Escherichia coli strain HB 101 containing recombinant pHBV 315 DNA was kindly provided by Dr.M.H.Yu (Genetic Engineering Center, KAIST). This plasmid contains the full-length HBV DNA as well as pBR 322 sequences. The bacterial cells were grown in 100 ml LB broth containing ampicillin (Bactotrypton 1%, Bacto-yeast extract 0.5%, NaCl 1%, pH 7.5, ampicillin 0.0035%) overnight with vigorous shaking. The plasmid was isolated by the modified method of Birnboim and Doly.⁸⁾ The cells were harvested at 4°C by spinning at 7,000 × g for 10 minutes. Ten ml of ice—cold solution I (50 mM glucose, 25mM Tris-HCl, pH8.0, 10mM EDTA) was added to resuspend the cell. The cells were harvested again, and resuspended with 3 ml of ice-cold solution I containing 4mg/ml lysozyme. Following incubation for 10 minutes at room temperature, solution II (0.2N NaOH, 1% SDS) was added and gently mixed. After 10 minutes on ice, 4.5 ml of cold 5M potassium acetate (pH 4.8) was added, and white precipitates developed. These were centrifuged at 9,000×g at 4°C for 20 minutes. The supernatant was collected and extracted with phenol. The aqueous phase was separated and precipitated with ethanol.

Instead of using ultracentrifugation for purification of plasmid DNA we employed the PEG precipitation method,⁹⁾ which is simpler and faster.

Purified pHBV 315 DNA was biotinylated by the nick translation method of Rigby et al.¹⁰⁾ Two μg of probe DNA was mixed in 50 μl of solution containing 50 mM Tris-HCl, pH7. 8, 10mM MgCl₂, 50μg/ml bovine serum albumin, 0.1 mM DTT, 20 μM of dATP, dGTP, dCTP (Sigma, St. Louis, MO.) and bio-11-dUTP (Bethesda Research Lab, Gaithersburg, MD.). In order to create nicks in the DNA, this mixture was treated with 0.2 ng of pancreatic DNase I (Sigma, St. Louis, MO.) at 37°C for 15 minutes. Four to five units of E. Coli DNA polymerase I (Pro-mega, Madison, WI.) were added and the reaction proceeded at 15°C for 3.5 hours. The reaction was stopped by adding 2μl of 0.5 M EDTA.

In a parallel reaction, ³²P-labelled HBV DNA probe and ³H-labelled probe were prepared by adding either alpha-³²P dATP (3,000 Ci/mM) (New England Nuclear) or ³H dATP (100 Ci/mM) instead of bio-11-dUTP in a solution containing 20 μM of the other 3 NTP’s.

Labelled HBV DNA was purified by precipitating twice with ethanol in sodium salt and ammonium salt.

Fifty μl of cleared serum samples and 50 μl of unlabelled probe of serially diluted concentration markers were treated with 1 M NaOH and 2M NaCl for 10 minutes, and then filtered through nitrocellulose paper mounted on the Hybridot system (BRL Inc. Gaithersburg, MD.) under vacuum. For neutralization, 200 μl of solution containing Tris-HCl, pH 7.4, 3M NaCl, were filtered again. The air-dried nitrocellulose filter paper was baked at 80°C in a vacuum oven for 2 hours. Prehybridization of the filter paper was carried out at 42°C for 1.5 hours in 50% deionized formamide 5× SSC (1×SSC = 0.015M sodium citrate, 0.15M NaCl), and Denhardt’s medium (0.1% ficoll, 0.1% BSA, 0.1% polyvinyl pyrrolidone) in a polyethylene bag. The filter was hybridized at 42°C for 18 hours with 3ml of 2×SSC containing labelled HBV DNA probe at a concentration of 0.5ng/ml. It was then washed three times in 0.2×SSC at room temperature and once at 40°C.

For detection of the biotinylated HBV DNA on the filter, the nitrocellulose was incubated for 15 minutes at 42°C in TTBS (0.1 M Tris-HCl, pH7.5, 0.1 M NaCl, 2mM MgCl₂, 0.05% Triton X-100) containing 3% crystalline grade BSA (Sigma, St. Louis, MO.). The air-dried nitrocellulose was baked at 80°C for 30 minutes, then rehydrated in TTBS containing 3% crystalline BSA for 20 minutes. The nitrocellulose paper was incubated in avidine-biotin-complex-alkaline phosphatase (Vector Lab. Burlingame, CA.) for 30 minutes in the dark. Color development was carried out by incubation of nitrocellulose in a freshly prepared mixture of substrates of BCIP (5-bromo, 4-chloro, 3-indolyl phosphate) and NBT (nitro blue tetrazolium). Following color development which usually takes 20 minutes to I hour, nitrocellulose was washed with 3 changes TTBS over 10 minutes and then baked at 80°C in a vacuum oven.

Signal detection of radioisotope-labelled HBV DNA was performed by autoradiography of the filter using Kodak X-OMAT cassetts with intensifying screen and Kodak XAR-5 film at −80°C. Exposure time for developing the film was 24 to 72 hours for the ³²P-labelled HBV DNA and 4 weeks for the ³H-labelled probe, respectively. Signal detection of the ³H-labelled HBV DNA was enhanced by using autoradiography enhancer (New England Nuclear).

HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc were assayed by commercial RIA kits (Abbott Lab. North Chicago, IL.).

In order to determine the sensitivity of the biotin-labelled HBV DNA probe assay, a control study using a radioisotope-labelled DNA probe was performed. The indicated amounts of pure unlabelled HBV DNA with serial dilutions were spotted directly on nitrocellulose paper in 50 μl volumes, followed by hybridization with labelled DNA and the signal detection by either enzymatic color development of ABC-AP with substrate or autoradiography of ³²P or ³H-labelling. As seen in Figure 1, the ³²P-labelled probe is the most powerful in resolution and quantitation of the lowest value of 40pg/50 μl The tritiated hybridization assay, however, yields poor in resolution to quantitate that value. By dotting recombinant lambda-HBV DNA to nitrocellulose and hybridizing with our biotinylated probe, a standard pattern was established and used to classify the quantity in serum according to the intensity of staining (Figures 1 and 2). The lowest detection limit of the biotinylated probe hybridization assay was found to be also 40pg/50μl but faint in intensity in comparison to the ³²P-labelled DNA hybridization assay. Contrarilly, the detection limit of tritiated probe was 100pg/50μl.

Of 31 HBsAg positive sera HBV DNA was positive in all of the samples (100%). Twenty five (73.5%) of 34 HBsAg negative sera were also positive in HBV DNA by dot blot hybridization utilizing the biotin-labelled probe (Table 1). Contrarily, nine sera revealed negative HBV DNA were also negative in HBsAg (Table 2).

Surprisingly, as seen in Table 3, all four sera with positive anti-HBs showed positive results in the HBV DNA assay. In fact, two of these four cases were sera from individuals whom had previously been vaccinated against HBV. Furthermore, half of the six sera which were negative in HBV-associated serological markers displayed positivity of HBV DNA.

Correlation of HBeAg/anti-HBe with HBV DNA was investigated in 19 sera of which HBsAg was negative but anti-HBc-positive. Of 13 sera with anti-HBe 10 (76.9%) cases revealed HBV DNA positivity, while four (66.7%) of six sera negative in anti-HBe were positive in HBV DNA (Table 4). This indicated, in turn, that HBV DNA could be detected regardless of the presence of HBsAg and irrespective of HBeAg/anti-HBe status.