The Use of Non-Isotopic DNA Probes to Detect Hepatitis B Virus Nucleic Acid by in Situ Hybridisation

Abstract

A non-radioactive in situ hybridisation method for the detection of hepatitis B virus nucleic acid in human liver has been developed. The method can be used on formalin fixed paraffin wax embedded tissue. Both biotin labelled and digoxigenin labelled DNA probes were investigated. The final method is based on the detection of a digoxigenin labelled probe with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The alkaline phosphatase is then visualised with an enzyme substrate reaction. The use of biotin labelled probes was unsuccessful due to high levels of background staining, probably caused by endogenous biotin in the liver sections. Using a digoxigenin labelled probe avoided the problem of endogenous biotin. The digoxigenin based method was optimised by exploring the following parameters of the method; proteinase K digestion, probe size, hybridisation temperature and post hybridisation rinse temperature, the use of a normal swine serum blocking step, using fluorescein and alkaline phosphatase labelled anti-digoxigenin antibodies, concentration of the anti-digoxigenin antibody and length of incubation in chromogen. Sections of liver from 23 individuals were investigated using the optimised method. These cases were also stained for HBsAg and HBcAg by an immunoperoxidase method. HBV DNA, when detected, was always in the presence of at least one other marker of HBV infection. Based on the in situ hybridisation and immunoperoxidase results the cases examined could be divided into four groups; 1) positive for cytoplasmic HBcAg and cytoplasmic HBV DNA (2 cases), these cases are probably supporting active viral replication, 2) positive for cytoplasmic HBV DNA but negative for HBcAg (4 cases), probably not supporting active viral replication, 3) HBsAg and/or HBcAg positive but HBV DNA negative (10 cases), may represent inactive disease, 4) negative for HBV DNA, HBsAg and HBcAg (7 cases). The sensitivity of the in situ hybridisation method used has not been determined. It is possible that the cases in groups 3 and 4 contain levels of HBV DNA below the level of sensitivity of this method.