An enzyme-linked immunosorbent assay with monoclonal antibodies for the determination of phosphorylated hepatitis B core protein (p21c) in serum

Journal of Virological Methods 72 (1998) 95 – 103

An enzyme-linked immunosorbent assay with monoclonal antibodies for the determination of phosphorylated hepatitis B core protein (p21c) in serum Sadakazu Usuda a, Hiroaki Okamoto b, Fumio Tsuda a, Takeshi Tanaka c, Yuzo Miyakawa d, Makoto Mayumi b,* a

Department of Medical Sciences, Toshiba General Hospital, Tokyo 140, Japan b Immunology Di6ision, Jichi Medical School, Tochigi-Ken 329 -04, Japan c Japanese Red Cross Saitama Blood Center, Saitama-Ken 338, Japan d Miyakawa Memorial Research Foundation, Tokyo 107, Japan

Received 5 November 1997; received in revised form 6 January 1998; accepted 6 January 1998

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of hepatitis B virus (HBV) core protein (p21c) using monoclonal antibody (mAb) directed to a phosphorylated C-terminal amino acid sequence that is not present in hepatitis B e antigen (HBeAg). HBV virions in the test serum were precipitated with horse polyclonal antibody to hepatitis B surface antigen (HBsAg), dissolved with Tween 20 and NaOH, and then neutralized. HBV core protein (p21c), released from HBV cores by this procedure, was sandwiched between immobilized mAb C33 directed to amino acids (aa) 133 – 140 of the core protein, fixed on a solid support and labeled mAb T2212 that recognizes aa 165–175, only when they are phosphorylated. The method was applied for the detection of phosphorylated p21c in sera from symptom-free carriers and patients with chronic hepatitis. The results indicated a higher extent of phosphorylation in p21c of HBV cores from symptom-free carriers than hepatitis patients. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Hepatitis B virus; Capsid; Hepatitis B e antigen; Monoclonal antibody

1. Introduction

* Corresponding author. Tel.: + 81 285 44 2111; fax: +81 285 44 1557.

The detection of hepatitis B virus (HBV) in sera from symptom-free carriers and patients with hepatitis B signals infectivity for HBV and reflects

0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00019-6

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disease activity. Usually, this is accomplished by detecting DNA of HBV either by dot-blot hybridization (Scotto et al., 1983) or amplification by the polymerase chain reaction (PCR) (Kaneko et al., 1989). However, these methods require radioisotopes or expensive equipment, thereby prohibiting a wide application to epidemiological and clinical studies. Since HBV contains DNA polymerase, the virus can be detected indirectly by determining the DNA polymerase activity associated with hepatitis B surface antigen (HBsAg) (Kaplan et al., 1973). This, also, is too cumbersome for routine use. Immunological methods to detect HBV would be more practical, because they are easy and inexpensive. The nucleocapsid of HBV is an icosahedron made of 180 molecules of the core protein measuring 21 kDa (p21c), and contains HBV DNA as well as DNA polymerase. Hence, immunological detection of p21c in serum would demonstrate HBV. Since p21c shares the N-terminal 149 amino acids (aa) with hepatitis B e antigen (HBeAg) polypeptide called p17e (Takahashi et al., 1983), p21c bears determinants of HBeAg named a and b (Imai et al., 1982). However, p21c possesses an additional epitope on its C-terminal sequence, in a phosphorylated state, which is not carried by p17e (Machida et al., 1991). The epitope is recognized by monoclonal antibody (mAb) named T2212 (Machida et al., 1991). An enzyme-linked immunosorbent assay (ELISA) was developed, with mAb T2212, for a specific detection of phosphorylated p21c. Utilizing this ELISA, phosphorylated p21c was determined in sera from symptom-free carriers and hepatitis patients, and then the extent of phosphorylation was compared after standardization of HBV DNA levels as determined by PCR.

2. Materials and methods

2.1. mAb mAb T2212 (Machida et al., 1991) has been raised against p21c isolated from human hepatoma tissues carried by nude mice (PLC/342) (Matsui et al., 1986). The mAb binds with an

oligopeptide representing aa 165–175 of p21c, only when it is phosphorylated; it does not bind with p17e that lacks aa 150–183 (Machida et al., 1991). mAb 19C1–8 and mAb C33 have been obtained by immunizing mice with p21c derived from HBV (Dane particles) purified from plasma of symptom-free carriers, and they are directed to aa 3–6 and 133–140, respectively (Usuda et al., 1997).

