Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.     

Binding parameters and idiotypic profile of the whole immunoglobulin and Fab' fragments of murine monoclonal antibody to distinct determinants of the human high molecular weight-melanoma associated antigen.

The nonspecific accumulation of radioactivity in bone marrow, liver, and spleen in patients with melanoma injected with radiolabeled whole IgG of anti-high molecular weight-melanoma associated antigen (HMW-MAA) monoclonal antibody (mAb) hampers the application of immunoscintigraphy to visualize melanoma lesions. This nonspecific background can be reduced by utilizing fragments which do not contain the Fc portion of anti-HMW-MAA mAb. Since the in vivo targeting to melanoma lesions of radiolabeled F(ab')2 and Fab' fragments of anti-HMW-MAA mAb is critically dependent on their binding parameters, we have analyzed the effect of fragmentation on the binding characteristics of the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2. The three mAbs recognize distinct and spatially distant determinants with a heterogeneous distribution on the pool of HMW-MAA molecules synthesized by melanoma cells. The determinant recognized by mAb TP41.2 is detectable on a markedly smaller population of HMW-MAA molecules than those recognized by mAbs 225.28 and 763.74. 125I-labeled F(ab')2 fragments of the three mAbs displayed an immunoreactive fraction similar to that of the whole IgG, while Fab' fragments displayed a lower one. Fragmentation of mAbs 225.28 and TP41.2 to F(ab')2 produced a 2- and 1.5-fold reduction in their association constants but did not cause a significant change in that of mAb 763.74. Cleavage of F(ab')2 fragments to Fab' fragments produced 2-, 40-, and 7-fold reductions in the association constants of mAbs 225.28, 763.74, and TP41.2, respectively. These changes qualitatively fit the predictions of theory for univalent and bivalent mAb binding, since mAbs 763.74 and TP41.2 appear to show bivalent binding to melanoma cells and mAb 225.28 to show univalent binding. The affinity constant of IgG, F(ab')2 and Fab' fragments of mAbs 225.28, 763.74, and TP41.2 displays an inverse relationship with the extent of their time-dependent release from the membrane of melanoma cells. Since no endocytosis of mAb could be detected, the latter results suggest that radioactivity remains bound to melanoma cells in vivo for a longer time following injection of F(ab')2 fragments than following that of Fab' fragments of each of the anti-HMW-MAA mAb tested. Radiolabeled Fab' fragments of mAbs 763.74 and TP41.2 displayed a marked reduction in their reactivity with some of the antiidiotypic mAb tested. The loss of some idiotopes is likely to be caused by changes in the conformation of the molecules associated with the fragmentation of IgG and by damage during the iodination procedure.     

Syngeneic anti-idiotype monoclonal antibodies to murine anticarcinoembryonic antigen monoclonal antibodies

BALB/c mice were immunized with either NP-3 or NP-4, two anticarcinoembryonic antigen murine monoclonal antibodies. Each animal produced anti-idiotype antibodies to the corresponding immunogen and no cross-reactivity between anti-NP-3 sera and anti-NP-4 sera was detected. Hybridomas were produced from these animals and two IgG1 anti-idiotype monoclonal antibodies were obtained: CM1 specific for NP-3 and CM11 specific for NP-4. CM1 and CM11 recognized determinants located within the antibody-combining site, since each anti-idiotype antibody inhibited the binding between the corresponding idiotype and carcinoembryonic antigens. Using an immunoblotting technique, neither CM1 nor CM11 reacted with isolated heavy or light chains of NP-3 or NP-4, whereas binding was observed with the intact molecule. This observation indicates that CM1 and CM11 are directed against conformational idiotypes resulting from the association of the variable regions of the heavy and light chains. Taken together, these results suggest that CM1 and CM11 might bear internal images of carcinoembryonic antigen epitopes, and that they are potential candidates as idiotype vaccines against colorectal tumors.     

Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies

The monoclonal antibodies 225.28S and 465.12 to human melanoma-associated antigens have been tested with a large variety of surgically removed skin lesions and malignant tumors as well as with a panel of cultured cell lines in serological and immunochemical assays. The antibody 225.28S reacts with a plasma membrane antigen while the antibody 465.12 detects a cytoplasmic antigen. Both antibodies fail to react with melanocytes from normal skin as well as benign skin lesions but react with nevi, melanoma cells and some skin carcinomas. Analysis with surgically removed tumors and cultured human cell lines indicated that the plasma membrane antigen is restricted to skin lesions whereas the cytoplasmic antigen is synthesized by tumor cells of various histologcal origins. The plasma membrane antigen is composed of two glyco-polypeptides of 280,000 and > 440,000 daltons, while the cytoplasmic antigen consists of 4 glycopolypeptides of 94,000, 75,000, 70,000 and 25,000 daltons. None of these components are bridged by disulfide bonds. The cytoplasmic antigen was readily detected in the spent culture medium of melanoma cell lines in the form of a major 94,000 dalton and a minor 72,000 dalton structure, while the plasma membrane antigen was detectable only after vastly increasing the sensitivity of the assay system.     

