Enzyme-immunoassay in diagnosis of hepatitis with emphasis on the detection of "e" antigen (HBeAg).

A brief survey of the application of enzyme-immunoassay (EIA) for the detection of hepatitis B surface antigen (HBsAg), hepatitis A, and their corresponding antibodies is given. The preliminary results of a similar EIA for detection of hepatitis B-related "e" antigen (HBeAg) and its antibody (anti-HBe) are reported. This EIA is much more sensitive than immunodiffusion: at least 128 times for HBeAg and at least 512 times for anti-HBe. HBsAg and its antibody do not interfere with the test. Only a few sera strongly positive for rheumatoid factor gave rise to false-positive results, as was demonstrated by a confirmatory test.     

Correlation of HBV DNA and monoclonal reactivity to HBsAg in serum of patients with HBV infection

Hepatitis B virus (HBV) DNA hybridization assay, a monoclonal radioimmunoassay (M-RIA) for hepatitis B surface antigen (HBsAg) and conventional polyclonal immunoassays for HBV associated antigens were used to study sera from patients on dialysis and with acute hepatitis B. HBV DNA was detectable in hepatitis B e antigen (HBeAg) negative patients with acute hepatitis but not in HBsAg+ HBeAg- dialysis patients. In acute hepatitis, HBsAg immunoreactivity by M-RIA could still be detected even though a commercial immunoassay for HBsAg, the AUSRIA II, and the HBV DNA assay were no longer positive. Unlike in acute HBV infection, serum HBV DNA was detectable in dialysis patients who were AUSTRIA II negative but M-RIA positive. Serial determination of HBsAg by M-RIA and HBV DNA revealed episodes of HBV DNA positivity months after both the HBsAg was no longer positive by polyclonal immunoassay. Thus, the M-RIA for HBsAg and the molecular hybridization technique for HBV DNA are sensitive and specific assays for the identification of potentially infectious individuals who would not have been characterized as such based on the results of conventional polyclonal immunoassays.     

Comparison of polyclonal and monoclonal antibodies in determination of glomerular deposits of hepatitis B virus antigens in hepatitis B virus-associated glomerulonephritides

The nature of hepatitis B virus (HBV) antigens in HBV-associated glomerulonephritides was investigated in 7 hepatitis B surface antigen (HBsAg) carriers with membranous nephropathy, 16 HBsAg carriers with mesangial IgA nephropathy, and 1 HBsAg carrier with a mixed picture of membranous and IgA nephropathies. Consecutive frozen sections of renal biopsy specimens were stained with polyclonal and monoclonal antibodies against HBV antigens. Glomerular capillary deposits of HBeAg and HBcAg were detected in 66% and 57% of renal biopsies from HBsAg carriers with membranous nephropathy by monoclonal and polyclonal antibodies, respectively. The discrepancy in the immunofluorescence findings resulted from the cross-reactivity of the polyclonal anti-HBcAg antiserum because it contains both anti-HBcAg and anti-HBeAg activities. Mesangial deposits of HBsAg were detected in 40% and 21% of renal biopsies from HBsAg carriers with mesangial IgA nephropathy by polyclonal and monoclonal antibodies, respectively. The authors' study confirms that HBeAg is the predominant HBV antigen deposited in HBV-associated membranous nephropathy, and glomerular HBsAg deposits are detected in some HBsAg carrier with mesangial IgA nephropathy. Careful testing and evaluation of each antibody are necessary to prevent misinterpretation.     

Monoclonal radioimmunoassay for hepatitis B surface antigen in sera of children with acute leukemia

Hepatitis B surface antigen (HBsAg) was detected by a monoclonal antibody radioimmunoassay in sera from five of 43 children (11.6%) with acute leukemia, who were negative by conventional assay. None of the nine positive sera had evidence of reactivity for HBV-DNA or DNA-polymerase activity. No correlation was found between the presence of HBsAg in serum by monoclonal RIA and the behaviour of anti-viral antibodies. Twenty-two children could be studied for liver HBsAg by immunofluorescence, and nine of them (40.9%) were positive, including three patients having HBsAg reactivity in serum. These data indicate that monoclonal antibodies increase the sensitivity of RIA for the detection of serum HBsAg in children with acute leukemia, who previously have frequently been found to have an atypical hepatitis B virus (HBV) serology.     

