Specific Antigenic Sites Research Articles

Site-specific serology unveils cross-reactive monoclonal antibodies targeting influenza A hemagglutinin epitopes.

Efficient identification of human monoclonal antibodies targeting specific antigenic sites is pivotal for advancing vaccines and immunotherapies against infectious diseases and cancer. Existing screening techniques, however, limit our ability to discover monoclonal antibodies with desired specificity. In this study, we introduce a novel method, blocking of binding (BoB) enzyme-linked immunoassay (ELISA), enabling the detection of high-avidity human antibodies directed to defined epitopes. Leveraging BoB-ELISA, we analyzed the antibody response to known epitopes of influenza A hemagglutinin (HA) in the serum of vaccinated donors. Our findings revealed that serum antibodies targeting head epitopes were immunodominant, whereas antibodies against the stem epitope, although subdominant, were highly prevalent. Extending our analysis across multiple HA strains, we examined the cross-reactive antibody response targeting the stem epitope. Importantly, employing BoB-ELISA we identified donors harboring potent heterosubtypic antibodies targeting the HA stem. B-cell clonal analysis of these donors revealed three novel, genealogically independent monoclonal antibodies with broad cross-reactivity to multiple HAs. In summary, we demonstrated that BoB-ELISA is a sensitive technique for measuring B-cell epitope immunogenicity, enabling the identification of novel monoclonal antibodies with implications for enhanced vaccine development and immunotherapies.

Antibodies targeting Crimean-Congo hemorrhagic fever virus GP38 limit vascular leak and viral spread.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen transmitted by tick bites, with no vaccines or specific therapeutics approved to date. Severe disease manifestations include hemorrhage, endothelial dysfunction, and multiorgan failure. Infected cells secrete the viral glycoprotein GP38, whose extracellular function is presently unknown. GP38 is considered an important target for vaccine and therapeutic design as GP38-specific antibodies can protect against severe disease in animal models, albeit through a currently unknown mechanism of action. Here, we show that GP38 induces endothelial barrier dysfunction in vitro, and that CCHFV infection, and GP38 alone, can trigger vascular leak in a mouse model. Protective antibodies that recognize specific antigenic sites on GP38, but not a protective neutralizing antibody binding the structural protein Gc, potently inhibit endothelial hyperpermeability in vitro and vascular leak in vivo during CCHFV infection. This work uncovers a function of the secreted viral protein GP38 as a viral toxin in CCHFV pathogenesis and elucidates the mode of action of non-neutralizing GP38-specific antibodies.

Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry.

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus.

Comprehensive N-glycosylation mapping of envelope glycoprotein from tick-borne encephalitis virus grown in human and tick cells

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

Identification of Dengue Virus Serotype 3 Specific Antigenic Sites Targeted by Neutralizing Human Antibodies

The rational design of dengue virus (DENV) vaccines requires a detailed understanding of the molecular basis for antibody-mediated immunity. The durably protective antibody response to DENV after primary infection is serotype specific. However, there is an incomplete understanding of the antigenic determinants for DENV type-specific (TS) antibodies, especially for DENV serotype 3, which has only one well-studied, strongly neutralizing human monoclonal antibody (mAb). Here, we investigated the human B cell response in children after natural DENV infection in the endemic area of Nicaragua and isolated 15 DENV3 TS mAbs recognizing the envelope (E) glycoprotein. Functional epitope mapping of these mAbs and small animal prophylaxis studies revealed a complex landscape with protective epitopes clustering in at least 6-7 antigenic sites. Potently neutralizing TS mAbs recognized sites principally in E glycoprotein domains I and II, and patterns suggest frequent recognition of quaternary structures on the surface of viral particles.

Antigenic Fingerprinting of Respiratory Syncytial Virus (RSV)-A-Infected Hematopoietic Cell Transplant Recipients Reveals Importance of Mucosal Anti-RSV G Antibodies in Control of RSV Infection in Humans.

Respiratory syncytial virus (RSV) infection causes significant morbidity in hematopoietic cell transplant (HCT) recipients. However, antibody responses that correlate with recovery from RSV disease are not fully understood. In this study, antibody repertoire in paired serum and nasal wash samples from acutely RSV-A-infected HCT recipients who recovered early (<14 days of RSV shedding) were compared with late-recovered patients (≥14 days of shedding) using gene fragment phage display libraries and surface plasmon resonance. Anti-F serum responses were similar between these 2 groups for antibody repertoires, neutralization titers, anti-F binding antibodies (prefusion and postfusion proteins), antibody avidity, and binding to specific antigenic sites. In contrast, nasal washes from early-recovered individuals demonstrated higher binding to F peptide containing p27. While the serum RSV G antibody repertoires in the 2 groups were similar, the strongest difference between early-recovered and late-recovered patients was observed in the titers of nasal wash antibodies, especially binding to the central conserved domain. Most importantly, a significantly higher antibody affinity to RSV G was observed in nasal washes from early-recovered individuals compared with late-recovered HCT recipients. These findings highlight the importance of mucosal antibodies in resolution of RSV-A infection in the upper respiratory tract.

