Beta Epitopes Research Articles

A reciprocal HLA-Disease Association in Rheumatoid Arthritis and Pemphigus Vulgaris.

Human leukocyte antigens (HLA) have been extensively studied as being antigen presenting receptors, but many aspects of their function remain elusive, especially their association with various autoimmune diseases. Here we discuss an illustrative case of the reciprocal relationship between certain HLA-DRB1 alleles and two diseases, rheumatoid arthritis (RA) and pemphigus vulgaris (PV). RA is strongly associated with HLA-DRB1 alleles that encode a five amino acid sequence motif in the 70-74 region of the DR beta chain, called the shared epitope (SE), while PV is associated with the HLA-DRB1*04:02 allele that encodes a different sequence motif in the same region. Interestingly, while HLA-DRB1*04:02 confers susceptibility to PV, this and other alleles that encode the same sequence motif in the 70-74 region of the DR beta chain are protective against RA. Currently, no convincing explanation for this antagonistic effect is present. Here we briefly review the immunology and immunogenetics of both diseases, identify remaining gaps in our understanding of their association with HLA, and propose the possibility that the 70-74 DR beta epitope may contribute to disease risk by mechanisms other than antigen presentation.

Immobilized amyloid precursor protein constructs: a tool for the in vitro screening of glial cell reactivity.

Astrocytes and microglia are closely associated with amyloid plaques in Alzheimer's disease (AD). Microglia constitute the first barrier surrounding plaques, although they seem to be unable to remove them efficiently. We evaluated the reaction of microglial cells from neonatal rats and mice to plaque mimetics. The C-terminal part of the amyloid precursor protein (APP) or amyloid peptide (A beta) was immobilized to either 60-microm or 2.8-microm beads and incubated with microglial cells. Beads of 60 microm, having approximately the size of senile plaques, were not phagocytosed, in contrast to 2.8-microm beads, which were phagocytosed by microglia but not by astrocytes. Once taken up by the cells, proteins immobilized to the beads were degraded rapidly, as confirmed by mass spectrometry and immunofluorescence with an antibody against beta-amyloid. On the other hand, no protein degradation was observed with 60-microm beads. Also, probably as a reaction to its incapability to phagocytose the beads, glia organized around the beads and started to proliferate. Cell proliferation was more pronounced when the beads contained the A beta epitope compared with the beads with an inert surface. This in vitro effect could be exploited to set up a screening assay for compounds that ameliorate the adverse reaction of microglia supposed to contribute to the pathogenesis of AD.

Magnetic resonance contrast enhancement of neovasculature with alpha(v)beta(3)-targeted nanoparticles.

Site-directed contrast enhancement of angiogenic vessels in vivo was demonstrated using antibody targeting of an MRI contrast agent to the alpha(v)beta(3) integrin, a molecular marker characteristic of angiogenic endothelium. The agent was tested in a rabbit corneal micropocket model, in which neovasculature is induced in the cornea using basic fibroblast growth factor. The targeted contrast agent consists of Gd-perfluorocarbon nanoparticles linked to alpha(v)beta(3) integrin antibody DM101. The animal group receiving the targeted contrast agent displayed a 25% increase in the average MR signal intensity after 90 min. Control groups in which the nanoparticles are either used alone, linked to an isotype-matched antibody, or linked to DM101 and administered following receptor blocking did not display MR contrast enhancement at similar dose levels. These findings indicate that the antibody-targeted agent enhances MR signal intensity in the capillary bed in a corneal micropocket model of angiogenesis, and is selectively retained within the angiogenic region via specific interaction with the alpha(v)beta(3) epitope.

The hololipoprotein complex of beta(2)-glycoprotein I and dicaproyl phosphatidylserine.

