The impact of hepatitis B surface antigen seroconversion on the durability of functional cure induced by pegylated interferon alpha treatment.

BACKGROUND: Hepatitis B surface antigen (HBsAg) loss is regarded as a pivotal criterion for assessing functional cure in patients diagnosed chronic hepatitis B (CHB). We conducted the research to investigate the real-world performance of HBsAg seroconversion in sustaining HBsAg loss. METHODS: This retrospective analysis confirmed 295 patients who attained HBsAg loss through combination therapy involving nucleos(t)ide analogues (NAs) and pegylated interferon alpha (peg-IFNα). Employing Kaplan-Meier estimates method to conduct survival analysis. The forest plot was used to visualize the results of multivariate Cox regression, and selected variables were included in the nomogram. RESULTS: HBsAg seroreversion was observed in 45 patients during follow-up periods, with a lower recurrence risk in patients with HBsAg seroconversion at the end of peg-IFNα therapy (EOT) (10.3% vs 37.3% at 96-week, P < 0.0001). Moreover, the sustainability of hepatitis B surface antibody (anti-HBs) in participants continuing therapy after HBsAg seroconversion was superior to those discontinued prematurely (72.5% vs 54.5% at 96 weeks, P = 0.012). Additionally, the former group was also relatively less likely to experience HBsAg reversion during long-term observation (8.4% vs 14.3% at 96 weeks, P = 0.280). Hepatitis B envelope antigen (HBeAg) status, anti-HBs status and consolidation treatment screened by multivariable analysis were utilized to construct a predictive model for HBsAg reversion. The concordance index(C-index = 0.77) and calibration plots indicated satisfactory discrimination and consistency of nomogram. CONCLUSIONS: HBsAg seroconversion was beneficial for sustaining functional cure in patients treated with peg-IFNα. Continuing consolidation therapy after HBsAg seroconversion also contributed to maintain HBsAg seroconversion and improve the durability of HBsAg loss. The nomogram illustrated its efficacy as a valuable instrument in showcasing survival probability of functional cure.

No Improvement of Hepatitis B Vaccination Response in Patients Dialysed with a Polymethylmethacrylate Membrane Compared to High-Flux Polysulfone: Results of the HEPADIAL Study.

BACKGROUND: An altered immune response and decreased vaccine response are observed in patients with chronic renal failure. A preliminary study of 15 non-immunised patients, despite appropriate previous hepatitis B vaccination, showed a 60% seroconversion rate after 3 months of dialysis with a polymethylmethacrylate (PMMA) membrane. This response was associated with circulating soluble CD40 (CD40s) decrease, a natural inhibitor of the humoral immune response. The aim of the study is to confirm these results in a randomised study. METHODS: We conducted a multicentre randomised intention-to-treat superiority clinical trial comparing polysulfone and a PMMA membrane in 2 parallel patient groups. The primary end point was the vaccine response rate, as defined by an anti-HBs antibodies titre of >10 IU/L, 1 month after the last vaccination with a double dose of Engerix B20®, performed at weeks 12, 16, 20, and 36. RESULTS: Twenty-five patients were randomised and included in an intention-to-treat analysis. They were dialysed on polysulfone (n = 11) or PMMA (n = 14) for 40 weeks. Fifty percent of the PMMA patients versus 54.5% of the polysulfone patients achieved seroconversion (p = 1.00). The median anti-HBs antibody titre in responders at week 40 was 496 (92-750) versus 395 (43-572) UI/mL for PMMA and polysulfone, respectively (p = 0.46). The median CD40s titre at week 12 was 306 (193-448) versus 491 (281-515) pg/mL (p = 0.21). The CD40s median variation between week 0 and week 12 was 5 (-105 to 90) versus 64 (-63 to 123) pg/mL (p = 0.55). The CD40s level at week 12 in non-responders was slightly inferior to that of the responders: median 193 (168-331) versus 413 (281-512) pg/mL (p = 0.08). CONCLUSION: We did not observe a better vaccine response with the PMMA membrane compared to high-flux polysulfone. The PMMA membrane did not decrease the CD40s more than the polysulfone membrane probably because the titre was previously low in the 2 groups.

Kinetics of hepatitis B surface antigen quasispecies during REP 2139-Ca therapy in HBeAg-positive chronic HBV infection.