2.2. Treatment of HBV core particles Core particles were isolated by ultracentrifugation from human hepatoma tissues (PLC/342) propagated in nude mice (Miyamoto et al., 1986). Following this, 10 ml of a solution of core particles (1 mg/ml), in 150 mM NaCl supplemented with 2% (vol/vol) Tween 20, were added with 50 ml of NaOH or HCl in increasing concentrations from 0.1 to 2 N, and allowed to stand at room temperature for 30 min. The reactant was neutralized by 50 ml of NaH2PO4 (0.125–2.5 M) or NaOH (0.1–2 N) in phosphate buffer (pH 7.2, 0.01–0.2 M). In all, 10 ml of core particle solution (1 mg/ml) in 150 mM NaCl were mixed with 1 ml of 0.2 M dithiothreitol (DTT), and left at room temperature for 1 h in the dark. Then, the reactant received 1 ml of 1 M iodoacetamide and left at 4°C for 1 h. After treatment, each reactant was adjusted to contain 1 ng/ml of core particles with Tris–HCl buffer (20 mM, pH 7.5) supplemented with 150 mM NaCl, 0.2% Tween 20, 0.1% (wt/vol) NaN3 and 10% (vol/vol) fetal calf serum (FCS).

2.3. ELISA for p21 c Wells of an immunoplate (Greiner, Frickenhausen, Germany) received 100 ml of phosphate buffer (10 mM, pH 7.5) containing 5 mg/ml of mAb C33, and were left at room temperature overnight. The plate was washed five times with 150 mM NaCl containing 0.05% Tween 20, and then unsaturated binding sites were quenched with 10% FCS in Tris–HCl buffer (20 mM, pH 7.5) containing 150 mM NaCl and 0.1% NaN3. The plate was washed and the test sample (100 ml)

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was delivered to a well, and agitated on a shaker (IKA Labortechnik, Janke and Kunkel, Staufen, Germany) for 90 min at room temperature and washed. Then, each well received 50 ml of Tris – HCl buffer containing 2.5 mg/ml mAb T2212 labeled with horseradish peroxidase and supplemented with 0.2% Tween 20, 0.02% (wt/vol) thimerosal, 25% FCS and 1% (vol/vol) mouse serum. The plate was agitated at room temperature for 90 min and washed, and each well received 100 ml of phosphate buffer (100 mM, pH 4.8) containing 0.6 mg/ml o-phenylenediamine, 0.0067% (vol/vol) H2O2 and 50 mM citrate. After 30 min, the reaction was terminated with 50 ml of 4 N H2SO4, and the absorbance at 492 nm (A492) was determined. The reactant in a well with A492 \2.5 was diluted 5-fold with 1.3 N H2SO4, and the obtained absorbance was multiplied by a factor of 5.

2.4. Sucrose density ultracentrifugation A 4-ml plastic tube received successively, 0.8 ml of 50% (wt/wt) sucrose, 1.5 ml of 30% sucrose and 1.5 ml of 15% sucrose in Tris – HCl buffer (10 mM, pH 7.5) containing 150 mM NaCl. A plasma sample positive for HBeAg and containing highly infectious HBV for chimpanzees (Shikata et al., 1977) was diluted to 1:2 and a 200-ml portion was layered onto the surface. The centrifugation was carried out at 300000 × g for 5 h with a Beckman SW60 rotor in the L7-65 centrifuge (Beckman, Palo Alto, CA). The tube was pierced at the bottom and 28 fractions with a size of 140 ml were obtained. A 1-ml portion of each fraction was added with 10 ml of 2% Tween 20 and 50 ml of 0.3 N NaOH, and left at room temperature for 30 min. Thereafter, it was neutralized with 50 ml of 0.375 M NaH2PO4. The reactant was subjected to ELISA for p21c with mAb. HBsAg was determined in 50 ml of a 1:500 dilution and HBeAg in 50 ml of a 1:50 dilution of each fraction by respective ELISA. Nucleic acids were extracted from 5 ml of each fraction, and HBV DNA was semi-quantitated by PCR in a one-fifth amount (corresponding to 1 ml of the fraction).