Pilot trial of murine monoclonal antibodies in patients with advanced melanoma

We have performed a pilot trial with two murine monoclonal antibodies (MAbs) directed against two surface membrane antigens, p97 (MAb 96.5) and a proteoglycan antigen (MAb 48.7) primarily expressed in human melanoma. Five patients with disseminated melanoma were studied, all of whom had multiple cutaneous metastases. Four patients received 212 mg each of antibodies 96.5 and 48.7, and one patient received 424 mg of antibody 96.5 alone. MAbs were administered in escalating doses over ten days in four patients and over six days in one patient. There was no clear treatment-related toxicity. Immunohistologic studies on biopsies taken two to 240 hours after treatment showed extensive binding of murine immunoglobulin to melanoma cells, but not to normal cells in the same section. The intensity of antibody binding was uniform across the diameter of the tumor nodules. In two patients, no murine immunoglobulin was detected in biopsies taken ten days after the last treatment. The mean initial elimination half-life (T1/2) of infused MAbs was 40.5 hours in two patients who received a combination of both antibodies and 53.0 hours in a third patient who received only antibody 96.5; none of these patients had previously been exposed to mouse immunoglobulin. The elimination T1/2 was 21 hours in a fourth patient, who three months previously had tumor imaging with 2-mg radiolabeled antigen-binding fragments (Fab) prepared from antibody 48.7. Serum from this patient appeared to contain anti-idiotypic antibodies which specifically bound Fabs of antibody 48.7. Three other patients also developed human anti-mouse antibodies. There were no objective tumor regressions, and no histologic changes were noted on biopsy.     

Immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma cell monoclonal antibody

The immunofluorescent localization of associated antigen of anti-human nasopharyngeal carcinoma (NPC) cell monoclonal antibody (McAb) was performed in different tissues (NPC, non-NPC tumor, chronic inflammation of nasopharyngeal mucosa, adult normal tissue and embryo tissue) with 5 control experiments. McAb (CN-1, Cs-C1) was produced by Department of Microbiology of our college and all the specimens were confirmed by pathology. The results revealed that Cs-C1 antigen was mainly found on the surface of NPC cells with a positive rate of 80.3%. Majority of the positive cells showed yellow-greenish linear fluorescence surrounding the cell membrane while some cells manifested granular fluorescence. In addition, Cs-C1 antigen was also found in a few embryo tissues, epithelial cells of the normal gastric mucosa and cells of the gastric cancer. It is suggested that Cs-C1 antigen be a kind of molecular structure, being gradually produced or increased in quantity during carcinogenesis, and belong to the tumor associated antigen. Cs-C1 antigen could be important in the study of carcinogenic mechanism of NPC cells and valuable to clinical immunodiagnosis.     

Human high molecular weight-melanoma associated antigen mimicry by mouse anti-idiotypic MAb MK2-23. Immunohistochemical analysis of the reactivity of anti-anti-idiotypic MAb with surgically removed melanoma lesions

The mouse anti-id MAb MK2-23 bears the internal image of the antigenic determinant defined by anti-HMW-MAA MAb 763.74. Active specific immunotherapy with anti-id MAb MK2-23 is associated with a statistically significant prolongation of survival in patients with malignant melanoma. Characterization of the immune response elicited by anti-id MAb MK2-23 may contribute to our understanding of the mechanisms underlying the apparent beneficial clinical effects of active specific immunotherapy with anti-id MAb MK2-23. Therefore, 8 HMW-MAA binding anti-anti-id MAbs elicited with MAb MK2-23 were characterized in their reactivity with a large panel of surgically removed benign and malignant tumors and of normal tissues. The 8 anti-anti-id MAbs displayed subtle differences in their immunoperoxidase staining of both benign and malignant lesions and of normal tissues. The diversity in the fine specificity of the 8 anti-anti-id MAbs is likely to reflect the few somatic mutations which occur in the amino-acid sequence of the variable regions of their heavy and light chains in the course of the immune response to MAb MK2-23. The reactivity patterns of the 8 anti-anti-id MAbs with the tissue substrates are similar, although not superimposable upon that of anti-HMW-MAA MAb 763.74 elicited with melanoma cells. This difference may reflect the imperfect mimicry by anti-id MAb MK2-23 of the antigenic determinant defined by anti-HMW-MAA MAb 763.74. © 1995 Wiley-Liss, Inc.     

Human high molecular weight-melanoma associated antigen mimicry by an anti-idiotypic antibody: characterization of the immunogenicity and the immune response to the mouse monoclonal antibody IMel-1

The mouse anti-idiotype (anti-id) monoclonal antibody (mAb) IMel-1 recognizes an idiotope in the antigen combining site of the immunizing anti-human high molecular weight melanoma-associated antigen (HMW-MAA) mAb 225.28. The mAb IMel-1 is able to induce an immune response against self cross-reacting HMW-MAA in rabbits that express HMW-MAA in normal tissues. Most of the rabbit anti-anti-id antibodies recognize a spatially distant determinant(s) from that defined by anti-HMW-MAA mAb 225.28. The immunogenicity of mAb IMel-1 is enhanced by its administration with the muramyl dipeptide-derived adjuvant. Anti-HMW-MAA antibodies were not detected in sera from rabbits immunized with HMW-MAA bearing cultured human melanoma cells. The differential immunogenicity of mAb IMel-1 and cell membrane bound HMW-MAA may account for the ability of anti-id mAb to induce anti-HMW-MAA immunity in patients who have not mounted such a response to HMW-MAA present in their lesions. Rabbit anti-HMW-MAA antibodies induced by anti-id mAb IMel-1 inhibited interactions of melanoma cells with elements of extracellular matrix. This may represent an additional mechanism by which anti-HMW-MAA immunity may affect the biology of melanoma cells in patients with melanoma immunized with anti-id mAb IMel-1.     

Cross-reactivity of mimotopes with a monoclonal antibody against the high molecular weight melanoma-associated antigen (HMW-MAA) does not predict cross-reactive immunogenicity

The high molecular weight melanoma-associated antigen (HMW-MAA) is highly expressed in advanced primary and metastatic melanoma. An epitope of the core protein of HMW-MAA is recognized by the murine monoclonal antibody (mAb) 225.28S. In this study, we aimed to characterize peptides that antigenically mimicked this epitope and to determine their efficacy as components of an HMW-MAA-based anti-melanoma vaccine. Therefore, we screened a constrained 10 mer phage display peptide library against mAb 225.28S. Selected phage-displayed peptides were then tested for their specificity for the antibody's antigen-binding site. DNA sequences coding for specific peptide ligands were determined. Binding of mAb 225.28S to HMW-MAA was inhibited in a dose-dependent manner by phage-displayed peptides from 51 to 83% and by synthetic peptides from 38 to 87%. Subsequently, the immunogenicity of the five mimotopes with the highest inhibition capacity was examined in rabbits. Immunizations with synthetic mimotopes conjugated to tetanus toxoid resulted in peptide-specific antibodies, but none of the highly antigenic mimotopes induced HMW-MAA cross-reactive antibodies. This report describes an example of disparity between antigenicity and cross-reactive immunogenicity, complicating the selection of potential vaccine candidates.     

New monoclonal antibodies specific for the guinea pig line 10 hepatocarcinoma

Monoclonal antibodies reactive to line 10 (L10) hepatocarcinoma cells, but not to L2C leukemia cells, of strain 2 guinea pig were produced and characterized. Complement-mediated cytolysis assay and quantitative absorption analysis revealed that one of the monoclonal antibodies, 3C4 antibody (IgG2a), was highly specific for L10. Neither normal guinea pig tissues including adult and fetal liver, nor other hepatomas of strain 2 guinea pig, line 1 (L1) and Hepatoma III, were reactive with the 3C4 antibody. Another antibody, 2E4 (IgM), was reactive to the liver, kidney and spleen cells but not to the thymocytes, lymph node cells, brain and red blood cells of the guinea pig. The 2E4 antibody reacted to Hepatoma III but not to L1 cells. Therefore, 3C4 antibody is specific to a tumor-associated antigen of L10 hepatoma, and 2E4 antibody is cross-reactive with a certain differentiation antigen of normal tissues. The 3C4 antibody showed antibody-dependent cytotoxicity. Since 3C4 antibody was different in the isotype and the antigen recognized from a monoclonal antibody (D3) which was reported from the NIH in the USA, 3C4 is a new monoclonal antibody reactive with L10 hepatocarcinoma.     

In vitro differentiation of human melanoma cells analyzed with monoclonal antibodies

Many monoclonal antibodies (MABs) have been produced against cell surface molecules of melanoma cells, and these reagents might help in the definition of stages of differentiation of the normal and the malignant cells. In an attempt to detect MAB-defined determinants that modulate with differentiation, we treated nonpigmented human melanoma cells with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 nM. Differentiation could be induced in all 4 cell lines, as evidenced by growth retardation, development of projections, and induction of melanin or of premelanosomes in the projections as detected by transmission electron microscopy. Of the 9 MAB-defined cell surface antigens, three were shown to modulate with TPA-induced differentiation, as assessed by fluorescence microscopy and fluorescence-activated cell sorter analysis. Antigens detected by MABs 15.75 and 15.95 decreased in every one of the four cells after TPA induction of differentiation. The proteoglycan defined by 225.28S increased slightly in one, showed no change in another, and decreased in the remaining two. These three MAB-defined molecules thus are linked to differentiation and might help in designing a scheme of differentiation of the melanocyte lineage.     

Endocytosis and degradation of murine anti-human CD3 monoclonal antibodies by normal and malignant T-lymphocytes

Treatment of lymphoid malignancies with monoclonal antibodies (mAbs) and immunoconjugates is a promising new immunotherapeutic approach. However, few published studies have examined in detail the subcellular fate of antibodies following binding to lymphocyte cell surface antigens. In this study, we have investigated the disposition of monoclonal anti-CD3 antibody 64.1 following binding to normal and malignant T-lymphocytes by using cellular radioimmunoassays and immunoperoxidase and immunogold electron microscopy. Anti-CD3 mAbs were predominantly cleared from the cell membrane at 37 degrees C by receptor-mediated endocytosis, although passive shedding of antibody was also observed. Internalized antibody was sequentially transferred from coated pits to receptosomes and eventually to lysosomes. Intralysosomal degradation appeared to be the ultimate fate of internalized radiolabeled mAbs and was followed by exocytosis of free 125I to the culture medium. Ammonium chloride and monensin were potent inhibitors of lysosomal degradation of 125I-anti-CD3 mAbs and caused intracellular trapping of radiolabeled antibodies. The rapid endocytosis, degradation, and exocytosis of antibody observed in these studies elucidate the mechanism of the improved efficacy of anti-CD3 immunoconjugates when used in conjunction with inhibitors of lysosomal action.     

Cross-reactivity between human hemopoietic cells and brain tumors as defined by monoclonal antibodies

Summary A battery of 24 monoclonal antibodies raised against human hemopoietic cells was tested in an indirect immunofluorescence technique on frozen sections of a variety of human neurogenic and non-neurogenic tumors. Twelve antibodies demonstrated some type of labeling of neurogenic tumors, frequently in patterns characteristic for benign and/or malignant gliomas and/or primitive neuroectodermal tumors (PNETs). Although also some cross-reactivity occurred in non-neurogenic tumors, the apparent operational specificity of some of our antibodies within the nervous system promises some aid in neuropathological tumor diagnosis; this was also demonstrated by combined use of some antibodies on smear preparations in which diagnosis by conventional stains was uncertain.This study confirms and expands previous data that sharing of antigenic determinants by hemopoietic cells and nervous system tumors is common. The significance of these cross- reactivities is at present a matter of speculation; cross-reacting autoantibodies might interfere with immune regulation in tumor patients, and an immune response might be initiated when glioma cells bearing la antigens present tumor-associated antigens to T cells.Presented in part at the Second International Symposium on Biology of Brain Tumours, London, October 1984.     

Fine specificity of high molecular weight-melanoma-associated antigen-specific cytotoxic T lymphocytes elicited by anti-idiotypic monoclonal antibodies in patients with melanoma