The ability of beta(2)-glycoprotein I (formerly referred to as apolipoprotein H) to act as an autoantigen for antibodies from patients with antiphospholipid syndrome is dependent upon its binding in vivo to anionic phospholipid surfaces or to surfaces in vitro which mimic their surface characteristics. The ability of the autoepitope(s) of beta(2)-glycoprotein I to be exposed by binding a short-chain (6-carbon), anionic phospholipid has not been explored. Here, we describe our studies of the hololipoprotein generated by reacting beta(2)-glycoprotein I with dicaproyl phosphatidylserine. The formation of the complex is accompanied by inhibition of beta(2)-glycoprotein I binding to phospholipid-coated polystyrene surfaces, with 50% reduction in binding occurring at about 10 mM. At concentrations >10 mM, dicaproyl phosphatidylserine also displaces beta(2)-glycoprotein I bound to anionic phospholipid surfaces. Physicochemical studies suggest that at DCPS concentrations >6 mM, the solutions are colloidal and that beta(2)-glycoprotein I forms a supramolecular complex with organized phospholipid structures. Using a standard human autoimmune anti-beta(2)-glycoprotein I plasma, as well as a series of six additional sera from patients with antiphospholipid syndrome, the complex did not generate a detectable epitope. We conclude that lipid binding, per se, is not sufficient for the presentation of the epitope(s) of beta(2)-glycoprotein I or its recognition by autoantibodies from patients with antiphospholipid syndrome.

Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein:antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization.

The BDC 2.5 T cell clone is specific for pancreatic beta-cell antigen presented by I-Ag7, and greatly accelerates diabetes when injected into 10-21-d-old nonobese diabetic (NOD) mice. The BDC 2.5 T cell receptor (TCR) has been solubilized as a TCR-IgG1 chimeric protein. All NOD mice immunized against BDC 2.5 TCR-IgG1 produced antibodies recognizing TCR C alpha/C beta epitopes that were inaccessible on the T cell surface. 56% of the mice produced antibodies against the BDC 2.5 clonotype that specifically blocked antigen activation of BDC 2.5 cells. We have used the adoptive transfer model of diabetes to demonstrate that maternal immunization with soluble TCR protects young mice from diabetes induced by the BDC 2.5 T cell clone.

The isoform-specific expression of the tri-iodothyronine receptor in osteoblasts and osteoclasts.

Hyperthyroidism is associated with an increase in both osteoblast and osteoclast activity. We have previously shown that, in vitro, osteoclasts do not respond directly to tri-iodothyronine to increase bone resorption but that the effect is mediated by another bone cell, probably the osteoblast. To investigate this issue further we have studied the isoform-specific expression of thyroid receptor (TR) protein in human osteoclasts derived from an osteoclastoma (giant cell tumour of bone) and the expression of TR mRNA and protein in the osteoblastic cell lines MG 63 and UMR 106. Three major TR receptor variants have been described; TR alpha 1 and TR beta are functional receptors whereas c-erbA alpha 2 is a non-functional variant. Northern blot analysis using [32P]-cDNA probes against human TR alpha 1, c-erbA alpha 2 and TR beta demonstrated specific binding of these probes to mRNA from MG 63 and UMR 106. mRNA for all three receptor variants was observed in both cell lines, in UMR 106 multiple mRNA transcripts were present for TR alpha 1 and TR beta. Immunohistochemical staining with antibodies recognizing a common TR alpha epitope and specific c-erbA alpha 2 and TR beta epitopes extended these observations by demonstrating receptor protein in both osteoblasts and osteoclasts. These findings are consistent with previous observations of TR expression in osteoblast-like-cells and are the first direct demonstration of TR in osteoclasts.

Usage of β1 Integrin Ligands by B Cells is Developmentally Regulated in Avian Bursa

Chicken B cell development takes place in a separate organ, the bursa of Fabricius, which provides the blood-borne stem cells, a microenvironment specialized for B cell maturation. Therefore, chicken can be used as a model to study specifically the molecules and interactions which control the development of the B cell compartment. In this work, we studied expression, localization and function of beta 1 integrins on maturing B cells and bursal stroma. The expression of beta 1 integrins on B cells increases during the embryonic development and beta 1 integrin-positive cells can be found both in the medulla and the cortex throughout the bursal development. The binding assays show that the attachment of B cells to stroma is mediated by beta 1 integrins. Binding to 10-day-old embryonic stroma is fibronectin-independent, whereas fibronectin-mediated binding takes place in an increasing manner during further embryonic maturation. After hatching fibronectin appears to be the main binding site for B cells. However, the epitope of beta 1 integrin which takes part in the adhesion of B cells to stroma and to fibronectin is not crucial for the homing of cells into the bursa in a cell transfer model. Our results indicate that the interaction of beta 1 integrins with their ligands is developmentally regulated in the bursa and suggest that B cell maturation may be partially controlled by this interaction.