Chronic HBV infection results in various clinical manifestations due to different levels of immune response. In recent years, hepatitis B treatment has improved by long-term administration of nucleos(t)ide analogues (NUCs) and peg-interferon. Nucleic acid polymers (NAPs; REP 2139-Ca and REP 2139-Mg) are new antiviral drugs that block the assembly of subviral particles, thus preventing the release of HBsAg and allowing its clearance and restoration of functional control of infection when combined with various immunotherapies. In the REP 102 study (NCT02646189), 9 of 12 patients showed substantial reduction of HBsAg and seroconversion to anti-HBs in response to REP 2139-Ca, whereas 3 of 12 patients did not show responses (>1 log reduction of HBsAg and HBV DNA from baseline). We characterized the dynamic changes of HBV quasispecies (QS) within the major hydrophilic region (MHR) of the 'pre-S/S' open reading frame including the 'a' determinant in responders and nonresponders of the REP 102 study and four untreated matched controls. HBV QS complexity at baseline varied slightly between responders and nonresponders (P = .28). However, these responders showed significant decline in viral complexity (P = .001) as REP 2139-Ca therapy progressed but no significant change in complexity was observed among the nonresponders (P = .99). The MHR mutations were more frequently observed in responders than in nonresponders and matched controls. No mutations were observed in 'a' determinant of major QS population which may interfere with the detection of HBsAg by diagnostic assays. No specific mutations were found within the MHR which could explain patients' poor HBsAg response during REP 2139-Ca therapy.

Plasmonic Hepatitis B Biosensor for the Analysis of Clinical Saliva.

A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.

An innate immune signature induced by AS01- or AS03-adjuvanted vaccines predicts the antibody response magnitude and quality consistently over time.

Background: Antibody-mediated protection can depend on mechanisms varying from neutralization to Fc-dependent innate immune-cell recruitment. Adjuvanted vaccine development relies on a holistic understanding of how adjuvants modulate the quantity/titer and quality of the antibody response. Methods: A Phase 2 trial (ClinicalTrials.gov: NCT00805389) evaluated hepatitis B vaccines formulated with licensed adjuvants (AS01B, AS01E, AS03, AS04 or Alum) in antigen-naïve adults. The trial investigated the role of adjuvants in shaping antibody-effector functions, and identified an innate transcriptional response shared by AS01B, AS01E and AS03. We integrated previously reported data on the innate response (gene expression, cytokine/C-reactive protein levels) and on quantitative/qualitative features of the mature antibody response (Fc-related parameters, immunoglobulin titers, avidity). Associations between the innate and humoral parameters were explored using systems vaccinology and a machine-learning framework. Results: A dichotomy in responses between AS01/AS03 and AS04/Alum (with the former two contributing most to the association with the humoral response) was observed across all timepoints of this longitudinal study. The consistent patterns over time suggested a similarity in the impacts of the two-dose immunization regimen, year-long interval, and non-adjuvanted antigenic challenge given one year later. An innate signature characterized by interferon pathway-related gene expression and secreted interferon-γ-induced protein 10 and C-reactive protein, which was shared by AS01 and AS03, consistently predicted both the qualitative antibody response features and the titers. The signature also predicted from the antibody response quality, the group of adjuvants from which the administered vaccine was derived. Conclusion: An innate signature induced by AS01- or AS03-adjuvanted vaccines predicts the antibody response magnitude and quality consistently over time.

The orchestration of cellular and humoral responses is facilitated by divergent intracellular antigen trafficking in nanoparticle-based therapeutic vaccine.

Therapeutic vaccination for the treatment of chronic hepatitis B is promising but has so far shown limited clinical efficacy. Herein, we employ polylactide nanoparticles (NPs) as the vaccine adjuvant and systematically explore their effect on activation of specific immunity and the underlying theoretical mechanisms. In vitro studies show that hepatitis B surface antigen (HBsAg) accumulates in antigen-presenting cells (APCs) to a larger content (270%) with the assistant of NP in comparison with the pure-antigen group. Besides the elevated costimulators (CD80/86) and increased major histocompatibility complex (MHC) II molecules, the MHC I molecules are also found upregulated. This result is mostly owing to the divergent antigen trafficking ways of NP-antigen in APCs, especially for the escape of exogenous HBsAg from the lysosomes to the cytosol. Interestingly, the MHC I level is downregulated in alum-antigen group, indicating a possible reason for its inefficiency in priming cellular response. Further in vivo experiments establish that NP-antigen group indeed enhances the CD8(+) CTL cytotoxicity and IFN-γ cytokine secretion. Meanwhile, specific antibody titer is also upregulated, and even surpasses that of the commercialized alum-antigen. All these results strongly support that NP-based antigen promotes an orchestration of cellular and humoral immune response, exhibiting favorable intrinsic properties to be applied in therapeutic vaccines.

