Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd ∼10-12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

A novel general and efficient technique for dissociating antigen in circulating immune complexes.

Circulating immune complexes (CICs) are produced during the immune response. It is more clinically important to establish a general and efficient CICs dissociation technique for the detection of antigens for CICs other than the detection of free antigens in the serum. Polyethylene glycol (PEG) two-precipitation separation and glycine-HCl as a buffer system were employed to develop a general and efficient buffer dissociation technique to separate CICs from serum and dissociate antigens from CICs. The measurement value of new PEG two-precipitation separation technique was higher than traditional PEG precipitation separation technique. There were slight differences in the dissociation conditions of HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC as compared to HBsAg-IC. The detection of antigens in HBsAg-IC, HCV Core-IC, HIV P24-IC, Ins-IC and TG-IC with this technique was superior to that with HCl Dissociation, Trypsin Digestion or Immune Complex Transfer technique. PEG two-precipitation dissociation technique may reduce macromolecular protein and the adhesion of free antigens during the co-precipitation, which increases the efficiency of separation and precipitation of CICs. This technique also avoids the damage of reagents to antigens, assuring the repeatability, reliability and validity. Thus, this technique is application in samples negative or positive for free antigens.

[ANALYSIS OF EFFECTIVENESS OF USINGJIgY FROM CHICKEN IN SANDWICH METHOD OF HBsAg TESTING].

AIM: Study antigen-binding ability of polyclonal antibodies (PCA) of chicken compared with monoclonal -antibodies (MCA) of mice in the model of interaction with HBsAg. MATERIALS AND METHODS: Mice MCA 18C8 and MKA F3/F4 (IgG) were used, effective in enzyme immunoassay sandwich method of HBsAg determination (with a minimal detection dose of 0.017 ng/ml), and affinity purified anti-HBsAg PCA of chicken (IgY), obtained from 2 immunized birds (PCA No. 1 and PCA No. 2). The ability of antibodies to bind HBsAg was evaluated by analytical sensitivity (slope of binding curve) of solid-phase enzyme immunoassay system using mice MCA and chicken PCA. RESULTS: PCA No. 2 has provided a statistically significant 40% increase of analytical sensitivity, compared with <

Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen.

BACKGROUND: Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules. RESULTS: Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface. CONCLUSIONS: Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.

An Overview of Hepatitis B Virus Surface Antigen Mutant in the Asia Pacific.

Hepatitis B virus infection is a serious health problem worldwide, and more than 350 million people are chronic carriers, constituting a major global threat. Southeast Asia and the Western Pacific have the highest levels of endemicity in the world, with an estimated seroprevalence ranging between 2% and 31%. Mutations in the hepatitis B surface antigen (HBsAg) have been reported in many parts of the world but are most common in Asian infants; such mutants have several clinical effects, such as the development of hepatocellular carcinoma. Diagnostic failures by commercial assays have reduced the diagnostic effectiveness of HBsAg detection. For example the substitution of an amino acid in the major hydrophilic region of the S gene reduces the binding of hepatitis B surface antibodies leading to immune escape. The safety of blood transfusion may be compromised by current screening tests due to escape from being neutralised by antibodies induced by HBsAg mutants, and undetectable levels of viral surface protein. Data on the epidemiology of HBsAg mutation in Asia Pacific are scant; however, this manuscript has reviewed the available information on the epidemiology of HBsAg mutation in Asia Pacific.

Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure.

Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25Å-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous "floor". Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis.

Virus trap in human serum albumin nanotube.

Infectious hepatitis B virus (HBV), namely Dane particles (DPs), consists of a core nucleocapsid including genome DNA covered with an envelope of hepatitis B surface antigen (HBsAg). We report the synthesis, structure, and HBV-trapping capability of multilayered protein nanotubes having an anti-HBsAg antibody (HBsAb) layer as an internal wall. The nanotubes were prepared using an alternating layer-by-layer assembly of human serum albumin (HSA) and oppositely charged poly-L-arginine (PLA) into a nanoporous polycarbonate (PC) membrane (pore size, 400 nm), followed by depositions of poly-L-glutamic acid (PLG) and HBsAb. Subsequent dissolution of the PC template yielded (PLA/HSA)(2)PLA/PLG/HBsAb nanotubes (AbNTs). The SEM measurements revealed the formation of uniform hollow cylinders with a 414 ± 16 nm outer diameter and 59 ± 4 nm wall thickness. In an aqueous medium, the swelled nanotubes captured noninfectious spherical small particles of HBsAg (SPs); the binding constant was 3.5 × 10(7) M(-1). Surprisingly, the amount of genome DNA in the HBV solution (HBsAg-positive plasma or DP-rich solution) decreased dramatically after incubation with the AbNTs (-3.9 log order), which implies that the infectious DPs were completely entrapped into the one-dimensional pore space of the AbNTs.