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2.5. Pre-treatment of sera for the determination of p21 c The test serum (50 ml) or a dilution thereof (50 ml), in phosphate buffer (20 mM, pH 7.2) containing 130 mM NaCl and 0.1% NaN3, was mixed with an equal amount of antibody to HBsAg (anti-HBs); horse polyclonal anti-HBs purified with affinity column of HBsAg, isolated from human plasma, was used at 60 mg/ml in the same buffer (passive hemagglutination titer 215). The mixture was incubated at 37°C for 2 h and left at 4°C overnight, and then spun in a microcentrifuge at 15000×g for 10 min, and the supernatant was discarded. The precipitate was suspended in 10 ml of 150 mM NaCl containing 2% Tween 20 and 0.1% NaN3 for 5 min by agitation. Then it was added with 50 ml of 0.3 N NaOH and agitated for 5 min at room temperature for further solubilization. The reactant was left at room temperature for 30 min, and neutralized with 50 ml of 0.375 M NaH2PO4. It was subjected to ELISA for p21c with mAb.

2.6. Serum samples Serum samples were obtained from 47 symptom-free carriers of HBsAg who were positive for HBeAg, and 125 patients with chronic hepatitis B including 64 positive for HBeAg. Controls were 120 serum samples without any serological markers of HBV infection.

2.7. Markers of HBV infection HBsAg was determined by reversed passive hemagglutination (MyCell; Institute of Immunology, Tokyo, Japan) or ELISA (Immunis HBsAg; Institute of Immunology). HBeAg was determined by ELISA (Immunis HBeAg/Ab EIA; Institute of Immunology). HBV DNA was determined by PCR in accordance with the method described previously (Okamoto et al., 1990). Briefly, serum or its fraction (5 ml) was treated with proteinase K and sodium dodecyl sulfate, and nucleic acids were extracted with phenol/chloroform. HBV DNA was amplified on extracted nucleic acids by PCR with nested primers deduced from the S

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gene. For semi-quantitating HBV DNA, nucleic acids were diluted 10-fold serially in Tris – HCl buffer (10 mM, pH 8.0) containing 1 mM ethylenediaminetetraacetic acid (disodium salt) and 20 mg/ml glycogen (Boehringer Mannheim, Mannheim, Germany), and tested by PCR. The highest dilution (10N) found positive for HBV DNA was estimated, which was converted to the titer per 1 ml of serum.

3. Results

3.1. Release of p21 c from HBV cores by chemical treatments Core particles derived from human hepatoma tissues (PLC/342) propagated in nude mice (Matsui et al., 1986) were treated with increasing concentrations of NaOH or HCl at room temperature for 30 min and then neutralized. Thereafter, the HBV core polypeptide (p21c) was determined by sandwiching between mAb C33 immobilized on a solid support and mAb T2212 (directed to a phosphorylated C-terminal sequence of p21c (aa 165 – 175) that is not present in HBeAg (Machida et al., 1991)) in ELISA (Fig. 1).

The core polypeptide (p21c) was released most efficiently by 0.3 N NaOH. Higher concentrations of NaOH prohibited the release of p21c. By contrast, the release by HCl was concentration-dependent. However, even the highest concentration of HCl (2 N) could release p21c in a level much lower than that accomplished by the optimal concentration of NaOH (0.3 N). Hence, 0.3 N NaOH was adopted for releasing p21c from core particles. Untreated core particles did not give significant A492 readings by ELISA. Reduction of core particles with 20 mM DTT did not release p21c appreciably. Reduction and alkylation released p21c in a level corresponding to only a few percent of that obtained by 0.3 N NaOH.

