Monoclonal radioimmunoassays for hepatitis B surface antigen: demonstration of hepatitis B virus DNA or related sequences in serum and viral epitopes in immune complexes.

High-affinity monoclonal IgM and IgG antibodies (anti-HBs) to hepatitis B surface antigen (HBsAg) have recently been developed and used by us in the construction of highly sensitive radioimmunoassays for the detection of HBsAg-associated determinants in serum. We now report that selected serum samples demonstrating monoclonal immunoreactive material also contain nucleic acid sequences complementary to hepatitis B virus (HBV) DNA by molecular hybridization analysis. In addition, monoclonal radioimmunoassays can detect viral determinants in HBsAg-anti-HBs immune complexes formed in anti-HBs excess; such determinants are undetectable by commercial radioimmunoassay. These and our previous studies suggest that there are HBV or "HBV-related" agents present in human serum that are detected by monoclonal antibodies but are not identified by conventional polyvalent antibodies.     

Immunodiagnosis of hepatitis B with high-affinity IgM monoclonal antibodies.

High-affinity monoclonal IgG and IgM antibodies to hepatitis B surface antigen (HBsAg) have been prepared and their functional capabilities explored by means of solid-phase radioimmunoassays. 125I-labeled HBsAg binding studies indicated that monoclonal IgM antibodies against HBsAg (anti-HBs) coupled to a solid-phase support quantitatively bound more HbsAg at a faster rate than conventionally prepared anti-HBs reagents or other high-affinity IgG monoclonal anti-HBs antibodies. Consequently, IgM anti-HBs was also radiolabeled, and an IgM-IgM radioimmunoassay was developed for the immunodiagnosis of hepatitis B. The lower limit of this assay was approximately 100 pg +/- 30 (SEM) of HBsAg per ml of serum. Compared to available commercial radioassays, preliminary studies have shown the IgM-IgM assay to have increased sensitivity, which improved the detection of a HBsAg-associated determinant in acute hepatitis and post transfusion hepatitis. It is probable that the multivalent interaction between monoclonal IgM anti-HBs and the polydeterminant HBsAg is important in augmenting the performance of this monoclonal assay.     

Clinical significance of enhanced detection of HBsAg by a monoclonal radioimmunoassay

We assessed the significance of the enhanced detection by monoclonal radioimmunoassay (M-RIA) of HBsAg in serum of patients with hepatitis B virus (HBV) infection. In acute HBV infection, the M-RIA detected HBsAg in the blood for a far longer period of time than previously recognized. In some patients, the "window phase" of HBV infection (defined as the presence of anti-HBc and the lack of detectable HBsAg and anti-HBs) was shortened or completely eliminated. Furthermore, 26% of individuals with anti-HBc as the only serologic marker of recent or past HBV infection were reactive only in the M-RIA. Analysis of patients tested for HBsAg because of suspected underlying liver disease revealed additional HBsAg-positive sera, unrecognized by polyvalent RIA, thus adding important information in the immunodiagnosis of HBV infection. The enhanced performance of the monoclonal RIA compared to conventional polyvalent RIA was due in part to the increased sensitivity of the assay and the observation that M-RIA may detect "hidden" HBV antigens in immune complexes.     

Detection of hepatitis B virus infection in hepatitis B surface antigen-negative hemodialysis patients by monoclonal radioimmunoassays

We studied 375 chronic hemodialysis patients for evidence of hepatitis B virus infection using first- and second-generation monoclonal radioimmunoassays. These assays employ high-affinity monoclonal antibodies produced against antigenic determinants that reside on hepatitis B surface antigen. Such assays have a lower limit of detection for hepatitis B surface antigen-associated determinants in serum of approximately 55 and 15 pg/ml, respectively. We found that 14 of 375 chronic hemodialysis patients were positive for hepatitis B surface antigen by both polyclonal and monoclonal radioimmunoassay. However, an additional 17, some of whom had chronic hepatitis and hepatocellular carcinoma, were identified as harboring hepatitis B virus infection only by the monoclonal radioimmunoassays. Thus the monoclonal radioimmunoassays improved the hepatitis B virus detection rate by 120% (3.7% vs. 8.3%). More importantly, 6 of the 17 monoclonal radioimmunoassay-reactive patients had no serologic evidence of recent or past hepatitis B virus exposure as shown by the absence of antibodies to the hepatitis B core and surface antigens in the blood. We conclude that there are hemodialysis patients with hepatitis B virus infection undetectable by conventional polyclonal radioimmunoassays.     