Prevalence of hepatitis B and C and HIV antibodies in children in a Romanian orphanage.

Two hundred and fifteen children in an orphanage in Romania were examined for serum markers of present or past hepatitis B and C virus and HIV infection. In total, 183 children (85.1%) had at least one marker (HBsAg, anti-HBs or anti-HBc) of hepatitis B virus infection. An HBsAg carrier state was diagnosed in 38 (20.8%) of the infected children. Among the carriers 24.3% were HBeAg carriers, 51.4% had anti-HBe and 24.3% had neither HBe antigen nor antibody. Nine children (4.2%) had antibodies to hepatitis C virus. All sera were negative in tests for HIV antibodies. False-positive reactions represented a considerable problem with these sera. Six percent of the sera gave false-positive reactions in indirect ELISA tests for hepatitis C and HIV. Sera giving false-positive reactions had rather high serum IgG levels. The results of this study indicate that these children have been heavily exposed to hepatitis B virus and to a certain degree to hepatitis C virus, while there were no cases of HIV infection in this orphanage.     

Characterization of private and cross-reactive idiotypes associated with human antibodies to hepatitis B surface antigen.

Four mouse monoclonal anti-idiotypic antibodies (anti-Id) were generated against human monoclonal and polyclonal antibodies to hepatitis B surface antigen (HBsAg). These monoclonal anti-Id, along with a polyclonal anti-Id raised in rabbits, were used to characterize the idiotype (Id) specificity of the human antibody response to HBsAg (anti-HBs). The anti-Id reagents identified distinct private and cross-reactive Id expressed on monoclonal and polyclonal human anti-HBs preparations respectively. The anti-Id recognized both HBsAg combining site and non-combining site related private Id, and HBsAg combining site related cross-reactive Id. The Id specificities recognized by two of the monoclonal anti-Id were associated with the H chain alone, whereas two of the monoclonal anti-Id, along with the polyclonal anti-Id appeared to recognize Id determinants associated with both isolated H and L chains. These data suggest that Id heterogeneity exists within the human antibody response to HBsAg. The knowledge that Id heterogeneity exists is of importance in understanding the observed variability in the immune response during hepatitis B virus infection.     

Assay of preS epitopes and preS1 antibody in hepatitis B virus carriers and immune persons

The diagnostical significance of the large hepatitis B surface protein with its preS1 attachment site and of anti-preS antibodies are not yet well known. We investigated the epitope of the preS1 attachment site to see whether it is a marker of viremia and whether antibodies against it occur in convalescents and vaccinees. For comparison, sera were also tested for the presence and relative amount of a preS2 epitope. The epitopes were detected by binding to specific monoclonal antibodies (mAb MA18/7 for the preS1 epitope and mAb Q19/10 for the preS2 epitope) at the solid phase of a sandwich enzyme-linked immunosorbent assay. Antibody against the preS1 epitope was detected by inhibition of binding to mAb MA18/7. This mAb inhibits attachment of preS1 antigen to hepatocytes and reacts with a subtypeindependent sequential epitope at the surface of hepatitis B virus between amino acid 29–36. This preS1 epitope occurs in most hepatitis B surface antigen (HBsAg) carriers, irrespective of viremia. Free preS2 epitope Q19/10 is present in samples with more than 8 g/ml total HBsAg and it is masked in sera with less HBsAg. Antibodies which compete with mAb MA18/7 for its viral preS1 epitope occur in one third of HBsAg carriers who were negative for hepatitis B e antigen. It also occurs in one third of convalescents and in most good responders to plasma-derived vaccines.     

Measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis

The occurrence of measles virus-specific IgM antibodies in sera from patients with chronic active hepatitis not caused by Hepatitis B virus was examined by a specific enzyme-linked immunosorbent assay (ELISA). Using whole serum, specific IgM antibodies were detected in 12 of 23 sera from patients with hepatitis B surface antigen (HBsAg)-negative chronic active hepatitis. In nine of these sera the finding of specific IgM antibodies was verified by separation of IgM by density gradient centrifugation and examination of the fractions by ELISA. Most of the sera from the patients with measles virus-specific IgM antibodies had an elevated level of specific IgG antibodies compared to the level of IgG found in control sera. The significance of these findings in view of a possible persistent measles virus antigen production in patients with chronic active hepatitis is discussed.     