HLA-A2-restricted CTLs, which lysed high molecular weight (HMW)-melanoma-associated antigen (MAA)(+) melanoma cells, were induced in patients with melanoma immunized with MELIMMUNE, a combination of the murine anti-idiotypic (anti-id) monoclonal antibodies (mAb) MEL-2 and MF11-30 (MW Pride et al., Clin Cancer Res 1998;4:2363.). In the present study we investigated whether CTL epitopes are present in anti-id mAb MF-11-30 and activate T cells to recognize HMW-MAA on melanoma cells. One candidate epitope in the mAb MF11-30 VH chain, VH (3-11), was selected based on the presence of HLA-A2 anchor residues and partial homology with the HMW-MAA epitope, HMW-MAA (76-84). Lymphocytes from HLA-A2(+)-immunized patients proliferated to VH (3-11) peptide and to a variant HMW-MAA peptide to a significantly greater extent than autologous lymphocytes stimulated with an irrelevant peptide and lymphocytes from nonimmunized patients. No proliferative response was detected to the wild-type HMW-MAA peptide (76-84). Significant increase in IFN-gamma production but not in interleukin 10 production in response to VH (3-11) and to variant HMW-MAA peptide (76-84) was observed in lymphocytes from the immunized patients. Stimulation of lymphocytes from HLA-A2(+) patients with the two peptides induced CTL, which lysed HMW-MAA(+)/HLA-A2(+) A375SM melanoma cells. This is the first report documenting the presence of immunogenic peptides in a murine anti-id mAb for a defined epitope expressed by a human melanoma-associated antigen. These results may be relevant for development of novel vaccines based on homology between anti-id mAb and tumor-associated antigen amino acid sequences.     

Radioimmunoimaging in malignant melanoma with monoclonal antibodies

Twenty-five patients with cutaneous melanoma were imaged with F(ab')2 fragments of antimelanoma monoclonal antibody (MoAb) 225.28S labeled with 99mTc and 131I or 111In. Out of 16 patients without evidence of metastatic lesions, 6 false-positive cases were observed. Only 4 out of 9 patients with known lesions showed positive findings (globally 8 of 19 metastatic sites). In conclusion, antimelanoma MoAb was of little help in radioimaging and stating this disease.     

Syngeneic antiidiotypic antisera to murine antihuman high-molecular-weight melanoma-associated antigen monoclonal antibodies

BALB/c mice were immunized with the murine antihuman high-molecular-weight melanoma-associated antigen monoclonal antibodies (MoAbs) 149.53, 225.28, 653.25, and 763.74. Antiidiotypic antibodies could be detected in bleedings obtained 3 days following the first booster, increased in titer in bleedings obtained following the second booster, and persisted at high level in bleedings obtained 38 days following the second booster. Cross-blocking studies with a panel of anti-melanoma-associated antigen, anti-HLA Class I, and anti-HLA Class II monoclonal antibodies showed that the antisera recognize private idiotypes. The latter are located within the antigen combining site, since antiidiotypic antisera specifically inhibited the binding of the corresponding immunizing anti-human high-molecular-weight melanoma-associated antigen monoclonal antibody to cultured human melanoma cells Colo 38 in a dose-dependent fashion. The spectrotype of the anti-MoAb 149.53 antiserum comprises eight major components in the range of pH 6.2 to 7.0; those of the anti-MoAb 225.28 antiserum and of the anti-MoAb 653.25 antiserum, two major components in the ranges of pH 6.4 to 6.6 and 6.5 to 6.7, respectively; and that of the anti-MoAb 763.74 antiserum, three major components in the range of pH 6.2 to 6.4.     

Synthetic peptides reactive with anti-human milk fat globule membrane monoclonal antibodies

Mammary mucins are increased in amounts in breast cancer patient sera, and most anti-breast cancer antibodies react with such mucins. One such mucin is found in human milk fat globule membrane and consists predominantly of O-linked sugars and a protein core. Partial complementary DNA clones for the protein core have recently been obtained. The nucleotide sequence is of interest as it contains a 60-base pair repeat, giving rise to a repeated 20-amino acid sequence (PDTRPAPGSTAPPAHGVTSA). Peptides with various lengths were synthesized using this sequence and the adjacent 4 amino acids (PDTR). Three anti-human milk fat globule membrane antibodies produced in our laboratory (BC1, BC2, and BC3) were tested to determine their reactivity with these synthetic peptides. Using three different assays (direct enzyme-linked immunosorbent assay test on peptides, direct enzyme-linked immunosorbent assay test on bovine serum albumin-conjugated peptides, and an inhibition test with the peptides in liquid, rather than solid phase), it was shown that APDTR was the minimum amino acid sequence required to form a reactive epitope with all 3 antibodies, although individual differences in the reactivities of the antibodies were noted. The addition of alanine (A) converted a nonreactive PDTR peptide to a reactive one, and the deletion of arginine (R) did the reverse; thus APDTR is the smallest peptide which reacts with these anti-human milk fat globule membrane antibodies.     

Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies

Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.