Evaluation of a new automated assay for hepatitis B surface antigen (HBsAg) detection VIDAS HBsAg Ultra

In a multicenter study a new automated screening assay, VIDAS HBsAg Ultra (long (L) and short (S) incubation protocol (Biomérieux, Marcy l'Etoile, France), was compared to a well established test (AxSYM HBsAg v2, Abbott Diagnostics, Wiesbaden, Germany) for the detection of hepatitis B virus (HBV) surface antigen (HBsAg). A total of 32 seroconversion panels, sera from the chronic phase of infection, dilution series of the WHO standard, S gene mutants (recombinant mutants and diluted and undiluted sera harbouring mutants with single or multiple amino acid (aa) substitutions, n = 40) and isolated anti-HBc positive samples were tested for the evaluation of sensitivity. Sera from HBsAg negative blood donors, pregnant women, hospitalized patients and potentially cross-reactive samples were investigated to determine the specificity of the new assay. The VIDAS HBsAg Ultra (L+S) had a higher sensitivity than the alternative assay for the detection of acute hepatitis B in seroconversion panels. The mean time of the diagnostic window was shortened with the VIDAS HBsAg Ultra (L) and (S) in comparison with the AxSYM HBsAg v2 by 1.06 and 0.66 days, respectively. The VIDAS HBsAg Ultra (L) did not detect one diluted sample out of six bearing the single aa G145R substitution, and two out of 12 diluted samples harbouring multiple aa substitutions. The analytical sensitivity of the assays varied from one surface mutant to another. While no false positive results were obtained with the VIDAS HBsAg Ultra (L+S) among potentially interfering samples, four false positives were detected with the AxSYM HBsAg v2. The respective values for sensitivity for the VIDAS HBsAg Ultra (L), (S) and the AxSYM HBsAg v2 were 99.07%, 97.87% and 94.14%. The specificities were 100% (VIDAS HBsAg Ultra L and S) and 99.6% (AxSYM HBsAg v2). In conclusion, the VIDAS HBsAg Ultra is highly sensitive and specific and represents an improvement for the detection of HBsAg in routine diagnostic laboratories.     

Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms

Mutations in hepatitis B virus surface antigen (HBsAg) involving amino acid substitution within the immunodominant "a" determinant may affect the performance of commercial HBsAg assays. The performances of four HBsAg assays that recently received Conformité Européene marking, Advia Centaur HBsAg (Bayer), Monolisa HBsAg Ultra (Bio-Rad), Liaison HBsAg (Dia Sorin), and Vidas HBsAg Ultra (bioMérieux), were compared with that of the routinely used HBsAg assay AxSYM HBsAg V2 (Abbott). Assays were evaluated for (i) analytical sensitivity performance with a national reference HBsAg panel (including 10 samples with calibrated HBsAg concentrations from 0.04 to 2.24 ng/ml) and (ii) the detection of HBsAg mutants by studying a panel of 35 HBsAg mutants (23 collected from patients and 12 recombinant mutants). The limits of detection of these assays were <0.15 ng/ml (from 0.089 to 0.121 ng/ml). The sensitivity performances for mutant virus detection varied, ranging from 37.1% to 91.4%. The lack of detection of these mutants by commercial assays was probably due to the epitope recognition of the anti-HBs assay reagents in the capture phase and in the conjugates. The prevalence and clinical impact of HBsAg mutants are under investigation. However, the manufacturers must be vigilant in the design of the assays in order to reduce the risk of missing a broad range of described S gene mutants.     