Latent hepatitis B virus (HBV) infection in systemic necrotizing vasculitis.

We have investigated hepatitis B virus (HBV) infection in systemic necrotizing vasculitis (SNV). Our approach included the detection of the viral surface antigen (HBsAg) with a radioimmunoassay employing monoclonal anti-HBs (m-RIA); in addition, HBV DNA was looked for in serum and peripheral mononuclear blood cells. Among 28 subjects with SNV, 12 were found to be positive for HBsAg with the conventional test (p-RIA) and 7 additional subjects had anti-HBc and/or anti-HBs. From the 16 HBsAg negative individuals, 9 had HBsAg epitopes identified in serum with the m-RIA test and 1 had a low amount of circulating viral DNA. In contrast, only 1 among 6 subjects with other systemic vasculitis showed a positive test for m-RIA and HBV DNA assays; this individual had acquired HIV infection through transfusions which were also probably the source of his HBV infection. HBV DNA sequences were identified in peripheral mononuclear blood cells of 9 from the 37 tested, including 2 individuals who were HBsAg positive only with m-RIA. Therefore, our study indicates a much higher rate of HBV infection in patients with polyarteritis nodosa than previously suspected.     

Incidence and significance of hepatitis B and A virus serologic markers in Greek cirrhotic patients

The incidence of serological markers of hepatitis B and A virus infection was studied by radioimmunoassay in 89 Greek cirrhotic patients. Controls consisted of 90 patients without liver disease. HBsAg was detected in 62 (69.5%) patients, anti-HBs in 35 (39.3%), anti-HBc in 60 (67.4%), HBeAg in 13 (14.6%), anti-HBe in 58 (65.1%), and the anti-HAV in 86 (96.6%). The corresponding figures for the control group were: HBsAg 4 (4.5%), anti-HBs 34 (37.7%), anti-HBc 41 (45.5%), HBeAg 3 (3.3%), anti-HBe 21 (23.3%), and anti-HAV 86 (95.5%). This high incidence of positive reactions in cirrhotic patients strongly suggests the possibility that HBV infection may be an important causative factor in the development of cirrhosis in Greece. No association could be established between hepatitis A virus infection and cirrhosis.     

IgM antibody to hepatitis B core antigen in Korean patients with hepatocellular carcinoma

Primary hepatocellular carcinoma (PHC) has been linked etiologically to persistent hepatitis B virus (HBV) infections by epidemiologic, serologic and molecular lines of evidence. To evaluate the frequency of IgM antibody to the viral core antigen (IgM anti-HBc) detected by a highly sensitive radioimmunoassay, we compared 110 Korean patients with PHC to a group of 63 age- and sex-matched control patients with other tumors. Results were correlated with those of commercially available HBV assays. IgM anti-HBc was found in 74 of 110 PHC patients (67%), but only 1 of 63 (1.6%) control patients. Although HBsAg was found in a larger percentage of PHC patients (81%), it was also present in more control patients (14%). Thus, IgM anti-HBc was more specifically associated with PHC than was the presence of HBsAg. IgM anti-HBc was found in 91% of PHC patients with detectable HBeAg and 74% of PHC patients with positive anti-HBe tests (p less than 0.04). The frequency of IgM anti-HBc was similar among HBsAg-positive PHC patients with and without anti-HBs, or those with low or high levels of serum alpha-fetoprotein. In 18 patients with PHC, IgM anti-HBc was further characterized by rate-zonal centrifugation of sera, all were found to have 19S IgM anti-HBc although 6 also had greater or equal IgM anti-HBc reactivity in the low molecular weight region. The presence of IgM anti-HBc in adult Korean HBsAg carriers may indicate an especially high risk for the development of PHC, and this should be evaluated in prospective studies.     

Evolution of hepatitis B serological markers in HIV-infected patients receiving highly active antiretroviral therapy.