Antigenic sites in influenza H1 hemagglutinin display species-specific immunodominance.

Hemagglutination inhibition (HI) titers are a major correlate of protection for influenza-related illness. The influenza virus hemagglutinin possesses antigenic sites that are the targets of HI active antibodies. Here, a panel of mutant viruses each lacking a classically defined antigenic site was created to compare the species-specific immunodominance of the antigenic sites in a clinically relevant hemagglutinin. HI active antibodies of antisera from influenza virus-infected mice targeted sites Sb and Ca2. HI active antibodies of guinea pigs were not directed against any specific antigenic site, although trends were observed toward Sb, Ca2, and Sa. HI titers of antisera from infected ferrets were significantly affected by site Sa. HI active antibodies of adult humans followed yet another immunodominance pattern, in which sites Sb and Sa were immunodominant. When comparing the HI profiles among different species by antigenic cartography, animals and humans grouped separately. This study provides characterizations of the antibody-mediated immune responses against the head domain of a recent H1 hemagglutinin in animals and humans.

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.

Networks link antigenic and receptor-binding sites of influenza hemagglutinin: Mechanistic insight into fitter strain propagation

Influenza viral passaging through pre-vaccinated mice shows that emergent antigenic site mutations on the viral hemagglutinin (HA) impact host receptor-binding affinity and, therefore, the evolution of fitter influenza strains. To understand this phenomenon, we computed the Significant Interactions Network (SIN) for each residue and mapped the networks of antigenic site residues on a representative H1N1 HA. Specific antigenic site residues are ‘linked’ to receptor-binding site (RBS) residues via their SIN and mutations within “RBS-linked” antigenic residues can significantly influence receptor-binding affinity by impacting the SIN of key RBS residues. In contrast, other antigenic site residues do not have such “RBS-links” and do not impact receptor-binding affinity upon mutation. Thus, a potential mechanism emerges for how immunologic pressure on RBS-linked antigenic residues can contribute to evolution of fitter influenza strains by modulating the host receptor-binding affinity.

Lipid rafts hinder binding of antibodies to the extracellular segment of the membrane-anchor peptide of mIgA

Membrane-bound IgA (mIgA) is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.

The lipid raft hinders antibodies from binding to the external segment of the membrane-anchor peptide of mIgA (52.19)

Abstract The mIgA is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα), comparing to the α chain of secreted IgA, contains an extra C-terminal membrane-anchor peptide, which encompasses an extracellular, a transmembrane, and an intracellular segments. The extracellular segment, referred to as mIg isotype-specific (migis-α) segment or extracellular membrane proximal domain (EMPD), has been proposed as a specific antigenic site for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α mAbs, such as MAb 29C11, which were specific to a peptide segment toward the N-terminal of migis-α. The mAbs bound strongly to synthetic peptides of migis-α and various recombinant proteins containing migis-α in ELISA. However, the mAbs did not bind to mIgA on B cell lines strongly in fluorescence flow cytometric analysis. After disrupting the lipid raft integrity of the B cells by a typical cholesterol extraction procedure, the mAbs could then bind to the treated B cells strongly. Immunopricipitation analysis with the mAbs indicated only when the B cell lines were solubilized by sufficiently strong detergents, such as sodium dodecyl sulfate (SDS), the mIgA could be pulled down by the mAbs. These results suggest that the migis-α region of mIgA in the BCR is associated with the lipid raft. Future studies will investigate inhibitory effects of mAb 29C11 on mIgA-expressing B cells.

Prevention of serious respiratory syncytial virus-related illness. II: Immunoprophylaxis

Respiratory syncytial virus (RSV) causes significant morbidity in very young children, preterm infants with and without chronic lung disease, and children with hemodynamically significant congenital heart disease. In the absence of a safe and effective vaccine, alternative means of protecting high-risk infants and young children from serious RSV illness have been studied. Clinical observations and animal model data over the past 30 years suggested that RSV immunoglobulin G (IgG) neutralizing antibodies might offer protection from severe RSV lower respiratory tract disease. Transfer of adequate amounts of IgG to the fetus does not occur efficiently until the third trimester of pregnancy, which helps to explain why premature infants are at high risk of serious RSV illness. Efforts shifted toward the prophylactic monthly administration of standard immunoglobulins and, later, of RSV-enriched immunoglobulin in selected high-risk infants and young children. Although this approach proved effective, RSV-enriched immune globulin was not suitable for all patients and administration was labor intensive. The development of palivizumab, a monoclonal antibody that can bind to a specific antigenic site on the virus and prevent cell-to-cell spread of infection has since become the mainstay of RSV illness prevention in preterm infants and those with significant congenital heart disease. Palivizumab, the only monoclonal antibody approved for the prevention of RSV lower respiratory tract disease must be administered monthly throughout the RSV season and does not always prevent serious RSV illness. Further research to develop more effective and less labor-intensive immunoprophylactic agents is ongoing.