[Anti-hepatitis B surface antigen titres in vaccinated dentistry students at Damascus University].

Dental practice carries considerable danger for acquiring bloodborne pathogens such as hepatitis B virus (HBV). Vaccination against this virus is an important approach to reducing the infection. Post-vaccination test to confirm the seroconversion is important also. Over the period 1 March-31 May 2010, we assessed the efficacy of HBV vaccination among 91 fourth-year dental students at Damascus University, who were vaccinated under the mandatory Faculty of Dentistry programme. Anti-HBsAg antibody titres were determined in the blood samples using an enzyme immunoassay to measure; > or = 10 IU/mm was considered an adequate response titer. Seven of the 91 dentistry students (7.7%) had anti-HBs antibody titre < 10 mlU/mL. The frequency of unresponsiveness was significantly higherwith smoking (P = 0.012) and alcohol consumption (P = 0.014). Anti-HBs test should be included in routine immunization services of the School of Dentistry at Damascus University.

[Effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation].

OBJECTIVE: To explore the effects of HBsAg pulsed dendritic vaccination on anti-HBs production in immunosuppressed rats after liver transplantation (LT). METHODS: Brown-Norway liver allografts were transplanted into Lewis recipients. The transplanted Lewis rats were injected with EK506 (2 mg/kg) and randomly divided into two groups: rats in HBsAg-DCs group (n = 15) were intraperitoneally injected with HBsAg pulsed DCs at 14 d and 28 d after LT, and rats in the HBsAg group (n = 15) were injected with HBsAg (200 mul) once a week for 12 weeks. Rats without any immunosuppressive treatment after LT served as controls (n = 5). IL-2 and IFN-gamma mRNA expression in spleen were analyzed by RT-PCR, serum IL-2, IFN-gamma and anti-HBs were detected by ELISA. RESULTS: High dose of FK506 resulted in the immunosuppressed in LT rats, as evident by low production of IL-2 and IFN-gamma, and without liver rejection compared to rats in the control group. HBsAg-DCs induced high titer of anti-HBs antibody, however, titer of anti-HBs were seldom detectable in the HBsAg group at 1, 2 and 3 mouth after vaccination. CONCLUSION: The capacity of HBsAg-DCs to induce anti-HBs in immunosuppressed rats suggested that DC vaccine may prevent HBV recurrence in liver transplanted patients.

[Effect of polyethylene glycol as adjuvant on hepatitis B virus DNA vaccine in vitro].

OBJECTIVE: To explore whether polyethylene glycol (PEG) as adjuvant could enhance the humoral and cellular immune responses on hepatitis B virus DNA vaccine. METHODS: C57BL/6J mice were immunized with PEG and pVAX-S2 or alone pVAX-S2. After these mice were finally immunized for 14 days, the anti-HBs IgG, T cell proliferation, the expression of cytokines and CTL in vivo were detected. RESULTS: Compared to mice immunized with alone pVAX-S2, PEG as adjuvant could increase the production of anti-HBs IgG and HBsAg specific T cell proliferation. In addition, the expression of IL-4, IFN-gamma in CD4+ T cells and IFN-gamma in CD8+ T cells was higher than control groups. The PEG/ pVAX-S2 groups could elicit significantly in vivo HBsAg specific CTL responses. CONCLUSIONS: PEG as adjuvant could enhance humoral and cellular immune responses, as well as in vivo CTL activity.

Survey of the level of anti-HBs antibody titer in vaccinated Iranian general dentists.

Hepatitis B is an infectious disease to which dentists are susceptible. The main aim of this study was to determine the level of antibody titer and immunity in vaccinated Iranian general dentists. A total of 861 general dentists were invited to participate in this study; 598 persons who could recall their history of vaccination and consented to have blood samples taken were recruited. Demographic and work-related data were recorded, and anti-Hepatitis B surface antigen (anti-HBs-Ag) evaluations were measured using the enzyme-linked immunosorbent assay (ELISA). Of the 598 participants, 35 (5.9%) were nonimmune (anti-HBs <10 IU/l), 101 (16.9%) were relatively immune (anti-HBs = 10-99 IU/l), and 462 (77.3%) were completely immune (anti-HBs > or =100 IU/l). Only 218 (36.5%) of the dentists knew their HBs antibody titer. Fourteen (2.3%) persons reported receiving one dose and 65 (10.9%) had received two doses. The number of those who had received the three recommended doses totaled 519 (86.8%), 491 (82.1%) of them receiving their vaccine on schedule. Age, city, pack-years of smoking, years of smoking, and the interval between the last vaccination and the commencement of the study had a significant relationship to the antibody titer level, whereas sex, marital status, place of practice, smoking, and vaccination schedule were not related. Only 36.5% of the general dentists had checked their antibody titer. We, therefore, recommend that dentists, as a potential high-risk group, should know their level of anti-HBs antibody titer so that those who require revaccination can get treatment.