BACKGROUND: Evolution of serological markers of hepatitis B virus (HBV) carriage or infection has rarely been investigated among human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: During the period 1997-2002, a total of 633 HIV-infected patients were tested for HBV serological markers at baseline, including hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs ), antibody to hepatitis B core antigen (anti-HBc), hepatitis C virus (HCV) antibody (anti-HCV) antibody, HCV RNA level, and HBV DNA level, all of which were retested at least 1 year apart. Medical records were reviewed to identify clinical characteristics associated with evolution of these serological markers. RESULTS: After a median duration of follow-up for 4.96 years, 161 patients (25.4%) had changes in HBV serological markers. Of 119 patients (18.8%) who tested positive for HBsAg at baseline, 6 (5.0%) developed anti-HBs, and 9 (7.6%) developed isolated anti-HBc. Of 270 patients (42.7%) who tested positive for anti-HBs, 18 (6.7%) lost anti-HBs. Of 179 patients (28.3%) in whom isolated anti-HBc had been detected, 73 (40.8%) developed anti-HBs, 18 (10.1%) lost all HBV markers, and 7 (3.9%) developed HBsAg. Of 65 patients (10.2%) who tested negative for all HBV markers, 13 (20%) developed anti-HBs, 13 (20%) developed isolated anti-HBc, and 4 (6.2%) developed HBsAg, indicating a high risk of HBV exposure. Patients in whom anti-HBc was detected at baseline were more likely to have acquired immunodeficiency syndrome (P=.008). Multivariate analysis revealed that an increase in the CD4 cell count after the commencement of HAART was significantly associated with persistence or subsequent development of anti-HBs in patients with anti-HBs or anti-HBc at baseline, respectively. CONCLUSIONS: Periodic measurements of HBV serological markers in HIV-infected patients are recommended, because new HBV infections and changes of HBV serological markers are not uncommon in patients with improved immunity after commencement of HAART.     

Hepatitis B virus reactivation and alemtuzumab therapy

Reactivation of hepatitis B virus infection in subjects receiving cytotoxic treatment for heamatological malignancies occurs in 21-53% of chronic HBsAg carriers and in an unknown number of HBsAg negative subjects harbouring occult HBV infection. Immunotherapy with alemtuzumab, a humanized monoclonal antibody against CD52 epitopes on lymphocytes cells produces deep immunosuppression. We describe two subjects with chronic lymphocytic leukaemia and occult HBV infection who developed a virological and biochemical flare of hepatitis B following immunotherapy with alemtuzumab. One of them developed full blown hepatitis with seroreversion from anti-HBs to HBsAg after four weeks of alemtuzumab therapy. Lamivudine (100 mg die) achieved a complete clinical recovery and HBV-DNA clearance from blood within 8 weeks. The second patient (HBsAg and HBV-DNA seronegative, anti-HBs and anti-HBc positive before treatment) was kept under prophylaxis with lamivudine up to three months after alemtuzumab. Two months after withdrawal of lamivudine, clinical and laboratory features of acute hepatitis B developed. Lamivudine therapy was restarted and a prompt recovery was obtained with HBsAg and HBV-DNA clearance.     

Serological Markers of Hepatitis B Virus Infection in Healthy Volunteer Blood Donors in Campania (Southern Italy)

The prevalence of hepatitis B virus (HBV) serological markers was determined in a prospective fashion by radioimmunoassay in 2,084 healthy volunteer blood donors. The results showed that 51.2% of the donors were positive for at least one marker, and the percentage of occurrence of each marker was: HBsAg 5.3, anti-HBs alone 1.7, anti-HBc alone 10.8, anti-HBs and anti-HBc 33.3. Because of the size of the problem this investigation strongly demands further studies on the potential role of blood positive for anti-HBc in transmitting HBV infection in our geographical area.     

Response to hepatitis B vaccine of persons positive for antibody to hepatitis B core antigen.

The significance of antibody to hepatitis B core antigen (anti-HBc) present in a person's serum without hepatitis B surface antigen (HBsAg) or its antibody (anti-HBs) is unknown. Serum specimens from 281 persons initially positive only for anti-HBc by enzyme immunoassay (EIA) were retested by radioimmunoassay (RIA), and of these, 177 (63%) remained positive for anti-HBc by both assays. Of these 177 persons, 3 were positive for HBsAg, and 72 possessed low levels of anti-HBs [less than 10 sample ratio units; (SRU's)]. When persons positive for anti-HBc by EIA and RIA were given one 20-micrograms dose of plasma-derived hepatitis B vaccine and tested for anti-HBs 1 month later, a booster response was observed in 14 of 41 (34%) persons with low level anti-HBs and 3 of 50 (6%) persons negative for anti-HBs. Of those positive only for anti-HBc by EIA but negative by RIA, only 3 of 37 (8.1%) showed a booster response. Of those who completed the three-dose immunization series and did not show a booster response, 63 of 80 (78.8%) developed anti-HBs levels greater than 10 standard ratio unit. The majority of persons with isolated anti-HBc will have a primary rather than a booster response to hepatitis B vaccine.     