Evaluation of a semi-automatic radioimmunoassay for hepatitis B surface antigen (HBsAg)

The recently developed semi-automatic Hepatube system^® was evaluated in comparison to another radioimmunoassay for the detection of hepatitis B surface antigen (HBsAg), the manual Ausria II-125 test^®. After incubation of serum in anti-HBs coated tubes, the Hepatube system uses a machine to wash the tubes and to add tracer. After a second incubation, tubes are washed again in the machine and are manually transferred to the γ counter. Two machines were used. Machine 1 had an undefined defect. Of 1490 samples tested, 69 (4.6%) gave false-positive results versus 11 (0.7%) in the Ausria II-125 test. Machine 2 had one false-positive result among 920 samples versus 5 in the Ausria II-125 test. The sensitivity was measured with reference panels from Wellcome and Abbott as well as in titration series. The Hepatube system was found to be a factor three less sensitive than the Ausria II-125 test. The Hepatube processor is easy to handle; radioactive material can be held at a distance during the whole procedure; waste material is limited and less voluminous than in the Ausria II-125 test.     

Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS HBsAg assay

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.     

Variable capacity of 13 hepatitis B virus surface antigen assays for the detection of HBsAg mutants in blood samples

BackgroundNatural variation and mutations in the envelope protein (S) of hepatitis B virus can translate into HBsAg variants no longer detectable by conventional HBsAg assays.ObjectivesThe aim of the study was to assess the performance of 13 commercial assays currently used for screening and clinical analysis of HBsAg variants.Study designThe limit of detection (LOD) for each assay was established using two reference standards (WHO HBsAg 00/588 and the SFTS French reference). Sensitivity was evaluated using different panels. Panel 1 included 25 recombinant HBs variants at three concentrations, panels 2 and 4 included 8 recombinant HBsAg variants and 9 wild-type proteins (genotypes A–F), respectively, panel 3 included 16 natural HBsAg variants.ResultsLODs ranged from 0.011 to 0.095 IU/ml with the WHO standard, and from 0.021 to 0.326 ng/ml with the French reference. The overall percentage of positive signals using HBsAg variants ranged from 62.9% to 97.9%. Three substitutions: T123, D144A and G145, were negative at all concentrations with at least one assay.DiscussionOur findings show that, although they fulfil CE requirements for analytical sensitivity (LODs below 0.13 IU/ml), HBsAg assays may vary in their capacity to detect HBsAg variants. This limit in diagnosis performance should encourage the health regulatory agencies to include HBsAg variant panels in the evaluation process.     

Evaluation of a new rapid test for the combined detection of hepatitis B virus surface antigen and hepatitis B virus e antigen

There are about 350 million chronic hepatitis B virus (HBV) carriers worldwide. A proactive approach to the management of this disease is likely to reduce the morbidity and mortality caused by HBV. This study aimed to evaluate the diagnostic performance of a novel tool for discriminating between infected and noninfected subjects, the hepatitis B sAg/eAg test (Binax Inc., Portland, Maine). The test is designed to rapidly and accurately detect both the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg). A cohort of 942 subjects was tested. The serum clinical sensitivity of the hepatitis B sAg/eAg test was 99.75 and 96.37% for HBsAg and HBeAg, respectively. Serum clinical specificity was 99.32% for HBsAg and 98.99% for HBeAg. Analytical sensitivity was satisfactory for the purposes of population screening. Visual evaluation showed that the test signals were stable for at least 3 h after the recommended evaluation time. No interference or cross-reactivity was observed with known interfering substances and virologic markers. These results indicate that the hepatitis B sAg/eAg test is well suited to the accurate detection of HBV carriers. In addition to the good clinical specificity and sensitivity of this test, its stability and user-friendly design mean that a correct performance, even under field conditions, is highly likely. Consequently, the hepatitis B sAg/eAg test has the potential to identify subjects who require HBV vaccination (HBsAg(-) and HBeAg(-)) and HBV-infected individuals who might benefit most from antiviral therapy (HBsAg(+) and HBeAg(+)).     