3.2. Distribution of p21 c in sucrose density fractions of plasma containing HBsAg and HBeAg A plasma positive for HBsAg as well as HBeAg, with an HBV DNA titer 109/ml and highly infectious for chimpanzees (Shikata et al., 1977) was subjected to ultracentrifugation in a sucrose density gradient. Fractions were tested for p21c, after solubilization with 2% Tween 20 and 0.3 N NaOH, as well as for HBsAg, HBeAg and HBV DNA (Fig. 2). The core polypeptide (p21c) appeared in fractions of high density and peaked with HBV DNA. These results attested to the solubilization of p21c from HBV virions containing HBV DNA. HBsAg distributed in fractions of lower density with a less sharp peak, while HBeAg dispersed widely in fractions of much lower density.

3.3. Determination of phosphorylated p21 c in sera

Fig. 1. Treatment with alkali or acid for releasing p21c from HBV cores. Core particles derived from human hepatoma tissues carried by nude mice were treated with increasing concentration of NaOH or HCl in the presence of Tween 20. p21c released into solution was determined by ELISA with mAb.

HBV particles in serum were precipitated with horse polyclonal anti-HBs. They were solubilized with 2% Tween 20 and 0.3 N NaOH, and then neutralized. Released p21c was determined by ELISA with mAb. The results of titrating p21c in three HBeAg-positive sera with high HBsAg hemagglutination titers (214 –215) and a high HBV DNA titer (108/ml) are shown in Fig. 3. The core polypeptide (p21c) in each serum was determined dose-dependently over A492 ranges less than four.

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Fig. 4. Determination of p21c in sera from 47 symptom-free carriers with ELISA with two different sets of mAb. Sera were diluted 20-fold in phosphate buffer (20 mM, pH 7.2) containing 130 mM NaCl and 0.1% (wt/vol) NaN3. A492 readings in ELISA with immobilized C33 and labeled T2212 and those in ELISA with immobilized C33 and labeled 19C1 – 8 are compared.

Fig. 2. Density gradient fractionation of a plasma sample. A plasma with high infectivity for HBV was subjected to ultracentrifugation in a density gradient in sucrose. Fractions were tested for HBsAg, HBeAg and HBV DNA, as well as for p21c after they had been treated with Tween 20 and NaOH.

When ELISA with C33 and T2212 was applied to 120 sera without any serological markers of HBV infection, they gave the mean9 S.D. A492

Fig. 3. Titration of p21c in sera from three HBeAg-positive carriers by ELISA with mAb.

reading at 0.01590.013 (range: 0.008–0.141), and a cut-off limit was set at 0.20. Since mAb T2212 recognizes p21c only when it is phosphorylated (Machida et al., 1991), unphosphorylated p21c would not be detected by ELISA with C33 and T2212. Total p21c, phosphorylated or unphosphorylated, was determined by ELISA with C33 and another mAb 19C1–8 (Usuda et al., 1997) that binds with a unphosphorylated region in p21c (aa positions from 3 to 6). ELISA with C33 and T2212 was applied to 47 HBeAg-positive sera from symptom-free carriers, and the results were compared with those by ELISA with C33 and 19C1–8, (Fig. 4). There was a close correlation of A492 readings between the two assays (r= 0.94). This indicated a constant extent of p21c phosphorylation in HBV cores in sera from symptom-free carriers. Although the ELISA with C33 and 19C1–8 could detect HBeAg polypeptide (p17e), the results would not have been affected by HBeAg in serum, since it should have been removed during the separation of HBV virions before the test. Likewise, antiHBe in sera would not have affected the detection of p21c, because antibodies were removed by this procedure, also.

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Table 1 Detection of phosphorylated p21c in sera from symptom-free carriers and patients with chronic hepatitis HBV DNA titer (10N/ml)

9 8 7 6 5 4 Total

Carriers

Patients

HBeAg (+)

HbeAg (+)

HBeAg (−)

N

p21c

N

p21c

N

p21c

10 29 7 1 — — 47

10 29 7 1

1 24 24 8 7 — 64

1 22 18 3 4

— 2 9 13 15 22 61

1 5 2 0 0 8

(100%) (100%) (100%) (100%)

47 (100%)