HBsAg clearance in chronic hepatitis B patients with add-on pegylated interferon alfa-2a to ongoing tenofovir treatment: A randomized controlled study.

BACKGROUND/AIMS: The ideal end point of treatment for chronic hepatitis B virus (HBV) infection is sustained off-therapy hepatitis B surface antigen (HBsAg) loss with or even without seroconversion to anti-HBs. We investigated the role of adding PEGylated interferon (PEG IFN) to ongoing tenofovir treatment in chronic HBV patients for achieving HBsAg clearance. PATIENTS AND METHODS: In this randomized controlled trial, chronic HBV patients who have been receiving tenofovir for >6 months with HBV viral load <2000 IU/ml were randomized into two groups. One group (add-on therapy) was given subcutaneous PEG IFN 180 mcg weekly for 12 months in addition to tenofovir. Patients in the other group received only tenofovir 300 mg orally on a daily basis. Patients in both groups were followed up for a total of two years, and patients in both groups were given tenofovir 300 mg daily indefinitely until they developed HBsAg clearance. RESULTS: Twenty-three patients were allocated to the PEG IFN and tenofovir (add-on therapy) group, and another 25 patients were recruited to the tenofovir monotherapy group. Before randomization, patients had received tenofovir for 1135 mean days (range203 to 1542 days). One patient (4.3%) in add-on therapy lost HBsAg and seroconverted. Within two years, mean HBsAg decreased significantly with add-on therapy (from 4753 IU/ml to 2402; P= 0.03); and it decreased from 5957 IU/ml to 4198; P= 0.09 in tenofovir monotherapy group. More patients in the add-on group developed serious side effects, with treatment discontinuation, and dose reductions (P = 0.3). CONCLUSION: PEG IFN and tenofovir add-on therapy was successful in achieving HBsAg clearance and seroconversion in 4.3% of the patients. Add-on therapy patients had a significant decrease in HBsAg levels in two years; and no significant decrease in HBsAg levels with the tenofovir monotherapy. With no significant HBsAg clearance, the utility of this combination regimen is questionable.

Effect of switching from treatment with nucleos(t)ide analogs to pegylated interferon α-2a on virological and serological responses in chronic hepatitis B patients.

AIM: To investigate the efficacy of switching to pegylated interferon-α-2a (PegIFNα-2a) treatment in nucleos(t)ide analog (NA)-treated chronic hepatitis B (CHB) responder patients. METHODS: A 48-wk prospective and retrospective treatment trial of NA-treated CHB patients who had received entecavir (ETV) for at least 48 wk and had serum hepatitis B virus (HBV)-DNA < 500 IU/mL, serum hepatitis B envelope antigen (HBeAg) < 100 S/CO, serum alanine aminotransferase, and aspartate aminotransferase levels < 2 × the upper limit of normal of 40 IU/L was performed. The effects on virological and serological responses and adverse reactions to 0.5 mg daily ETV for 48 wk vs switching to PegIFNα-2a were compared. Forty-four patients were randomized to be switched from NA treatment to the PegIFNα-2a group, and 44 patients were simultaneously randomized to the ETV group. RESULTS: After 48 wk of therapy, the decrease in hepatitis B surface antigen (HBsAg) levels was greater in the PegIFNα-2a group than in the ETV group (3.1340 log10 IU/mL vs 3.6950 log10 IU/mL, P = 0.00). Seven patients who were anti-HBs-positive at baseline achieved HBsAg loss when switched to PegIFNα-2a (15.91% vs 0%, P = 0.018). The HBeAg serological conversion rate was higher in the PegIFNα-2a group than in the ETV group; however, the difference was not significant because of the small sample sizes (34.38% vs 21.88%, P = 0.232). In the PegIFNα-2a group, patients with HBsAg levels < 1500 IU/mL at baseline had higher HBeAg seroconversion and HBsAg loss rates at week 48 than those with HBsAg levels ≥ 1500 IU/mL (HBeAg seroconversion: 17.86% vs 62.5%, P = 0.007; HBsAg loss: 41.67% vs 6.25%, P = 0.016). Moreover, patients with HBsAg levels < 1500 IU/mL at week 24 had higher HBsAg loss rates after therapy than those with HBsAg levels ≥ 1500 IU/mL (36.84% vs 0%, P = 0.004). However, there were no statistically significant differences in HBeAg seroconversion rates (47.06% vs 25.93%, P = 0.266). CONCLUSION: NA-treated CHB patients switched to sequential PegIFNα-2a achieved highly potent treatment termination safely.