Determination of the hepatitis B virus surface antigen (HBsAg) by immunoenzyme analysis

Studies aimed at the development of a variant of ELISA for the determination of hepatitis B virus surface antigen (HBsAg) showed the assay to be most effective when a modified periodate method of conjugation of highly purified horseradish peroxidase with the IgG-fraction of antiserum to HBsAg was used. With the resulting conjugates, the "sandwich"-test on polystyrene solid phase could detect HBsAg in concentrations up to 5 ng/ml which is several thousand-fold higher in sensitivity than counter immunoelectrophoresis, one order of magnitude more sensitive than the passive hemagglutination test, and comparable with the radioimmunoassay (RIA). The specificity of the method was confirmed by positive results of the neutralization test. By this method, HBsAg in the sera of hepatitis B patients was detected as frequently as by the RIA.     

Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay

We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.     

Characterization of the 3rd International Standard for hepatitis B virus surface antigen (HBsAg)

BackgroundHBsAg is the most important marker for laboratory diagnosis of HBV infection. Validation and quality control of HBsAg tests requires International Standards (IS). Recently the 2nd IS was replaced by the 3rd IS. Both IS are made from plasma-derived hepatitis B vaccines, but production and geographical origin are different.ObjectiveCharacterization of the HBsAg in the source material (SM) for the 3rd IS and comparison with the 2nd IS and native HBsAg.Study designThe SM was analyzed using solid-phase immunoassays, quantitative immune electrophoresis, ultracentrifugation, immunoblotting and HBV DNA sequencing.ResultsThe plasma-derived HBsAg of the SM originated from at least two different HBV strains, both of subgenotype (sgt) B4, typical for Vietnam. The HBsAg subtype was heterogeneous with ayw1 and adw2. The HBsAg concentration was 23,700 IU/ml as determined by solid-phase immunoassay; immune electrophoresis calibrated with sgt B2 revealed a concentration of 24,500 IU/ml while calibration with sgt D1 provided lower values. Proteins in the SM are heterogeneous in size containing only traces of preS. The protein subunits are partially cross-linked.ConclusionsThe antigenicity of the 3rd IS is suitable for HBsAg calibration in laboratory tests. In contrast to the 2nd IS, the 3rd IS is representative for a highly endemic region. Similar to the 2nd IS and different from native HBsAg, preS domains are depleted, protein subunits are partially cross-linked and the HBsAg particles are partially aggregated in the 3rd IS. The HBV subgenotype differences between the two IS may lead to variations in different quantitative assays.     

Polyalbumin receptors on hepatitis B virus and on 22 nm hepatitis B surface antigen (HBsAg)2 particles

Receptors for polymerized human serum albumin ( pHSA ) were studied by solid-phase radioimmunoassay on different hepatitis B surface antigen (HBsAg) particles subpopulations prepared both from hepatitis B e antigen (HBeAg) and from anti-HBe-positive sera. HBsAg particles in HBeAg-positive serum showed higher expression of the receptor compared with HBsAg particles from anti-HBe-positive serum. Analysis of different morphological forms of virus particles was performed after separation by density-gradient ultracentrifugation. Maximum receptor expression was detected in HBV particles containing fractions while the 22-nm HBsAg particles had significantly lower receptor activity. These observations support the hypothesis of a pathogenetic role of the pHSA receptor in mediating virus access to hepatocytes. Indeed, the higher pHSA binding activity on HBV particles could allow selective attachment of the infectious virion to liver cells that bear similar albumin receptors on their surface.     

Cholesterol requirement of hepatitis B surface antigen (HBsAg) secretion

Hepatitis B virus-infected patients secrete enormous quantities (50-300 microg/ml) of hepatitis B surface antigen (HBsAg) in their serum. One hypothesis for this synthetic effort is that these lipoprotein particles serve to adsorb neutralizing antisurface antibodies. We have shown that insulin suppresses the expression of HBsAg in human hepatoma cell Hep3B cells. We further studied the signaling pathway of insulin on the inhibition of HBsAg. Using a fungal metabolite, lovastatin, to block the p21Ras signaling pathway of insulin, we found that lovastatin inhibited the secretion of HBsAg into culture medium in Hep3B cells; however, the involvement of p21Ras-MAPKs was excluded in this effect. The cholesterol depletion from the membrane, leading to the destabilization of rafts, was the mechanism for the lovastatin inhibition of HBsAg secretion. However, lovastatin has no effect on the secretion of infectious viral Dane particles. Herein, we show for the first time that cholesterol is required for HBsAg secretion.     