Serological association of lupus autoantibodies to a limited functional domain of 28S ribosomal RNA and to the ribosomal proteins bound to the domain.

Site-specific anti-RNA antibodies were sought in 120 sera of patients with autoimmune diseases by ribonuclease-protection assay using six fragments covering 28S ribosomal RNA (rRNA) as antigens. Fifteen of 90 sera from patients with systemic lupus erythematosus (SLE), but none of 30 sera of the other autoimmune diseases, provided a 60 nucleotide fragment within a region termed the 'GTPase domain' of 28S rRNA. These sera had potency to precipitate 0.42-69.3 nmol of the RNA domain per ml serum, which was higher than 15 control sera of healthy donors. No other specific antigenic site was detected in 28S rRNA under conditions used. All of the 15 sera having this anti-RNA antibody showed reactivity to ribosomal P proteins (anti-P), and two of them contained an additional antibody to ribosomal protein L12. These results suggested a strong association of the production of these three antibodies. Since P and L12 proteins form a stable complex with the GTPase domain, this serological association may result from an immune response to epitopes clustered on a single RNA-protein complex domain in ribosomes.

Eu-Chelate Construct Ultrasensitive Time-Resolved Fluoroimmunoassay-Assay of Pepsinogen I

Eu-chelate were used to construct a two-site sandwich-type assay for pepsinogen I (PGI) with time-resolved flu-oroimmunoassay (TRFIA) as a detection technique. On the noncompetitive assay, captured monoclonal antibodies (McAbs) coated on wells were directed against a specific antigenic site on the PGI. Another McAbs, called as labeling McAbs, were prepared with the Eu-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-N, N, N, N-tetraacetie acid and directed against a different antigenic site on the PGI. The fluorescence counts of bound Eu 3+ -McAbs were measured with the auto DELFIA 1235 system. The PGI in sera from healthy volunteers were determined by PGI-TRFIA. The within-run and between-run CVs of the PGI-TRFIA were 1.9% and 4.7%, respectively, and the recovery rate was 102.65%. The assay had a detection limit of 0.05 μg·L −1. The PGI-TRFIA provided a linear response from 3.5 to 328 μg·L −1. The cross-reacting rate with pepsinogen II was negligible. The linear correlation of PGI-TRFIA and radioimmunassay measurements resulted in a correlation coefficient of 0.977. The means of healthy volunteers were 154 ± 43 μg·L −1 for serum PGI. The availability of a highly sensitive, reliable, and convenient method for quantifying PGI will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastritis and severe atrophic gastritis.

Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications

A fast and highly sensitive assay for pepsinogen I (PG I) and pepsinogen II (PG II) by using time-resolved fluoroimmunoassay (TRFIA) detection technique has been developed for the determination of serum PG I and PG II against gastrointestinal diseases. On the noncompetitive assay, one monoclonal antibody (McAb) coated on wells was directed against a specific antigenic site on the PG I or PG II. The McAb, called as labelling McAb, was prepared with the europium-chelate of N-( p-isothiocyanatobenzyl)-diethylenetriamine- N, N, N, N-tetraacetic acid and directed against a different antigenic site on the PG I or PG II molecule. After bound/free separation by washing, the fluorescence counts of bound Eu 3+–McAb were measured. The levels of PG in sera from patients or healthy volunteers were determined by PG I and PG II TRFIA using the autoDELFIA 1235 system. The measurement ranges of PG I-TRFIA were 3.5–328.0 μg L −1 and those of PG II-TRFIA were 2.0–55.0 μg L −1. The within-run and between-run CVs of the PG I-TRFIA were 1.9% and 4.7%, respectively, and those of PG II-TRFIA were 2.1% and 3.8%, respectively. The recovery rates of PG I-TRFIA and PG II-TRFIA were 102.7% and 104.6%, respectively. The detection limitations of PG I and PG II were 0.05 μg L −1 and 0.02 μg L −1, respectively. The dilution experiments showed the percentage of expected value of PG I-TRFIA was 93.2–102.3% and of PG II-TRFIA was 97.3–110.6%. The cross-reacting rate between PG I and PG II was negligible. The linear correlation of radioimmunoassay (RIA) and TRFIA measurements resulted in a correlation coefficient as 0.926 of PG I and as 0.959 of PG II. The europium-labelling McAbs were stable for at least one year at −20 °C, and the results of the TRFIA with same reagents were reproducible over one year as well. The means of 1600 healthy volunteers were 162.4 ± 52.1 μg L −1 for serum PG I, 11.7 ± 6.8 μg L −1 for serum PG II, and 13.8 ± 7.4 for the PG I/PG II ratio. The normal ranges of Serum PG I levels for healthy volunteers were 58.2–266.6 μg L −1, and those of serum PG II levels were less than 25.3 μg L −1. The availability of a highly sensitive, reliable, and convenient PG-TRFIA method for quantifying PG will allow investigations into the possible diagnostic value of this analysis in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastric ulcer and gastritis. The sensitivity and reproducibility of the assay were satisfactory for clinical applications.

Specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in SARS patients

Background: Severe acute respiratory syndrome (SARS) is an infectious disease which was caused by a novel coronavirus (SARS-CoV). SARS has caused an outbreak in the world during 2003 and 2004,...

LRRK2 in Parkinson's disease and dementia with Lewy bodies

BackgroundMutations in LRRK2 encoding leucine-rich repeat kinase 2 are thus far the most frequent genetic cause associated with autosomal dominant and idiopathic Parkinson's disease (PD). To examine whether LRRK2 is directly associated with neuropathology of PD and other related disorders, we analyzed LRRK2 in brains of patients affected by PD and dementia with Lewy bodies (DLB) using highly specific antibodies to LRRK2.ResultsWe demonstrated that anti-LRRK2 antibodies strongly labelled brainstem and cortical Lewy bodies, the pathological hallmarks of PD and DLB, respectively. In addition, anti-LRRK2 also labelled brain vasculature, axons, and neuronal cell bodies. Interestingly, the immunocytochemical profile of LRRK2 varied with different antibodies depending upon specific antigenic sites along the LRRK2 protein. All anti-LRRK2 antibodies tested that were raised against various regions of LRRK2, were found to be immunoreactive to recombinant LRRK2 on Western blots. However, only the antibodies raised against the N-terminal and C-terminal regions of LRRK2, but not the regions containing folded protein domains, were positive in immunolabeling of Lewy bodies, suggesting a differential exposure of specific antigenic sites of LRRK2 on tissue sections.ConclusionWe conclude that LRRK2 is a component of Lewy bodies in both PD and DLB, and therefore plays an important role in the Lewy body formation and disease pathogenesis. Information on the cellular localization of LRRK2 under normal and pathological conditions will deepen our understanding of its functions and molecular pathways relevant to the progression of PD and related disorders.

A modified Trastuzumab antibody for the immunohistochemical detection of HER-2 overexpression in breast cancer

The immunohistochemical determination of HER-2 to identify patients with advanced breast cancer candidates for Trastuzumab treatment proved neither accurate nor fully reliable, possibly because none of the current reagents detects the specific antigenic site target of Trastuzumab. To circumvent this problem, we conjugated the NH2 groups of Trastuzumab with biotin, and the compound obtained, designated BiotHER, was added directly to tissue sections. Biotin-labelling was revealed with horseradish peroxidase-conjugated streptavidin. Specificity and sensitivity of BiotHER immunostaining with respect to HER-2 amplification were tested on 164 breast carcinoma samples. BiotHER staining was detected on the tumour cell membrane of 12% of all specimens and in 49% specimens with gene amplification, while absent in nonamplified tumours. Predictivity of BiotHER status with respect to the clinical outcome was analysed in 54 patients with HER-2 amplified advanced breast cancer treated with Trastuzumab plus chemotherapy. BiotHER staining, detected in 50% of tumours with HER-2 amplification, was an independent predictor of clinical outcome. In fact, BiotHER positivity was independently associated with increased likelihood of tumour response and reduced risk of tumour progression and death. Biotinylated Trastuzumab can thus be used for immunohistochemical detection of HER-2 overexpression in breast cancer, and has the potential to identify patients likely to benefit from Trastuzumab treatment.

A New Epitope of CYP2D6 Recognized by Liver Kidney Microsomal Autoantibody from Japanese Patients with Autoimmune Hepatitis

Liver-kidney microsomal antibodies type 1 (LKM-1) are a diagnostic marker for autoimmune hepatitis type II (AIH-II). However, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The autoantigen to anti-LKM-1 has been identified to be CYP2D6. To identify the specific antigenic site of CYP2D6 for LKM-1 serum, we established an ELISA with peptides spanning the entire sequence of CYP2D6. