Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg

In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.     

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.     

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.     

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.     

The analytic sensitivity and mutant detection capability of six hepatitis B surface antigen assays

Hepatitis B virus surface antigen (HBsAg) mutants occur in clinical specimens. We studied the analytic sensitivity and ability to detect recombinant and native mutants of 6 HBsAg assays. The ARCHITECT, AUSZYME MONOCLONAL and AxSYM assays (Abbott Diagnostics, Abbott Park, IL), the ADVIA Centaur assay (Bayer Diagnostics, Tarrytown, NY), and the Test System 3 and VITROS ECi assays (Ortho Clinical Diagnostics, Raritan, NJ) showed comparable sensitivity with wild-type HBsAg. The ARCHITECT, AUSZYME, and AxSYM assays detected all mutants that were tested. The Test System 3 and VITROS ECi assays failed to detect mutants with amino acid substitutions at positions 143, 144, and 145, which are located in the immunodominant "a" determinant. The ADVIA Centaur failed to detect substitutions at position 145 and showed negative or very low positive results for substitutions at position 143. The inability to detect HBsAg mutants may lead to misdiagnosis of hepatitis B virus infection. Further studies on the prevalence of HBsAg mutants and the ability of commercial assays to detect them are needed.     

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.     

乙型肝炎病毒表面抗原基因点突变对HBsAg抗原性的影响

目的 研究乙型肝炎病毒表面抗原(HBsAg)常见基因点突变对HBsAg抗原性的影响,了解我国目前常用的HBsAg检测试剂对HBV S基因突变株的检测灵敏性,减少漏检,有效控制乙肝病毒(HBV)感染的传播.方法 构建HBsAg重组野毒株和重组变异株表达质粒,分别将重组野毒株HBsAg表达质粒pSS1adw2及pSS1adr和重组变异株HBsAg表达质粒pSS1adw2-145Arg、pSS1adr-126 Ser和pSS1adr-126 Asn转染COS-7细胞,进行瞬时转染.采用市售HBsAg ELISA检测试剂盒对细胞上清进行抗原性检测.结果 野毒株HBsAg和两种126位变异株HBsAg具有较好的抗原性;145位点突变后、导致HBsAg的抗原性下降.结论 推测是由于145位点变异影响了"a"抗原决定簇的空间结构,从而降低了其与抗-HBs的结合能力.     

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.     

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.     

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.     

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.     

Labeling of hepatitis B virus surface antigen (HBsAg) synthesized in a HBsAg-producing hepatoma cell line.

Hepatitis B virus surface antigen (HBsAg) could be studied until recently only by isolating it from the blood of carriers, thus making incorporation of radioactive precursors into this protein(s) impossible. The isolation of a cell line producing HBsAg [Alexander et al, 1978] has eliminated this obstacle. The cell line was therefore used for labeling HBsAg either with 35S-methionine or with 35S-cystine. HBsAg was purified by pelleting the component and by isopycnic centrifugation in CsCl gradients. HBsAg-positive fractions (as determined by solid-phase radioimmunoassay) were isolated from the gradients and analyzed in sodium dodecyl sulfate-containing polyacrylamide gels. It was found that although HBsAg contains substantial amounts of 35S-cystine, very little 35S-methionine was incorporated into this protein. In contrast, both labels were found in other structures having a buoyant density of about 1.3 gm/cm3 in CsCl. It was concluded that HBsAg is very low in methionine, and therefore this amino acid should not be used for labeling HBsAg in cells or in a cell-free system. Analysis of 35S-cystine-labeled HBsAg-positive material (buoyant density about 1.2 gm/cm3 in CsCl) revealed five proteins with molecular weights in the range of 48,000-82,000.     

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.     

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(+)).     

Hepatitis B surface antigen (HBsAg) subtype-specific antibodies in persons vaccinated against hepatitis B

One hundred sixty-three persons immunised against hepatitis B with a vaccine containing HBsAg either of adw or ayw subtype were examined for antibodies against the a, d, and y determinants of HBsAg. Sera were tested for antibodies against HBsAg adw and HBsAg ayw separately by a solid-phase radioimmunoassay using polystyrene beads coated with HBsAg of either adw or ayw subtype, and the relative amounts of antibodies against the single determinants were calculated. After the third immunisation, all vaccinees had antibodies against the common determinant a. A quantitative evaluation showed that on average about 50% of HBsAg-specific antibodies were directed against the a determinant, and about 50% against d or y, respectively. However, as only anti-a is protective against cross-infection with other HBsAg subtypes, the degree of immunity of a person vaccinated against hepatitis B should be evaluated by the determination of antibodies to a rather than antibodies against total HBsAg.     

A novel deletion mutant of hepatitis B virus surface antigen.

HBsAg is the most important serological marker for acute or chronic hepatitis B. Nevertheless, there are reports of HBsAg-negative virus carriers, either with anti-HBc as the only marker for hepatitis B virus (HBV) infection or even positive for anti-HBs and anti-HBc. We report isolates from a patient, in which a deletion in the HBs-gene was associated with persisting viremia in the presence of anti-HBs. The 62-year-old female, infected most likely by her husband, had detectable markers of chronic active hepatitis B, such as HBsAg, HBeAg, and anti-HBc-IgM, for 2 years. The patient then seroconverted to anti-HBs, although HBeAg and anti-HBc-IgM remained detectable. At this time, semiquantitative polymerase chain reaction showed about 10(4) viral genomes per milliliter of serum. Direct sequencing of the amplified products revealed a major population of DNA molecules with a deletion of nucleotide 31 of the HBs-gene, which up to now has not been described. This deletion led to a frame-shift and introduced a stop-codon after 21 amino acids of the sHBsAg. We suspect that this deletion, and the resulting HBsAg lacking the major epitopes recognized by specific antibodies, could favor ongoing viral replication, despite the presence of anti-HBs. However, because the reading frame of the polymerase was also severely damaged by this deletion, it is assumed that a minor population of intact genomes was present to help in the formation of virus particles.     

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.     

T-cell subsets in the hyporesponsiveness to hepatitis B surface antigen (HBsAg) and antigen-specific suppressor lymphocytes in chronic hepatitis B virus (HBV) infection

In contrast to convalescent hepatitis B patients, who exhibit the ability to elicit a specific immune response to HBsAg, patients with chronic hepatitis B virus (HBV) infection are markedly hyporesponsive to HBsAg and show a decrease in the normal ratio of OKT4-positive (helper/inducer) to OKT8-positive (suppressor/cytotoxic) lymphocytes. In this study the role of OKT4-positive and OKT8-positive cells on cellular immune response to HBsAg was evaluated in patients with chronic HBV infection and the ability of such patients to develop antigen-specific suppressor lymphocytes after in vitro sensitization to HBsAg. Elimination of OKT8-positive cells markedly improved the in vitro lymphocyte proliferative response to HBsAg without altering the reactivity of cells from the same donor to PPD or Candida. In contrast, the degree of responsiveness to HBsAg was not affected by the depletion of OKT4-positive cells. In vitro co-culture experiments, performed in the seven chronically HBV-infected patients who showed a proliferative response when their PBM were cultured with purified HBsAg or PPD, have demonstrated that lymphocytes from chronic HBV carriers, stimulated with HBsAg, inhibit the response of autologous PBM to HBsAg but not to the unrelated antigen PPD.