3.4. Different extents of p21 c phosphorylation in circulating HBV between symptom-free carriers and hepatitis patients Differences were noted in the detection of phosphorylated p21c between symptom-free carriers and hepatitis patients (Table 1). Phosphorylated p21c was detected in all 47 HBeAg-positive carriers, but less often in 48 of the 64 (75%) hepatitis patients with HBeAg. This was not attributable to different HBV DNA titers in them, because the detection of phosphorylated p21c was less frequent in patients than carriers who had the same HBV DNA titers. Furthermore, phosphorylated p21c was detected only in eight of the 61 (13%) hepatitis patients negative for HBeAg, albeit most of them had lower HBV DNA titers. A492 readings in ELISA for phosphorylated p21c are compared between symptom-free carriers and patients, who were positive for HBeAg and had comparable HBV DNA titers (Table 2). Both for sera with HBV DNA titers at 107 and 108, respectively, symptom-free carriers revealed higher A492 readings for phosphorylated p21c than patients; the difference was significant in comparison of sera with an HBV DNA titer at 108. Differences in A492 readings for phosphorylated p21c paralleled those in HBsAg hemagglutination titers, between symptom-free carriers and patients, which were significant both for sera with HBV DNA titers at 107 and 108.

(100%) (92%) (75%) (38%) (57%)

48 (75%)

(50%) (56%) (15%) (0%) (0%) (13%)

4. Discussion There are no practical methods to determine serologically HBV virions. HBeAg (Magnius and Espmark, 1972) may serve this purpose, since its detection is closely associated with markers of HBV virions such as HBV DNA and HBsAg-associated DNA polymerase (Miyakawa and Mayumi, 1985). However, the lack of physical association of HBeAg with HBV makes it merely an indirect marker of virions. In practice, high titers of HBV DNA can exist in sera not only with HBeAg, but also in those with antibody to HBeAg (Hadziyannis et al., 1983; Bonino et al., 1986). In the circulation, the HBV core polypeptide (p21c) exists only in the viral core which is coated with HBsAg. As such, the detection of p21c would reflect the presence of HBV. A method based on the same principle has been developed for the detection of hepatitis C virus in serum (Kashiwakuma et al., 1996). For immunological detection of p21c, however, it should be noted that p21c bears HBeAg determinants such as a and b (Takahashi et al., 1983). HBeAg polypeptide (p17e) is made of 10 aa coded for by the precore region and 149 aa encoded by the C gene. Therefore, p17e shares 81% of the amino acid sequence with p21c. Machida et al. (1991) reported an mAb (T2212) which is directed to an oligopeptide spanning aa 165–175 in the C-terminus of p21c that are not

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Table 2 Comparison of A492 readings for phosphorylated p21c and HBsAg titers between symptom-free carriers and hepatitis patients who had same HBV DNA titers in serum HBV DNA titer (10N/ml)

Sera from

N

p21c readinga (A492) mean 9S.D. (range)

HBsAg titer (2N) mean 9S.D. (range)

7

Carriers Patients Carriers Patients

7 24 29 24

44.2 956.9 (0.3 – 160.1) 2.2 92.8 (0 – 9.6) 62.3 9 37.6 (1.6 – 142.4)c 5.0 96.2 (0.1 – 26.1)

12.991.8 10.4 9 1.9 14.4 91.0 11.392.8

8

(11 – 15)b (4 – 13) (12 – 16)c (4 – 15)

Serum samples with A492 reading \4.0 were diluted 20-fold in phosphate buffer (20 mM, pH 7.2) containing 130 mM NaCl and 0.1% (wt/vol) NaN3. Then they were subjected to ELISA, and obtained A492 readings were multiplied by a factor of 20. b Higher than patients (PB0.01). c Higher than patients (PB0.001). a