Retrained Generic Antibodies Can Recognize SARS-CoV-2.

The dramatic impact novel viruses can have on humans could be more quickly mitigated if generic antibodies already present in one's system are temporarily retrained to recognize these viruses. This type of intervention can be administered during the early stages of infection, while a specific immune response is being developed. With this idea in mind, double-faced peptide-based boosters were computationally designed to allow recognition of SARS-CoV-2 by Hepatitis B antibodies. One booster face is made of ACE2-mimic peptides that can bind to the receptor binding domain (RBD) of SARS-CoV-2. The other booster face is composed of a Hepatitis B core-antigen, targeting the Hepatitis B antibody fragment. Molecular dynamics simulations revealed that the designed boosters have a highly specific and stable binding to both the RBD and the antibody fragment (AF). This approach can provide a cheap and efficient neutralization of emerging pathogens.

Construction of a humanized antibody to hepatitis B surface antigen by specificity-determining residues (SDR)-grafting and de-immunization.

We previously constructed a humanized antibody, HuS10, by grafting the complementarity-determining regions (CDRs) of a parental murine monoclonal antibody into the homologous human antibody sequences. This process is termed CDR grafting. Some residues that were thought to affect the CDR loops and stabilize the structure of the variable regions were retained in the framework region. HuS10 exhibited in vivo virus-neutralizing activity, but its murine content had the potential to elicit immune responses in patients. In this study, to minimize the immunogenic potential of HuS10, we replaced 17 mouse residues in HuS10 with the comparable human residues using specificity-determining residue (SDR)-grafting and de-immunization methods. The resultant humanized antibody, HzS-III, had the same affinity and epitope specificity as HuS10 and had reduced immunogenic potential, as assessed by T-cell epitope analysis. Thus, SDR grafting in combination with de-immunization may be a useful strategy for minimizing the immunogenicity of humanized antibodies. In addition, HzS-III may be a good candidate for immunoprophylaxis of HBV infection.

Survival after orthotopic liver transplantation: the impact of antibody against hepatitis B core antigen in the donor.

Liver transplantation using grafts from donors with antibody against hepatitis B core antigen (anti-HBc) increases the recipients' risk of developing hepatitis B virus (HBV) infection post-transplantation. Our aim was to assess whether using such grafts was associated with reduced posttransplantation survival and whether this association depended on recipients' prior exposure to HBV on the basis of their pretransplantation serological patterns. Data were derived from the United Network for Organ Sharing on adult, cadaveric, first-time liver transplants performed between 1994 and 2006. Among recipients who did not have HBV infection before transplantation, those with anti-HBc-positive donors had significantly worse unadjusted posttransplantation patient survival than recipients with anti-HBc-negative donors [hazard ratio, 1.35; 95% confidence interval (CI), 1.21-1.50]. However, after adjustments for other predictors of posttransplantation survival, including donor age, donor race, and recipient underlying liver diseases, patient survival was not significantly different between the 2 groups (hazard ratio, 1.09; 95% CI, 0.97-1.24). Among recipients without antibody against hepatitis B surface antigen (anti-HBs), use of anti-HBc-positive donor grafts was associated with a trend toward worse survival (adjusted hazard ratio, 1.18; 95% CI, 0.95-1.46), whereas no such trend was observed among recipients positive for anti-HBs. In conclusion, in patients without HBV infection before transplantation, using anti-HBc-positive donors was not independently associated with worse posttransplantation survival. Matching these donors to recipients with anti-HBs pre-transplantation may be especially safe.

Hepatitis B surface antibody purification with hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-tyrosine methyl ester) particles.

Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-tyrosine methyl ester) particles were prepared for the purification of hepatitis B surface antibody from human plasma. N-methacryloyl-L-tyrosine methyl ester was chosen as a complexing agent for hepatitis B surface antibodies. Hepatitis B surface antibody imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-tyrosine methyl ester) particles were characterized by surface area measurements, swelling test, scanning electron microscopy, elemental analysis, and Fourier transform infrared spectroscopy. Ethylene glycol (1.0M) was used as desorption agent. Adsorption studies were performed from hepatitis B surface antibody and anti-hepatitis A antibody positive human plasma. Effects of antibody concentration, contact time, N-methacryloyl-L-tyrosine methyl ester content and temperature on the adsorption capacity were investigated. The amount of hepatitis B surface antibody adsorbed per unit mass increased with increasing hepatitis B surface antibody concentration, then reached saturation. Maximum hepatitis B surface antibody adsorption amount was 21.4 mIU/mg. Adsorption process reached the equilibrium in 60 min. Competitive adsorption of hepatitis B surface antibody, total anti-hepatitis A antibody and total immunoglobulin E was investigated for showing the selectivity. Hepatitis B surface antibody-imprinted particles could adsorb hepatitis B surface antibody 18.3 times more than anti-hepatitis A antibody and 2.2 times more than immunoglobulin E. It can be concluded that hepatitis B surface antibody-imprinted particles have significant selectivity for hepatitis B surface antibody.

Validation of a new alternative for determining in vitro potency in vaccines containing Hepatitis B from two different manufacturers.

Facing the discontinuation of the Auszyme kit, an alternative is needed for determining the in vitro potency of Hepatitis B surface antigen in vaccines. An inhibition ELISA has already proven to be reliable, but not in vaccine combinations. We validated this method by the evaluation of monovalent and combined vaccines from two different manufacturers. All validation parameters fulfilled the defined acceptance criteria. There was some interference with Hepatitis B potency, mainly produced by the whole cell Pertussis component, but it was not significant. A significant correlation between the Auszyme kit and the in vivo method was observed. We demonstrated that our ELISA is suitable for evaluating HB antigen in vaccines and could be considered as a potential alternative to the Auszyme kit.

Fabrication, characterization, and application of potentiometric immunosensor based on biocompatible and controllable three-dimensional porous chitosan membranes.

A novel three-dimensional porous chitosan membrane material was prepared as a matrix to encapsulate hepatitis B surface antibody (HBsAb) for fabrication of immunosensors. The porous chitosan matrix was prepared by electrodepositing a designer nanocomposite solution of chitosan-encapsulated silica nanoparticle hybrid film on an ITO electrode, and then removing the silica nanoparticles with HF solution. Using HBsAb as a model, the potentiometric immunosensor was constructed by linking HBsAb molecules to the three-dimensional porous chitosan film using glutaraldehyde as a cross-linker. Scanning electron microscopy was used to investigate the surface morphology of the three-dimensional porous chitosan films. Cyclic voltammograms and electrochemical impedance spectroscopy were used to probe the interfacial properties of the immunosensor. Results showed that the fabricated immunosensor with three-dimensional porous structure possessed high surface area, good mechanical stability, and good hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized protein and increased the protein loading. Therefore, the present immunosensor exhibits a wide linear range from 6.85 to 708 ng mL(-1) with a low detection limit of 3.89 ng mL(-1) for the detection of hepatitis B surface antigen (HBsAg). This work implied that the biocompatible and controllable three-dimensional porous chitosan membrane possessed potential applications for biosensing.

A novel method for sensitive, low-cost and portable detection of hepatitis B surface antigen using a personal glucose meter.