Sites that bind polymerized albumin on hepatitis B surface antigen particles: detection by radioimmunoassay.

Antibodies to polymerized human albumin (poly-HSA) could not be detected by using sensitive methods (enzyme-linked immunosorbent assay and radioimmunoprecipitation) in sera from chronic carriers of hepatitis B surface antigen (HBsAg) or in serial bleedings from one chimpanzee infected with type A hepatitis virus and one infected with non-A, non-B hepatitis virus. By a solid-phase radioimmunoassay, receptor sites for poly-HSA could be detected on HBsAg particles from sera containing either hepatitis B "e" antigen (HBeAg) or anti-HBe. Blocking experiments showed that monomeric HSA did not bind to this receptor. In general, the HBsAg particles from sera with HBeAg had more poly-HSA receptor sites or relatively more particles carrying this receptor compared with HBsAg from sera with anti-HBe. Microtiter plates coated with poly-HSA bound HBsAg from sera containing HBeAg with greater efficiency than did anti-HBs coupled to a solid phase (Ausria II beads), whereas with sera positive for anti-HBe, the two assays were equally sensitive. Decreased ability of HBsAg to bind to poly-HSA was seen in some sera which had been stored for a few years at 4 degrees C, whereas the binding to anti-HBs was unaffected. It is possible that polymers of albumin on the surface of hepatocytes could function as receptors for hepatitis B virus.     

Development of a second generation monoclonal immunoradiometric assay. Increased sensitivity leads to enhanced detection of hepatitis B viral infection

We have developed and employed a second generation monoclonal immunoradiometric assay (M2-IRMA) using antibodies of high affinity for epitopes that reside on hepatitis B surface antigen (HBsAg). This assay is capable of detecting as little as 15 pg/ml of HBsAg in serum. Improvements in sensitivity over a first generation immunoradiometric assay (MI-IRMA) was achieved by increasing the sample volume and time of incubation, and subjecting the reaction to a mechanical rotary devise. We then studied 164 subjects with chronic hepatitis, 105 with cirrhosis, 67 with hepatocellular carcinoma, six with acute hepatitis A, seven with acute hepatitis B, 167 chronic carriers of hepatitis B virus (HBV) and 235 healthy individuals from Japan and compared the results of the M2-IRMA to a conventional polyclonal radioimmunoassay (P-RIA). By using a more sensitive assay design (M2-IRMA), a significant number of additional cases of HBV infection heretofore unsuspected in the etiology of chronic liver disease were identified. We conclude that improvement in assay sensitivity for HBsAg is important in the serologic diagnosis of HBV in patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma.     

Comparison of two techniques for detecting antibody to HBsAg during a hepatitis B vaccine study

Two assays for the detection of antibody against hepatitis B surface antigen (anti-HBs) were compared. The first was a direct sandwich radioimmunoassay (RIA) which detects, in principle, antibody against any epitope of hepatitis B surface antigen (HBsAg). The second assay was an inhibition enzyme-linked immunosorbent assay (ELISA). In this assay a fixed amount of HBsAg which can be blocked by anti-HBs is measured in a direct sandwich test. Prevaccination screening sera (n = 191) and follow-up sera obtained from high risk groups (n₁ = 85; n₂ = 41) during two hepatitis B vaccine studies were compared in RIA and ELISA. In prevaccination sera either HBsAg or anti-HBs were detected by ELISA. Full agreement between the results of RIA and ELISA for anti-HBs was obtained in sera containing more than 10 IU/1 anti-HBs. Both tests showed variable results at low titres. Experiments with monoclonal anti-HBs indicated that ELISA is less sensitive for subtype specific antibodies (anti-d, anti-y), which may explain that there were consistent differences between RIA and ELISA in a minority of cases.     

Non-productive infection of human newborn blood mononuclear cells with herpes simplex virus: effect on T cell activation, IL-2 production and proliferation.

IgG subclasses of antibodies to the hepatitis B surface antigen (HBsAg) in sera from 40 healthy infants immunized with the vaccine against hepatitis B virus (HBV) were detected using an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The infants were born to asymptomatic HBsAg-positive mothers. Total serum IgG subclasses were also tested to exclude a deficiency of certain subclasses in these infants but their distribution was the one expected according to age. In contrast, IgG subclass antibodies to HBsAg were predominantly IgG1 and IgG4. The collected data indicate that infants produce significantly higher levels of IgG1 and IgG4 than IgG2 and IgG3 in response to the vaccine for HBV. The IgG4 response to anti-viral vaccinations is uncommon. The role of that IgG4 subclass is not yet clear: even if an anaphylactic role was suggested, no adverse reactions were observed in vaccinated children.     

A study of the antigenicity and immunogenicity of a new hepatitis B vaccine using a panel of monoclonal antibodies

The successful prevention of infection with hepatitis B virus (HBV) has been achieved by vaccination with purified hepatitis B surface antigen (HBsAg). The ability of a novel synthetic HBV envelope antigen vaccine (Hep B-3, Hepagene ™; Medeva), which contains part of the pre-S1 and the complete pre-S2 regions and the whole of the S region and was produced in a mammalian cell line, to induce antibodies required for a protective immune response is of importance. In this study, the use of a panel of monoclonal antibodies known to bind to epitopes within the common “a” determinant has demonstrated that the epitopes present on this new vaccine are comparable to those found with plasma-derived HBsAg. In addition, the epitope specificity of the antibodies induced by this vaccine was examined and shown to accord well with previous results obtained using both a plasma-derived vaccine and a recombinant vaccine prepared in yeast. J. Med. Virol. 54:1–6, 1998. © 1998 Wiley-Liss, Inc.     

Extensive trial of a monoclonal immunoenzyme test system for the detection of HBsAg in donors and patients

The enzyme immunoassay system for hepatitis B diagnosis developed on the basis of monoclonal antibodies to hepatitis B virus surface antigen (HBsAg), EIAmca, was demonstrated to detect twice as many antigen-containing donor sera than DHA test, and 9-10 times more than CIEP. By means of EIAmca, HBsAg could be detected in a larger number of patients' sera and in higher dilutions of the sera. This attests to a higher sensitivity of the developed EIAmca system as compared with diagnostic systems of CIEP, DHA test and EIApca.     

Can the serological status of “anti‐HBc alone” be considered a sentinel marker for detection of “occult” HBV infection?

Some individuals have "occult" infection with hepatitis B virus (HBV), defined as presence of HBV genome in the serum or liver tissue without HBV surface antigen (HBsAg) in the serum. The aim of this study was to investigate whether serum antibodies against HBV core antigen in isolation ("anti-HBc alone") are a useful marker of "occult" HBV in patients with or without hepatitis C virus (HCV) infection. "Anti-HBc alone" was detected in the sera of 119/6,544 (1.8%) asymptomatic outpatients referred to the diagnostic laboratory for routine testing for viral hepatitis, 62/607 (10.2%) drug users, and 42/195 (21.5%) patients with hepatocellular carcinoma. Using three in-house nested-PCR amplification assays to detect HBV preS-S (S), precore-core (C), and Pol viral regions, respectively, "occult" HBV sequences were found in 9 of the 223 sera (4.0%) with "anti-HBc alone." The highest prevalence of "occult" HBV sequences (5.9%) was detected in "anti-HBV alone" sera of individuals referred to the diagnostic laboratory without HCV antibodies. Direct sequencing of all PCR products confirmed the specificity of the PCR reactions and revealed the predominance of HBV genotype D. The data presented in this study suggest that detection of "anti-HBc alone" could reflect unrecognized "occult" HBV infection and that physicians should consider investigating such patients with HBV molecular tests.     

Simultaneous use of poly- and monoclonal antibodies as enzyme tracers in a one-step enzyme immunoassay for the detection of hepatitis B surface antigen.

A one-step third generation enzyme immunoassay (EIA) was developed for the detection of hepatitis B surface antigen (HBsAg) in human serum or plasma using a polyclonal (Pab-HBsAg) and two monoclonal antibodies to HBsAg (Mab1-HBsAg and Mab2-HBsAg). In this assay, the solid phase is coated with Mab1-HBsAg and the specimen is incubated simultaneously with peroxidase labelled Pab-HBsAg and Mab2-HBsAg. If HBsAg is present in a specimen it will form sandwich complexes with capture and tracer antibodies. The hook effect, observed in some HBsAg detection tests when a high concentration of HBsAg is present, was minimized in this assay by increasing the concentration of the peroxidase-labelled Mab2-HBsAg. The sensitivity of this assay for HBsAg/ay and HBsAg/ad subtypes in a standard (2 h incubation) procedure was 0.6 and 0.3 ng/ml and in an overnight (16-22 h incubation) procedure 0.2 and 0.15 ng/ml, respectively. Strong elimination of the hook effect was observed with specimens containing high levels of HBsAg compared with test results using peroxidase-labelled Pab-HBsAg alone as enzyme tracer. This EIA offers a procedure, with a high specificity and wide range of sensitivity for the detection of HBsAg in human sera or plasma.     

Identification of a dominant immunogenic epitope of the nucleocapsid (HBc) of the hepatitis B virus

Four monoclonal antibodies to hepatitis B core antigen are described. The antibodies bind to the same or a very closely related epitope. Antibodies to this dominant epitope are present in the sera of patients with either acute or chronic hepatitis B virus (HBV) infection. A high percentage of inhibition of the binding of these antibodies to the core antigen by these four monoclonal antibodies suggests that the core antigen has a restricted antigenicity in man. Radiolabeled or peroxidase labeled forms of these monoclonal antibodies can be used to assay IgM and total anticore in serum.     

Significance of "anti HBC alone" serological profile in 284 patients suspicious of being infected with hepatitis B virus

The "anti-HBc alone" serological profile is a frequent finding in hepatitis B virus infections, but little is known about its clinical significance. The aim of this study was to explore the 'anti-HBc alone' serological profile obtained by immunosorbent assay (ELISA) in 284 patients suspicious of being infected with hepatitis B virus. Sera were screened for following serological markers: HBs Ag, anti-HBc and anti-HBs antibodies using immunoradiometric assay (IRMA) and for HBV DNA using polymerase chain reaction. Among 284 studied sera with 'anti-HBc alone' serological profile, 124 were positives for anti-HBs antibodies by IRMA and corresponding to a recovered form of hepatitis B. Nineteen sera were negatives for anti-HBc antibodies, suggesting false positive results by ELISA. Two sera were found positives for HBs Ag by IRMA, which are related to authentic hepatitis B. HBV DNA was positive in 4 sera, suggesting occult hepatitis B. This study indicates that "anti-HBc alone" serological profile is most often correlates with recovered hepatitis B infection, but it can mask an occult hepatitis B.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

IgM antibodies in sera from patients with chronic active hepatitis reacting with the measles virus matrix protein and nucleoprotein

The antigenic specificity of measles virus IgM antibodies in sera from patients with chronic active hepatitis not caused by hepatitis B virus has been examined. An immunosorbent column containing antihuman IgM covalently bound to Sepharose was used to pick up IgM from the sera. Radiolabelled measles virus antigens were then allowed to react with the IgM antibodies. The immune complexes were eluted and analysed by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis. Four sera from patients with hepatitis B surface antigen [HBsAg]-negative chronic active hepatitis with high measles virus haemagglutination inhibition [HI] and complement fixation [CF] antibody titres and positive enzyme-linked immunosorbent assay [ELISA] for measles-virus-specific IgM were examined. The results were compared with those obtained using sera from patients with an acute measles virus infection and from healthy controls. In both patient groups, IgM antibodies with specificity against the matrix protein represented the major portion of the measles virus IgM. IgM antibodies against the measles virus nucleoprotein and probably against host-cell-derived actin were also present. The patient sera contained only traces of IgM antibodies with specificity against the measles virus haemagglutinin or fusion protein. No specific IgM antibodies were found in sera from healthy controls.