Methodology Study Of Clealring Hepaitits B Virus Through Immunoadsorption Assays

Hepatitis B（HB）is a major problem of liver disease worldwide,which is caused by hepatitis B virus（HBV）infection,including acute and chronic hepatitis,some chronic HB patients even lead to cirrhosis or cancer of the liver.Currently there are about 400 million people who has been infected by HBV,and more than 75%is located in the Asian-Pacific region.The number of deaths caused by the HBV is above 1 million each year.Now,the main treatments for hepatitis B are vaccine and drug therapy,the vaccine is the most effective method for the prevention of HBV,at the same time,the drug therapy including interferon similar drugs and nucleic acid drugs.However,there are many problems,these drugs mainly reduce the viral activity and inhibit viral replication,while long-term therapy may produce drug resistance.So,the treatment of hepatitis B and the clearance of HBV is still the focus of the current research highlights of human health.Immunoadsorption is a new therapy in the past years,mainly for the treatment of disease which is difficult to cure through the conventional therapies.The preliminary study shows that the HBV surface antigen（HBs Ag）can be bound by anti-HBs monoclonal antibodies by an immunosorbent assay.Through the way of fixing the antibody,antigen-specific adsorption of certain substances having the ability of adsorbing on the carrier by adsorption performance.Selectively clear out the toxic proteins and endogenous pathogenic factors from blood,so as to achieve the purpose of treating disease and purify the blood.Now,the immunoadsorption has been used to treat autoimmune disease in clinical trials,such as systemic lupus erythematosus,myasthenia gravis,rheumatic diseases and so on.In this study,we find that the HBV surface antigen（HBs Ag）can be bound by anti-HBs antibodies in an immunosorbent assay.The anti-HBs Ag monoclonal antibody（anti-HBs）was immobilized on Sepharose 6 Fast Flow（FF）beads to produce a Sepharose 6 FF-anti-HBs column.Extracorporeal circulation of plasma through this system was able to clear HBV and toxic viral proteins from the plasma.The hydroxyl groups is around the surface of the Sepharose 6 FF beads,which was activated by chemical crosslinking agents,such as brominated cyanide（CNBr）,carbonyldiimidazole（CDI）,epichlorohydrin（ECH）,sodium periodate（Na IO4）and other activating agent,last,verify the sodium perioddate oxidation as the highest fixing way,the antibody fixing rate was 79%,and the adsorption capacity of activated microspheres is 0.96 mg/ml.The methods of sodium periodate oxidation as the best activating way for the high fixing rate,high stability et al.For the adsorption validation study,through the fixing of three different antibodies,anti-HBs,anti-Pre-S1,and HBIG as the ligands and through the plasma adsorption test,the HBs Ag and HBV DNA number of copies were recorded.The three ligands have the ability of adsorbing HBV,and the anti-HBs has the highest rate of adsorbing,the adsorption capacity 11.9μg/ml.Meanwhile,to analysis the stability of the adsorbent fall off,we found that the microspheres have a low falling rate.For the sterilization method,we have choosen the epichlorohydrin,irradiation and intermittent wet and heat sterilizaiton to test the adsorbing rate of the adsorbent.We found the irradiation intensity determine the adsorption efficiency,the higher intensity,the lower adsorption efficiency,and the recovery rates of antibody activity at different intensity are 85%at 30 k Gy and 60.9%at 10 k Gy while the intermittent wet and heat sterilization is 89.8%,so,this methods would be the best way to keep the antibody activity of immunosorbent.The immunoadsorption have reduced the HBs Ag concentration and DNA copies,and the HBs Ag is the important indicator of HBV screening,related reducing rate is 80%around.Other indicators of HB were tested after adsorption treatment,such as HBs Ab,HBe Ag,HBe Ab,HBc Ab;the ion change in plasma were determined:Na⁺,K⁺,Cl^-,Ca²⁺;last,the protein,as total protein creatinine,total cholesterol,serum blbuminand son.All these indicaters are still in the range of normal reference value,which keep the balance of the plasma.So,this strategy could be a part of the combination therapy or separate therapy to surpass the viral activity and clear the HBV for the goal of alleviating and curing the patients.