Serologic markers of hepatitis A and B in the population of Bali, Indonesia

A total of 343 sera from Balinese subjects in different age groups and geographic locations were tested by radioimmunoassay (RIA) for serum antibodies to hepatitis B surface antigen (anti-HBs) and hepatitis B core antigen (anti-HBc); most sera were also tested for hepatitis B surface antigen (HBsAg), and for antibody to hepatitis A virus (anti-HAV). One hundred percent of the adult population was found to have anti-HAV, with antibody acquisition beginning in early childhood and reaching a level of 95% by the age of 10 years. Antibodies to hepatitis B virus were also frequent in young children, rapidly peaking to near 80% in older children and adolescents, then declining to a plateau that fluctuated between 40% and 60% throughout adult life. Overall, anti-HBc (49%) was detected slightly more often than anti-HBs (45%), but the relative frequencies of the 2 antibodies varied considerably from group to group. Despite these high antibody prevalences, HBsAg was detected in only 1.5% of the general population, and in no woman of child-bearing age. In utero infection is thus far less likely to account for the early acquisition of antibody to hepatitis B virus than inapparent percutaneous transmission occurring under conditions of close personal contact.     

Value of screening for markers of hepatitis in dialysis units

The value of monitoring the serum activity of SGOT, as well as markers of hepatitis B virus and hepatitis A virus infections, in the patients and staff of two dialysis units has been assessed retrospectively. Sera were checked each month for SGOT and HBsAg on 406 patients and 170 staff members over a 4-year period. Anti-HBc, anti-HBs, and anti-hepatitis A antibodies were assayed on the stored sera. Only 30% of the patients had normal SGOT values (less than 65 units per ml) on all occasions. Most of the abnormal values were less than 100 units per ml and could not be explained. Viral hepatitis was a reasonable explanation for only half of those instances where the SGOT value was greater than 100 units per ml. Hepatitis A virus contributed nothing to the problem of dialysis-associated liver disease. Testing for HBsAg alone missed approximately 40% of the hepatitis B events acquired in the unit. Only two of these episodes were of epidemiologic importance, however, because the rest were recognized only after there was serologic resolution of the infection. There was a high frequency of potentially "false positive" reactions with all the antibodies tested. It is not cost-effective to monitor dialysis patients and staff regularly with anti-HBc, anti-HBs, or antibodies against hepatitis A. Initial screening with anti-HBc and anti-HBs on entry to the unit is of value but weak positive results must be interpreted with caution since approximately half of such results will prove to be nonspecific.     

Anti-HBc screening in Indian blood donors：Still an unresolved issue

AIM：To study the seroprevalence of antibody to hepatitis B core antigen （anti-HBc） in healthy blood donors negative for HBsAg and to evaluate whether anti-HBc detection could be adopted in India as a screening assay for HBV in addition to HBsAg. METHODS： A total of 1700 serum samples collected from HBsAg-negative healthy blood donors were tested for the presence of anti-HBc antibody （IgM ＋ IgG）. All samples reactive for anti-HBc antibody were then investigated for presence of anti-HBs and for liver function tests （LFTs）. One hundred serum samples reactive for anti-HBc were tested for HBV DNA by PCR method. RESULTS： Out of 1700 samples tested, 142 （8.4%） blood samples were found to be reactive for anti-HBc. It was signif icantly lower in voluntary （6.9%） as compared to replacement donors （10.4%, P = 0.011）. Seventy- two （50.7%） anti-HBc reactive samples were also reactive for anti-HBs with levels 〉 10 mIU/mL and 70 （49.3%） samples were non-reactive for anti-HBs, these units were labeled as anti-HBc-only. These 142 anti-HBc reactive units were also tested for liver function test. HBV DNA was detected in only 1 of 100 samples tested. CONCLUSION： Keeping in view that 8%-18% of donor population in India is anti-HBc reactive, inclusion of anti- HBc testing will lead to high discard rate. Anti-HBs as proposed previously does not seem to predict clearance of the virus. Cost effectiveness of introducing universalanti-HBc screening and discarding large number of blood units versus considering ID NAT （Individual donor nuclic acid testing） needs to be assessed.     

Serologic markers of hepatitis A and B in chronic active hepatitis.