Seroprevalence of Hepatitis B Infection in Nigeria: A National Survey.

Hepatitis B virus (HBV) infection accounts for about 1 million deaths worldwide annually. This study was to determine the prevalence, distribution of HBV, and factors associated with infection in an apparently healthy population in Nigeria. A cross-sectional study among the general population was conducted employing a multistage sampling technique. Data on demographic, social, and behavioral indicators were collected using questionnaires and blood samples tested for HBV seromarkers. Descriptive, bivariate, and multivariate analyses were done. Prevalence of hepatitis B infection was 12.2% (confidence interval [CI] = 10.3-14.5). Of the participants, more than half, 527 (54.6%), had evidence of previous exposure to HBV, while 306 (31.7%) showed no serologic evidence of infection or vaccination. Only 76 (7.9%) participants showed serologic evidence of immunity to HBV through vaccination. Factors associated with testing positive for HBV infection were dental procedure outside the health facility (odds ratios [OR] = 3.4, 95% CI = 1.52-7.70), local circumcision (OR = 1.73, 95% CI = 1.17-2.57), and uvulectomy (OR = 1.65, 95% CI = 1.06-2.57). With logistic regression, only dental procedure outside the health facility (adjusted OR = 3.32, 95% CI = 1.38-7.97) remained significant. This first national survey on seroprevalence of hepatitis B describes the epidemiology and high prevalence of HBV infection in Nigeria and highlights the need for improved vaccination against HBV.

The fixation of anti-HBsAg on plastic surfaces.

Serum samples were found to be capable of desorbing as much as 40% of the antibody to hepatitis B surface antigen (anti-HBsAg) adsorbed to plastic surfaces. This previously unreported loss could affect the accuracy of the assay, so chemical fixation was examined as a means for preventing antibody desorption during a 'sandwich' radioimmunoassay for HBsAg. Methods for fixing the anti-HBsAg were developed with glutaraldehyde and ethylchloroformate. Both methods prevented antibody desorption from polyvinylchloride and polystyrene without affecting immunoreactivity in radioimmunoassay. A combined glutaraldehyde-ethylchloroformate method resulted in stronger fixation that fully resisted the sera that caused the greatest desorption. It was found that only polymerized glutaraldehyde fixed anti-HBsAg to plastic; the monomer was ineffective. Anti-HBsAg fixed microtiter plates could be stored for at least 4 weeks without loss of sensitivity in radioimmunoassays. These methods could be adapted for use in other assays where the prevention of protein desorption from the solid phase is an important consideration.

Precipitability and composition of HBsAg-anti-HBs immune complexes formed in the presence of complement. A model of circulating immune complex analysis.

Immune complexes (IC) of partially purified HBsAg and human anti-HBs were prepared at different antigen/antibody ratios in the presence of complement in normal human serum (NHS), and under conditions not allowing complement activation in buffers or in NHS containing 10 mM EDTA (NHS-EDTA). Commercial preparations of the radiolabelled antigen and antibody were used. IC formed in NHS were not significantly precipitated even after incubation for 24 h at 4 degrees C, whereas a typical precipitation curve was observed with complexes formed in the absence of complement. Thus, complement activation was found to markedly and permanently inhibit precipitability of HBsAg-anti-HBs immune complexes (HBsAg-IC). HBsAg-IC were precipitated from sera with 3.5% polyethylene glycol (PEG), boiled in sodium dodecyl sulphate (SDS)-urea buffer, and analysed by SDS-polyacrylamide slab gel electrophoresis (SDS-PAGE). With complexes formed in the presence of complement, about one-sixth of the antibody activity was found in high molecular weight fractions corresponding in size to IgG oligomers. By contrast, with complexes formed without complement, no significant amount of antibody was found in these fractions. With blotting technique and radiolabelled anti-human-C3 antibody, it was demonstrated that anti-HBs was covalently bound to C3b fragments in IC formed in the presence of complement and was in the high molecular weight fractions.