Selective killing of T cells by immunotoxins directed at distinct V beta epitopes of the T cell receptor.

The potency and specificity of anti-T cell receptor (TcR)-directed immunotoxins were studied in two T cell leukemia lines, HPB-ALL and Jurkat, and in primary T cells. Immunoconjugates were synthesized using anti-CD3, or distinct anti-V beta antibodies cross-linked to CRM9, a binding site-mutant of diphtheria toxin. All TcR-expressing cells display the CD3 complex on the plasma membrane. HPB-ALL cells express the V beta 5 gene product in the beta subunit of the TcR, while Jurkat cells express V beta 8. V beta expression in primary T cells isolated from buffy coats is heterogeneous. Primary T cell populations expressing specific V beta epitopes in the TcR were generated by plating CD3+ T cells on V beta-specific antibody-coated flasks or by positive immunomagnetic selection. Immunotoxins directed against the invariant CD3 epsilon epitope target and kill all T cells. Immunoconjugates targeted at distinct anti-V beta epitopes are specific for cells that express the corresponding gene product in the TcR. The results demonstrate the ability of anti-TcR-based immunotoxins selectively to kill T cells with defined V beta epitopes. These reagents may be clinically useful in disorders mediated by autoreactive T cell populations exhibiting V beta restriction and in the treatment of clonal TcR-expressing lymphomas.

Immunological recognition and clinical significance of nicked human chorionic gonadotropin in testicular cancer

We studied the physical properties, immunological recognition, and clinical significance of nicked human chorionic gonadotropin (hCGn) in testicular cancer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the beta-subunit of hCGn (hCG beta n) dissociated into two peptides, and, by reversed-phase chromatography, hCG beta n was found to be less hydrophobic than hCG beta. Immunologically, hCGn lacked two epitopes specific for holo-hCG (c1, c2), whereas at least five hCG beta epitopes (beta 1-beta 5) were preserved, and, as a result, recognition of hCGn by different hCG assays varied widely. In 309 sera and 88 urine samples from patients with seminomatous or nonseminomatous testicular cancer, hCG-only, hCG+hCGn, and hCG+hCGn+hCG beta+hCG beta n+hCG beta core-fragment assays gave parallel results. hCGn was more abundant in urine than in serum samples. In conclusion, hCGn lacks two conformationally dependent epitopes of hCG, causing a change in hydrophobicity and explaining its failure to react in certain holo-hCG assays. Recognition of hCGn, however, does not seem to be crucial in the routine use of serum hCG as a tumor marker in patients with testicular cancer.

Isolation and Characterization of a Rat Testis Glycosphingolipid Based on Gangliotetraosylceramide and Having a Blood Group B Determinant Extended with β3-Linked N-Acetylgalactosamine1

Monoclonal antibody 14.2, directed against the GalNAc beta 3Gal alpha 3Gal beta epitope, was used to monitor the isolation of a novel glycosphingolipid from rat testes. Structural analysis was performed by mass spectrometry, proton NMR spectroscopy, gas chromatography-mass spectrometry after degradation, and exoglycosidase digestion. It was concluded that the structure was [formula: see text] This is the first report on an extended blood group B determinant.

Experimental autoimmune panencephalitis and uveoretinitis transferred to the Lewis rat by T lymphocytes specific for the S100 beta molecule, a calcium binding protein of astroglia.