Sera from 44 patients with a well-documented diagnosis of chronic active hepatitis (CAH) were analysed for antibodies to hepatitis A virus (anti-HAV), hepatitis B surface antigen (HBsAg) and anti-HBs, e-antigen (HBeAg) and anti-HBe, as well as antibodies to hepatitis B core antigen (anti-HBc). Twenty-two patients had serologic evidence of hepatitis A infection. The frequency of anti-HAV was low in patients under 50 years of age (21%) but high among older patients (72%). There was, however, no significant difference between patients and age-matched controls regarding the prevalence of anti-HAV in serum. Markers for hepatitis B virus were found in 10 patients or 23% as compared with about 10% in Swedish blood donors. The results indicate that hepatitis A virus is of little importance in the pathogenesis of CAH and confirm the association between hepatitis B virus and development of chronic active hepatitis.     

Hepatitis,Epidemiology and Liver Function in Hemophiliacs in Sweden

ABSTRACT The epidemiology of viral hepatitis and liver function were studied in a retrospective survey of 69 patients with moderate and severe hemophilia A and B, and with severe von Willebrand's disease. Forty-nine patients were on prophylactic self-therapy and 20 on episodic treatment by medical personnel. Serologic markers of viral hepatitis (HBsAg, anti-HBs, anti-HBc, anti-HAV, and in some cases HBeAg and anti-HBe) and liver function tests (ASAT, ALAT, IgG) were followed for up to 12 years. There was a history of clinical hepatitis in 19%, and 96% showed some serologic evidence of exposure to hepatitis B virus. Only one patient was a HBsAg carrier. The prevalence of elevated ASAT and/or ALAT was 65% and the incidence 96%. In 68% of the patients there had been a transaminase elevation for more than 6 months. The clinical picture, serologic markers or liver function tests showed no significant difference between the types of hemophilia, amounts and modes of therapy, or age groups. The chronic hepatitis seen in our hemophiliacs seemed to be a slowly or non-progressive disease.     

Isolated antibody to hepatitis B core antigen in patients with chronic hepatitis C virus infection

AIM: To evaluate the prevalence of isolated anti-HBc in patients with chronic hepatitis C virus (HCV) infection, and its relation to disease severity. METHODS: We screened all patients with chronic HCV infection referred to King Faisal Specialist Hospital and Research Center for hepatitis B surface antigen (HBsAg), antibody to hepatitis B surface antigen (anti-HBs), and anti-HBc. One hundred and sixty nine patients who tested negative for both HBsAg and anti-HBs were included in this study. RESULTS: Pathologically, 59 had biopsy-proven cirrhosis and 110 had chronic active hepatitis (CAH). Of these 169 patients, 85 (50.3%) tested positive for anti-HBc. Patients with CAH had significantly higher prevalence of isolated anti-HBc than patients with cirrhosis, 71 (64.5%) and 14 (23.7%) respectively (P < 0.001). Twenty-five patients were tested for HBV DNA by qualitative PCR. The test was positive in 3 of them (12%; occult HBV infection). CONCLUSION: Isolated anti-HBc alone is common in Saudi patients with chronic HCV infection, and is significantly more common in those with CAH than those with cirrhosis. Therefore, a screening strategy that only tests for HBsAg and anti-HBs in these patients will miss a large number of individuals with isolated anti-HBc, who may be potentially infectious.     

Hepatitis B virus pre-S gene-encoded antigenic specificity and anti-pre-S antibody: relationship between anti-pre-S response and recovery

A solid-phase radioimmunoassay involving specific antibody was developed for determination of the pre-S gene-encoded epitopes of hepatitis B virus and anti-pre-S antibody in sera of hepatitis B patients. The reaction for pre-S determinants associated with HBsAg was quantitatively inhibited by soluble, polymerized human serum albumin, and the lower limit of the assay was about 1.6 ng of HBsAg per ml. Continuous expression of pre-S-coded antigenic sites on HBsAg particles in chronic hepatitis B patients seropositive for HBeAg or anti-HBe shows that these determinants may be considered as a marker of chronicity during hepatitis B virus infection. The anti-pre-S antibody was determined by inhibition of the reaction for pre-S determinants. This antibody, different from anti-HBs, was detected during HBsAg antigenemia in patients recovering from acute type B hepatitis, before anti-HBs response. Kinetics of synthesis of anti-pre-S antibody in the course of acute type B hepatitis, followed by elimination of HBsAg and recovery, suggest the possible role of this antibody in the immunological clearance of infective hepatitis B virus particles.     

Sensitivity and specificity of Anti‐HBc screening assays – which assay is best for blood donor screening?

Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.