Application of impedance spectroscopy for monitoring colloid Au-enhanced antibody immobilization and antibody-antigen reactions.

We used colloidal Au to enhance the amount of antibody immobilized on a gold electrode and ultimately monitored the interaction of antigen-antibody by impedance measurement. Self-assembly of 6 nm (diameter) colloidal Au onto the self-assembled monolayers (SAMs) of 4-aminothiophenol modified gold electrode resulted in an easier attachment of antibody. The redox reactions of [Fe(CN)6](4-)/[Fe(CN)6](3-) on the gold surface were blocked due to the procedures of self-assembly of 4-aminothiophenol and antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.4 with various concentrations of antigen at 37 degrees C for 30 min. The antibody recognition layers and their interactions with various concentrations of antigen could be detected by measurements of the impedance change. The results show that this method has good correlation for detection of Hepatitis B virus surface antigen in the range of 0.5-200 microg/l and a detection limit of about 50 ng/l.

Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

Demonstration of HBsAg as the antigen component in circulating immune complexes detected by peg-solid phase test.

The development of an enzyme-linked immunosorbent assay to identify HBsAg as the antigen component within circulating immune complexes using immobilized polyethylene glycol (PEG) is described. The method utilizes, on one hand, the ability of PEG to bind stably to plastic supports and, on the other, to precipitate circulating macromolecules. This method is easily performed, very cheap, quick and, above all, it helps define the biological nature of the immune complexes. HBsAg can be revealed as the antigen component of HBsAg/anti-HBs soluble immune complexes at concentrations of at least 20 ng/ml and either in antigen or antibody excess. Our results indicate that HBsAg circulates in a complexed form in 47% of HBsAg chronic carriers and in 10.7% of patients with liver disease who are positive for serum antibody to hepatitis B surface antigen (anti-HBs) and to core antigen (anti-HBc). None of the other groups of patients in the study had circulating HBsAg in the complexed form.

Effect of nonspecific interactions of the hepatitis B surface antigen with the solid phase on its detection by two-site immunoradiometric assay.

Two-site immunoradiometric assay (Austria II-125, procedure B, Abbott) was used to detect hepatitis B surface antigen (HBsAg) in chronic carrier sera from clinically healthy subjects or from kidney transplant or hemodialysis patients. The titration curves of some high-titered sera showed a prozone-like effect; this corresponded in two such sera LB and MA, to congruent to 80% inhibition of HBsAg detection in undiluted serum relative to a 1:800 dilution. The nature of the inhibition in the above two sera was investigated. We found that the inhibition depended on the time of first incubation but not on the affinity of capture antibodies. Nonspecific adsorption of HBsAg to the solid phase during the first incubation with inhibitory sera and elution of the thus adsorbed antigen during the second incubation, with the resulting neutralization of 125I-labeled anti-HBs, appeared to cause the inhibition of detection observed. The inhibition was prevented by addition of the detergent Triton X-100 to serum or by elution of adsorbed antigen prior to the second incubation, thus indicating that further improvements of the HBsAg immunoradiometric assay may be possible.

A novel immunosensor based on immobilization of hepatitis B surface antibody on platinum electrode modified colloidal gold and polyvinyl butyral as matrices via electrochemical impedance spectroscopy.

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of platinum electrode modified with colloidal gold and polyvinyl butyral (PVB) as matrices to detect hepatitis B surface antigen (HBsAg) via electrochemical impedance spectroscopy (EIS). The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that K(4)[Fe(CN)(6)]/K(3)[Fe(CN)(6)] reactions on the platinum electrode surface were blocked due to the procedures of self-assembly of HBsAb-Au-PVB. The binding of a specific HBsAb to HBsAg recognition layer could be detected by measurements of the impedance change. A new strategy was introduced for improving the sensitivity of impedance measurements via the large specific surface area and high surface free energy of Au nanoparticles and the encapsulated effect of polyvinyl butyral. The results showed that this strategy caused dramatic improvement of the detection sensitivity of HBsAg and had good linear response to detect HBsAg in the range of 20-160 ng.ml(-1) with a detection limit of 7.8 ng.ml(-1). Moreover, the studied immunosensor exhibited high sensitivity and long-term stability.