Evaluation of assay methods for hepatitis B surface (HBsAg) antigen and its antibody (anti-HBS) in viral hepatitis B (VHB)-HBsAg-positive

The sensitivity of methods for the detection of HBsAg and its anti-HBs was compared in serial 1200 sera samples from 30 patients with VHB-HBsAg-positive. HBsAg was tested by gel-diffusion (GD), counter-immunoelectrophoresis (CIE), reversed haemogglutination (rHA), radioimmunoassay (RIA), and enzyme immunoassay (EIA). RIA and EIA methods are statistically significantly more sensitive compared with the other methods (P<0.0005). By these methods the minimal concentrations of HBsAg in sera can be proved. Although there is no statistically significant difference in the sensitivity between RIA and EIA, the latter is more sensitive if the subtype ay-HBsAg is considered (12 sera samples). In 24 patients the subtype was ay, in two ad, and in four it could not be differentiated.In 70% of patients anti-HBs was proved by RIA and in 10% by CIE, i.e., in 73% and 9% of sera samples, respectively. In 117 sera samples of these patients the sensitivity of RIA and EIA was compared for determination of anti-HBs. No statistically significant difference between the methods for determination of anti-HBs was found (50.42% 40.17%). No immune response to HBsAg has been observed in 9 cases, but 6 of them have remained permanent carriers of this antigen.     

Cellular immunity in 18 cirrhotic African carriers of hepatitis B virus surface antigen (HBsAg)

The cell mediated immunity has been studied by 3 different tests on a population including 32 african patients with cirrhosis; amongst those, 18 were HBs Ag carriers and 14 were Hbs Ag-. In those 2 groups, the intradermal tuberculin test and the DNCB cutaneous test showed a striking imminutary deficiency in half cases. The lymphoblastic transformation induced by PHA gave less striking results.     

Antigenic and physicochemical characterization of the 2nd International Standard for hepatitis B virus surface antigen (HBsAg)

BackgroundStandard preparations for HBsAg are required for quality control of test kits and clinical studies on HBsAg quantitation. WHO provides purified heat inactivated HBsAg diluted in negative defribinated plasma as 2nd International Standard (IS) for quality control of tests.ObjectiveStudy of possible alterations of antigenicity, protein composition, size and density of the heat inactivated source material (SM) for the 2nd IS.Study designNative HBsAg and SM were examined by quantitative immune electrophoresis (QIE), SDS-PAGE, ultracentrifugation and gel chromatography. HBV DNA was sequenced and the HBsAg geno/subtype derived.ResultsThe SM contained 97,600 International Units HBsAg/ml in QIE which agreed very well with the previous evaluations by WHO using 10 different assays. In SDS-PAGE, SM showed on a strong background the small HBs proteins but no preS proteins. SM had a more heterogeneous density than native HBsAg and contained particle aggregates. The HBsAg geno/subtype of SM was A2/adw2.ConclusionsThe IS has very good HBs antigenicity, but it lacks the preS domains, has modified HBs proteins and is partially aggregated. While it has been proven very useful for quality control of tests, certain inconsistencies due to the altered structure of its HBsAg cannot be excluded.     

Inhibition ELISA for hepatitis B surface antigen (HBsAg) using monoclonal idiotype-anti-idiotype interaction

Monoclonal anti-idiotypic antibody (Anti-Id) to the common (a) epitope of hepatitis B surface antigen (HBsAg) were raised and used to detect serum HBsAg in an inhibition enzyme-linked immunosorbent assay (inhibition ELISA). HRP-labelled Id 8D-3-6 was reacted with Anti-Id 4-8H coated on the solid phase in the presence of HBsAg. The ability of the antigen to inhibit the binding of labelled Id 8D-3-6 to anti-Id 4-8H was determined and the results correlated well with those obtained by radioimmunoassay. This assay requires only one washing step, takes 2 h and covers the range 10 ng/ml to 1 microgram HBsAg/ml. The inhibition ELISA is a more convenient, rapid and relatively sensitive assay which can be used to measure the level of a wide range of serum HBsAg.     

Comparative studies of the immunogenic activity of hepatitis B surface antigen (HBsAg) and HBsAg polypeptides

Three hepatitis B surface antigen (HBsAg) preparations were compared: purified intact 22-nm HBsAg particles; HBsAg-derived, sodium dodecyl sulfate (SDS)-denatured P25 + GP30 polypeptide pool; and nondenatured P25 + GP30 micelles. The micelles had the same polypeptide composition as the P25 + GP30 pool. The immunogenicity in mice of each preparation, administered either in saline suspension or adsorbed to aluminum gel, was compared. The SDS-denatured polypeptides were less immunogenic than intact HBsAg particles, whereas the micelles were more immunogenic. High anti-HBs titers were observed in mice immunized with micelle preparations in either saline suspension or adsorbed to aluminum gel for as long as 200 days after a booster inoculation, administered 26 days after the primary dose.     

Detection of human antibodies to hepatitis B surface antigen (HBsAg) by an enzyme-immunoassay for HBsAg.

We studied the feasibility of detecting antibody to hepatitis B surface antigen (anti-HBs) by an inhibition assay using the reagents of an enzyme-immunoassay for HBsAg (Hepanostika). Several modifications of the basic assay were investigated. Sensitivity was greatest when the test sample was incubated with a predetermined amount of HBsAg before the usual procedure of HBsAg detection. The presence of anti-HBs in the test sample was shown by a reduction of the solid-phase bound enzyme label. Results were obtained with a dilution series of serum samples containing anti-HBs, the anti-HBs Reference Panel of the American Bureau of Biologics, sera of hepatitis B patients, and sera of two individuals passively immunised with anti-HBs. The enzyme-immunoassay method showed at least the same sensitivity as passive haemagglutination. It was less sensitive than a commercially available radioimmunoassay (Ausab). There are no indications that non-specific reactions occur frequently. This study also revealed that the antigenaemia of acute hepatitis-B patients can be interrupted by a transient seroconversion.     

A phage with high affinity for hepatitis B surface antigen for the detection of HBsAg

An M13 phage, called PHH2, with the ability to bind HBsAg was isolated from a coat protein III display library. The region of the HBsAg polypeptide to which PHH2 binds was determined. The HBsAg binding phage was used in an assay referred to as "PHALISA", an abbreviation for phage-linked immune-absorbent assay. This assay was at least 20-100 times more sensitive in the detection of HBV antigen than conventional enzyme-linked immune-absorbent assays (ELISA). The application of this method for screening and detection of specific protein is discussed.     

Comparative study of methods of detection of hepatitis 'B' surface antigen (HBsAg)

The serum samples were collected from 52 patients of acute viral hepatitis and 235 hospital staff from Kasturba Hospital for Infectious Diseases. HBsAg was detected in their sera by counter-immuno-electrophoresis (CIEP), reverse passive hemogglutination (RPHA) and by micro-enzyme-linked-immunosorbent assay (ELISA) technique. Among the patients, HBsAg was detected in 12 cases (23%) by CIEP, in 18 cases (34%) by RPHA and in 23 patients (45%) by ELISA. In the hospital staff, HBsAg was detected in 4 samples (1.7%) by CIEP, in 8 samples (3.5%) by RPHA and in 32 samples (13.5%) by ELISA. Thus ELISA was found to be the most sensitive technique in detecting HBsAg.