of the various factors on antibody preparation were studied in this experiment. Furthermore,the different methods were used to purify the mAb and the better methods to purify HBcAg monoclonal antibody were established by comparison and parameters optimization,which laid the foundation for clinical application to rapidly diagnose and treat the patients with chronic hepatitis B,and provided a certain reference value to purification of other monoclonal antibodies.Some experimental results are as follows:1.The effects of inducer,age and gender of mice on the product and activity of ascites antibody were studied.The production of ascites can be raised by the pretreatment of female mice of 10～12 weeks with freund’s incomplete adjuvant.The recovery rate and purity of mAb purified by CA-AS were higher than AS.2.The purification of mAb from mouse ascites was studied by using hydrophobic interaction chromatography.The optimized loading condition was pH7.0,20 mmol/L PB+1.2 mol/L（NH₄）₂SO₄,and satisfactory results were obtained using（NH₄）₂SO₄ gradient elution,from 1.2 mol/L（NH₄）₂SO₄ to 0 mol/L（NH₄）₂SO₄,12CV,followed by distilled water,0.5mol/L NaOH,70%ethanol.With this method,purity up to 75% was achieved with recovery of 80%.3.The purity and recovery of mAb could up to 95%and 75% respectively when the ascites sample was diluted one folds with butter（pH7.0,20 mmol/L PB+3 mol/L NaCl） prior to loading on r-protein A affinity chromatography and then eluted with pH3.0,0.1mol/L glycine-HCl.4.The purification of mAb from mouse ascites was studied by using IMAC（Nickel）.The sample was loaded on the IMAC column using a phosphate buffer（pH8.0,20 mmol/L PB）containing 0.5 mol/L NaCl, followed by elution with a pH stepwise elution（pH6.0、5.5、5.0、4.5、4.0）.With this method,purity up to 85%was achieved with recovery of 80%.The biological activity of purified mAb in this experiment did not decline.