shared by p17e. mAb T2212 binds with the undecapeptide only when it is phosphorylated (Machida et al., 1991). Hence, they deduced that Ser168 and Ser170 in the undecapeptide would be phosphorylated in the authentic HBV core, which has been confirmed later (Liao and Ou, 1995). Furthermore, a protein kinase is present in the HBV core which can phosphorylate serine residues (Albin and Robinson, 1980), and phosphorylation reduces the capacity of HBV core to encapsidate the pregenomic RNA (Kann and Gerlich, 1994). In addition, phosphorylation deprives the affinity of p21c for HBV DNA (Machida et al., 1991), and interferes with the localization of p21c in the nucleus (Liao and Ou, 1995). A method was developed for the detection of phosphorylated p21c by sandwiching between immobilized mAb C33, which is directed to an octapeptide representing aa 133 – 140 (Usuda et al., 1997), and mAb T2212 labeled with horseradish peroxidase. Before the detection of phosphorylated p21c in sera, the peptide should be separated from HBeAg or antibodies and released from the viral core. This was accomplished by precipitating HBV virions with high-titer anti-HBs, followed by solubilization with detergent and alkali. Tween 20 and NaOH served for the solubilization; NaOH was found superior to HCl for releasing p21c from cores, and a concentration at 0.3 N was found optimal. The ELISA with mAb T2212 could detect phosphorylated p21c in sera from symptom-free

carriers and patients with chronic hepatitis B. Differences were found between HBeAg-positive sera from symptom-free carriers and those from hepatitis patients. Sera from symptom-free carriers revealed higher levels of phosphorylated p21c than those from hepatitis patients, even when they were corrected for HBV DNA titers. Moreover, HBV cores from hepatitis patients without HBeAg in serum disclosed lower levels of phosphorylated p21c than the patients with HBeAg at a given HBV DNA titer. It was noted, also, that sera from symptom-free carriers contained higher levels of HBsAg than those from hepatitis patients, in comparison between those with same HBV DNA titers. Differences in the extent of p21c phosphorylation between symptom-free carriers and hepatitis patients may be due to the following factors. First, the state of phosphorylation may differ among them. The higher the extent of phosphorylation, the higher would become the ratio of phosphorylated p21c to HBV DNA. The extent of phosphorylation would be estimated by determination of total p21c to be compared with phosphorylated p21c. Total p21c, either phosphorylated or non-phosphorylated, would be determined by a similar procedure in ELISA with mAb directed to non-phosphorylated regions of p21c. This was accomplished by ELISA with immobilized mAb C33 and labeled mAb 19C1–8 that is directed to an epitope borne by aa 3–6 of p21c; the epitope is in the N-terminus of p21c and does not contain serine or threonine (Usuda et al., 1997).

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There was a close correlation (r =0.94) between the amounts of phosphorylated p21c (determined by ELISA with C33 and T2212) and total p21c (determined by ELISA with C33 and 19C1 – 8) in HBV cores from sera of 47 symptom-free carriers. Hence, the proportion of phosphorylated p21c to total p21c would be constant in HBV cores in the circulation of symptom-free carriers. This suggests the possibility that the state of phosphorylation may be constant also for hepatitis patients, and it would be decreased especially for the patients without HBeAg in serum, thereby creating a low rate of phosphorylated p21c on the basis of HBV DNA. This needs to be evaluated by determining the total amount of p21c by ELISA with mAb 19C1–8, also, in sera from hepatitis patients. Secondly, the majority of HBV particles in sera from symptom-free carriers are found empty by electron microscopy (Takahashi et al., 1976). The degree of emptiness in hepatitis patients, however, might be less than that in symptom-free carriers. A low efficiency of encapsidating the pregenomic RNA in symptom-free carriers, in turn, would be attributable to excessive phosphorylation of p21c which interferes with the encapsidation (Kann and Gerlich, 1994). If such were the case, the presence of phosphorylated p21c in high levels may be responsible for the empty core. Thirdly, deletion of nucleotides occurs frequently in the C gene sequences from patients with chronic hepatitis B (Wakita et al., 1991). Since a C-terminal, arginine-rich sequence of p21c is not required for the assembly of HCV core (Gallina et al., 1989), a deletion within this region may result in cores which would be found negative by ELISA for phosphorylated p21c. However, HBV DNA encapsidated in core particles may not necessarily be that encoding p21c; it could be argued that complementing, non-defective HBV would be coding for authentic p21c for the core particle assembly (Okamoto et al., 1993). This view is supported by reported sites of deletion which cluster in the center of the C gene and rarely occur in the coding sequence for the arginine-rich region (Wakita et al., 1991). The ability to detect phosphorylated p21c, the ELISA with mAb T2212 may have a number of applications, not only for detection of HBV viri-

ons by serological means, but also for studies of the mechanism and effect of p21c phosphorylation.

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