The pathogenic potential of autoimmune T cell responses to nonmyelin autoantigens was investigated in the Lewis rat using the astrocyte-derived calcium binding protein S100 beta, as a model nonmyelin autoantigen. The Lewis rat mounts a vigorous RT1B1 (major histocompatibility complex class II) restricted autoimmune response to an immunodominant S100 beta epitope (amino acid residues 76-91). The adoptive transfer of S100 beta-specific T cell lines induced a severe inflammatory response in the nervous system, but only minimal neurological dysfunction in naive syngeneic recipients. The inability of S100 beta-specific T cell transfer to induce severe disease was associated with a decreased recruitment of ED1+ macrophages into the central nervous system (CNS) in comparison with that seen in severe experimental autoimmune encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein (MBP)-specific T line cells. Moreover, unlike encephalitogenic MBP-specific T cell lines, S100 beta-specific T cell lines exhibited no cytotoxic activity in vitro. Histopathological analysis also revealed striking differences in the distribution of inflammatory lesions in MBP- and S100 beta-specific T cell-mediated disease. In contrast to the MBP paradigm, S100 beta-specific T cell transfer induces intense inflammation not only in the spinal cord, but throughout the entire CNS and also in the uvea and retina of the eye. In view of the distribution of lesions throughout the grey and white matter of the CNS we propose to term this new model experimental autoimmune panencephalomyelitis (EAP) to differentiate it from EAE. These experiments demonstrate for the first time that nonmyelin CNS autoantigens can initiate a pathogenic autoimmune T cell response, although the nature of the target autoantigen profoundly influences the clinical and histopathological characteristics of the resulting autoimmune disease. This is not simply a consequence of the distribution of the autoantigen, as both MBP and S100 beta are coexpressed in many areas of the CNS, but reflects differences in the capacity of different regions of the CNS to process and present specific autoantigens. This new model of T cell-mediated autoimmune CNS disease exhibits a number of similarities to multiple sclerosis (MS), such as its mild clinical course and the involvement of areas of the brain and eye, which are absent in myelin-mediated models of EAE. Nonmyelin autoantigens may therefore play an unexpectedly important role in the immunopathogenesis of inflammatory diseases of the CNS.

Correlation between cellular rejection of cardiac allografts and quantitative changes among T-cell subsets identified by V beta epitope expression.

Cellular rejection of an allograft is mediated in part by peripheral blood T cells. We tested the hypothesis that quantitative changes in T-cell subsets can be detected in the peripheral blood and that these changes correlate with rejection. T-cell subset analysis was performed by flow cytometry using monoclonal antibodies recognizing six isotypic epitopes of the T-cell receptor beta-chain variable (V) region. These analyses were done at 7-day (mean) time intervals. Fluctuations within a given subset were determined by dividing the number of positive cells observed by the number of positive cells found on the previous analysis. For healthy volunteers observed over a period of 30 days, 119 of 120 subset ratios (99.2%) fell between 0.5 and 2.0. For patients, 57 of 240 subset ratios (23.8%) fell outside of this range (P < .004, chi 2). The occurrence of the abnormal ratios coincided more closely with cellular rejection (mean +/- SD, 7.7 +/- 6.2 days from a positive biopsy; median, 5 days; range, 0 to 28 days) than did the occurrence of normal subset ratios (mean +/- SD, 14.4 +/- 10.9 days from a positive biopsy; median, 11 days; range, 0 to 44 days; P < .005 by Mann-Whitney U test). Regression analysis confirmed a significant (P < .001, R = .91) temporal association between cellular rejection and abnormal subset fluctuations. No correlation was found between abnormal subset ratios and either vascular rejection or use of high-dose prednisone. T-cell subset measurement may be a method of noninvasive monitoring of cellular rejection after transplantation and may provide insights into the physiology of graft rejection with the potential for the development of more specific immunosuppressive therapy.

Beta-Amyloid precursor epitopes in muscle fibers of inclusion body myositis.