Hepatitis B virus (HBV) infection is the major public health problem leading cause of death worldwide. The most important diagnostic marker for this infection is hepatitis B surface antigen (HBsAg). In this study, a novel, inexpensive, portable and sensitive ELISA method was designed and investigated for diagnosis of HBsAg based on the functionalized Fe3O4 and Al2O3 nanoparticles, with the strategy for detecting the concentration of glucose using a cheap and accessible personal glucose meter (PGM). The ELISA system was constructed using hepatitis B antibody against HBsAg immobilized on streptavidin coated magnetic iron oxide particles (S-Fe3O4) as the capture antibody (Ab1). In addition, another hepatitis B antibody against different epitope of HBsAg (Ab2) and glucoamylase both were immobilized on Al2O3 nanoparticles. After formation of the sandwich immune complex between Ab1 and Ab2 immobilized on S-Fe3O4 and Al2O3 NPs, respectively, through HBsAg, starch was converted into glucose using glucoamylase. Then, the glucose concentration was measured using PGM. The concentration of HBsAg was calculated based on the linear relation between the concentrations of HBsAg and glucose. Under optimal conditions, this assay showed detection limit values of 0.3 to 0.4 ng ml-1 for "ay" and "ad" subtypes of HBsAg, respectively. The results indicate that the designed assay is comparable to the commercial kits in terms of sensitivity, on-site, specificity, cost, simplicity, portability and reproducibility. The presented method can be used in disadvantaged areas of the world and blood transfusion centers. To the best of our knowledge, this is the first report of using PGMs for HBSAg detection.

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens.

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the "a" region. METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the "a" determinant at positions 120 (P-->S), 123(T-->N) and 161 (M-->T) were found to affect reactivity of these mAbs. CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

Validation of a new ELISA method for in vitro potency assay of hepatitis B-containing vaccines.

The discontinuation of the Auszyme kit used by vaccine manufacturers and national control laboratories to determine the Hepatitis B surface antigen (HBsAg) content of hepatitis B vaccines has led GlaxoSmithKline (GSK) to develop an alternative inhibition ELISA method. Validation of this ELISA was performed according to The International Conference of Harmonization and reproducibility was assessed in a feasibility study with four Official Medicines Control Laboratories (OMCLs). The dose response curve demonstrated linearity (R2>0.99) in the range of 60-360 ng/ml HBsAg. The repeatability (CV<7%), intermediate precision (CV<10%) and accuracy (91-113% recovery) were similar to the Auszyme method. The commercial antibodies used in the assay were shown to contain antibodies that bind to a protective epitope of HBsAg and the specificity of the method for HBsAg was demonstrated. There was a good concordance with the Auszyme method, although the ELISA yielded higher results (25.3 vs. 24.4 micro.g/ml for Engerix-B (n=64), 28.9 vs. 27.0 micro.g/ml for Twinrix (n= 69) and 25.5 vs. 21.6 micro.g/ml for Infanrix penta (n=62)). The method was successfully transferred to the four OMCLs. It has been demonstrated that the ELISA is suitable for its intended purpose with hepatitis B-containing vaccines from GSK and thus could be used for these vaccines by national control laboratories and authorities. Further validation studies should focus on the use of this ELISA with vaccines from other manufacturers.

Cloning, expression and identification of the gene of human single-chain variable fragment antibody against Hepatitis B virus surface antigen.

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis B virus surface antigen (HBsAg) was panned with HBsAg immobilized on microtiter plate wells. After five rounds of panning 30 phage clones specific to HBsAg were obtained and one selected clone was sequenced. It was found to consist of 789 bp and its amino acid sequence and specifically detected the respective antigen in the patients but not in healthy persons.

Liver grafts from hepatitis B surface antigen-positive donors: A review of the literature.

The scarcity of available organs and the gap between supply and demand continue to be the main limitations of liver transplantation. To relieve the organ shortage, current transplant strategies have implemented extended criteria, which include the use of liver from patients with signs of past or present hepatitis B virus (HBV) infection. While the use of liver grafts from donors with evidence of past HBV infection is quite limited, some data have been collected regarding the feasibility of transplanting a liver graft from a hepatitis B surface antigen (HBsAg) positive donor. The aim of the present work was to review the literature regarding liver transplants from HBsAg-positive donors. A total of 17 studies were identified by a search in Medline. To date, HBsAg positive grafts have preferentially been allocated to HBsAg positive recipients. The large majority of these patients continue to be HBsAg positive despite the use of immunoglobulin, and infection prevention can only be guaranteed by using antiviral prophylaxis. Although serological persistence is evident, no significant HBV-related disease has been observed, except in patients coinfected with delta virus. Consistently less data are available for HBsAg negative recipients, although they are mostly promising. HBsAg-positive grafts could be an additional organ source for liver transplantation, provided that the risk of reinfection/reactivation is properly prevented.

Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv.

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1 M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precursor revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation.

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.