For the production of hepatitis B surface antigen using the mouse cell lines, the cotransformation plasmid pKHBV-3 containing pre-S and S gene was constructed by using the hepatitis B virus DNA isolated from plasma of chronic hepatitis B carriers.
As the transformants secreting hepatitis B surface antigen from mouse LTK-cell lines were isolated by hypoxanthine, aminopterin, thymidine(HAT) selection and hepatitis B surface antigen assay, hepatitis B surface antigen secreting clone(MLB5) was selected and it was confirmed secreting the 1 μg of hepatitis B surface antigen per $10^6$ cells per day.
Hybridomas which are morphologically myeloma type and secrete hepatitis B surface antigen were selected by fusion between MLB5 and myeloma cell lines. KBH3 clone among the hybridomas was able to grow in mouse ascites and secreted hepatitis B surface antigen in the ascitic fluids. Also, KBH3 clone successfully secreted the hepatitis B surface antigen, while the MLB5 clone failed to produced them. The growth of KBH3 showed much faster growth kinetics than that of MLB5.
Mouse monoclonal $IgG_1$ antibody to human chorionic gonadotropin from ascites fluid was purified by one-step hydroxylapatite chromatography. The yield of the purified monoclonal antibody was over 90% and essencially free of contaminating mouse IgG found in ascites fluid.
Hepatitis B surface antigen in the culture media of MLB5 clone was purified by immunoaffinity chromatography using the monoclonal antibody to hepatitis B surface antigen (anti-HBs) and characterized with techniques of electron microscopy, UV-scanning spectrum and sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. Hepatitis B surface antigen of MLB5 showed similar size, shape and same pattern of UV-spectrum such as hepatitis B surface antigen purified from hepatitis B carrier blood was shown.
The polypeptides of hepatitis B surface antigen as the component of 22 nm particles were chromatographed by using the Ultrogel AcA 44 gel filtration. They showed the hepatitis B surface antigen activities and intensified the antigenicity in egg lecithin solution.
A new format for agarose solid-phase enzyme immunoassay(ASEIA) has been developed in which a monoclonal anti-HBs-coupled agarose, incorporated into a 1 ml disposable plastic syringe, regulates the sample and the enzyme reaction. The sensitivity of the technique was equivalent to a commercially available enzyme immunoassay kit and was able to detect 1 μg/1 of hepatitis B surface antigen.