Sporadic inclusion body myositis (IBM) and hereditary inclusion body myopathy (hIBM) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers that contain 15- to 21-nm cytoplasmic tubulofilaments (CTFs). Those vacuolated muscle fibers also contain abnormally accumulated ubiquitin and beta-amyloid protein (A beta), and they contain amyloid in beta-pleated sheets as indicated by Congo red and crystal violet positivity. Using several well-characterized antibodies, we have now demonstrated that, in addition to A beta, two other epitopes, N-terminal and C-terminal, of the beta-amyloid precursor protein (beta PP) are abnormally accumulated in IBM vacuolated muscle fibers and similarly in hIBM. At the light microscopy level, immunoreactivities of N- and C-epitopes of beta PP closely colocalized with A beta and ubiquitin immunoreactivities. However, by immunogold electronmicroscopy, even though N-, C-, and A beta epitopes of beta PP and ubiquitin colocalized at the amorphous and dense floccular structures, only A beta was localized to the 6- to 10-nm amyloid-like fibrils and only ubiquitin was localized to CTFs. beta PP immunoreactive structures were often in proximity to CTFs, but CTFs themselves never contained beta PP immunoreactivities. The fact that A beta but not C- or N-terminal epitopes of beta PP localized to the 6- to 10-nm amyloid-like fibrils suggests that free A beta might be generated during beta PP processing and, after aggregation, may be responsible for the amyloid present within IBM muscle fibers. Our study demonstrates that three epitopes of beta PP accumulate abnormally in diseased human muscle, and therefore this phenomenon is not unique to Alzheimer's disease, Down's syndrome brain, and Dutch-type cerebrovascular amyloidosis.

High-level expression and in vitro mutagenesis of a fibrillogenic 109-amino-acid C-terminal fragment of Alzheimer's-disease amyloid precursor protein

We amplified DNA encoding the 3' 109 codons of Alzheimer's-disease amyloid precursor protein (APP) inclusive of the beta protein (A beta) and cytoplasmic domains from cDNA using oligonucleotide primers designed to facilitate cloning into the T7 expression vector pT7Ad23K13. We also modified this construct to generate recombinant molecules incorporating two recently described APP mutants by site-directed mutagenesis. Both native C109 (deletion construct inclusive of the C-terminal 109 residues of APP) and constructs with a single mutation at codon 642 (T-->G, resulting in a substitution of glycine for valine) or a double mutation at codons 595 (G-->T, substituting asparagine for lysine) and 596 (A-->C, substituting leucine for methionine) were expressed in Escherichia coli to levels of 5-20% of total bacterial protein after induction. The major constituent of expressed C109 protein had an apparent molecular mass of 16-18 kDa by SDS/PAGE and appeared to be the full-length construct by size and N-terminal microsequencing. Also present was a 4-5 kDa species that co-purified with C109, constituting only approximately 1% of expressed protein, which was revealed by Western-blot analysis with antibodies specific for A beta epitopes and after biotinylation of purified recombinant C109. This fragment shared N-terminal sequence with, and appeared to arise by proteolysis of, full-length C109 in biosynthetic labelling experiments. C109 spontaneously precipitated after dialysis against NaCl or water, and with prolonged (> 20 weeks) standing was found by electron microscopy to contain a minor (< 5%) fibrillar component that was reactive with antibodies to a C-terminal epitope of APP. Recombinant C109 appears to duplicate some of the biochemical and physicochemical properties of C-terminal A beta-inclusive fragments of APP that have been found in transfected cells, brain cortex and cerebral microvessels.

Cell biological studies with monoclonal and polyclonal antibodies against human casein kinase II subunit beta demonstrate participation of the kinase in mitogenic signaling.

