Journal of Virologicalklethods, 2 (1981) 349-356 @ Elsevier/North-Holland Biomedical Press 349 A MODIFIED TECHNIQUE FOR THE DETECTION OF HEPATITIS B VIRUSSPECIFIC DNA POLYMERASE C.T. FANG, N. NATH*, M. PIELECH and R.Y. DODD Transmissible Diseases and Immunology Laboratory, American Red Cross Blood Services Labora tories. 9312 Old Georgetown Road, Bethesda, MD 20014, U.S.A. (Accepted 17 February 1981) A modified and improved technique for the detection of hepatitis B virus-specific DNA polymerase activity is described. DNA polymerase is released from Dane particles by mixing samples with the detergent Nonidet P-40 and p-mercaptoethanol. After incubation of pretreated samples with a reaction mixture containing tritiated thymidine-methyl-5’-triphosphate (‘H-TTP), DNA is precipitated onto a trichloroacetic acid (TCA)-treated paper. Unincorporated ‘H-TTP is then chromatographically eluted with a 5% TCA solution and precipitated counts are determined. A sample is considered positive for DNA polymerase if the incorporated counts are significantly higher than the counts of a group of negative control samples. The modifications include pretreatment of the paper with TCA, chromatographic elution of un- incorporated ‘H-TTP with TCA solution, prefiltration of the sample through bacteriological filters, and use of sound statistical methods for evaluation of data. These changes have led to a highly re- producible, reliable and sensitive technique. The coefficient of variation of negative control samples from various test runs was in the range of 2.7.-8.5%. A linear relationship between incorporated counts and DNA polymerase concentration was shown. A total of 419 serum samples from asymptomatic HBsAg-carrying blood donors were tested. Twenty-three (5.5%) of these were found to contain detectable DNA polymerase activity, All 23 samples also contained HBeAg. INTRODUCTION The discovery of hepatitis B surface antigen (HBsAg) in 196.5 by Blumberg et al. provided a diagnostic marker for the screening of asymptomatic carriers of hepatitis B virus (HBV). HBsAg represents excess viral coat material and its level in blood seems un- related to the severity of the disease. Indeed, some carriers who have high titers of cir- culating HBsAg show slight or no liver damage (Barker and Murray, 1971; Feinman et al., 1973). * To whom correspondence and requests for reprints should be addressed.