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active (alpha and/or alpha') and two regulatory (beta) subunits. It has been suspected that, among numerous other cellular functions, CKII might play a role in the control of mitogenic signaling. To test for such a role and its mechanism in intact cells, monoclonal antibodies (mAbs) were generated against CKII beta using a recombinant protein containing amino acids 20-200 of human CKII beta. The CKII beta-specific mAb with the highest reactivity, mAb IVG6 (classified as IgG1 with kappa light chains), was purified to homogeneity. It recognized a CKII beta epitope comprising the amino acids 140-156, a basic and highly conserved region. In addition, polyclonal antibodies (pAbs) were raised and made monospecific by affinity purification. pAbs-mediated quantitative immunofluorescence microscopy of human IMR-90 fibroblasts and/or Western blots of cell fractions revealed (i) CKII beta was present in exponentially growing cells at a 2-3-fold higher level than in quiescent cells, (ii) CKII beta was localized predominantly in the nucleus of cells (3-15-fold cytoplasmic level depending on cellular state and assay used), and (iii) the nuclear/cytoplasmic ratio of CKII beta was higher by a factor of 2 in exponentially growing cells. Consequently, mitogenic stimulation of quiescent cells by fetal calf serum doubled the nuclear/cytoplasmic ratio of CKII beta. The increase occurred within the 1st h of stimulation. The translocation of CKII beta into the nucleus was inhibited when mAb IVG6 was injected into the cytoplasm at the time of mitogenic stimulation. This microinjection also significantly inhibited the cell proliferation. The data imply that cytoplasmic CKII participates in the transmission of mitogenic signals by translocation into the nucleus.

Distribution of SP 1, immunoreactivity among different plasma proteins: real molecular heterogeneity or adsorption of SP 1-beta to other plasma proteins?

Three SP 1-containing factors from pooled term pregnancy sera were subjected to crossed immunoelectrophoresis. New patterns as far as electrophoretic mobilities and shapes of the immunoprecipitates were revealed. The appearance of an additional anodic radioimmunoassayable activity in agarose electrophoresis of mixed SP 1-alpha and SP 1-beta suggested a binding capacity of SP 1-alpha for SP 1-beta determinants. In the serum of a single patient at the third trimester of pregnancy we also found two SP 1 variants, possessing little radioimmunological reactivity and with crossed immunoelectrophoretic characteristics quite different from those of the ‘usual’ alpha and beta SP 1 forms. These results suggest that, in this particular case, the overall SP 1 production cannot be evaluated by competitive binding assay and, that in general, SP 1 is a complex antigen the heterogeneity of which can be determined following adsorption of some beta epitopes to another serum protein.

A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

Human and murine B-cells recognize the HBeAg/beta (or HBe2) epitope as a linear determinant

The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), deduced from the genome of the HBV ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to the beta (or HBe2) epitope of hepatitis B e antigen (HBe/b mAbs; 57 8 , 78 3 , 141 158 and 141 207 ). Cross-competition between the mAbs with a mAb to the HBe/alpha epitope (or HBel) and an anti-HBc mAb showed that all the HBe/b mAbs specifically inhibited human anti-HBe/b binding. Screening the HBc/e peptides showed that all anti-HBe/b mAbs recognized a peptide covering the residues 126–135. Three of the mAbs, 78 3 , 141 152 and 141 207 , had a less restricted reactivity than the other two, suggesting the recognition of the HBe/b as a discontinuous determinant. Fine mapping of the region aa 126–135 was performed by synthesizing decapeptides with nine overlapping aas, covering residues aa 121–140. All mAbs, except 78 3 , reacted with the linear sequence TPPAYR, at residues 128–133. An additional set of peptides was synthesized, where the six aas within the epitope 128–133 were substituted in turn by the other 19 possible aas. By this approach, the essential aas for mAb 7 8 were found to be the sequence of PPA at residues 129 131 , and for mAb 141 158 the sequence PP-Y, at residues 129, 130 and 132, respectively. Human recognition of the linear HBe/b epitope was investigated by using a peptide covering residues 121–140 (p 33). Thirty-one sera from chronic carriers of HBsAg, of which seven were positive for HBeAg and the remaining 24 for anti-HBe, were investigated. Of the sera with HBeAg, two had low levels of anti/-HBe/b in the p 33 assay. Out of the sera with anti-HBe, eight were positive in the p 33 EIA. Thus, murine monoclonals and human sera may recognize the HBe/b epitope as a linear determinant residing around aa 130.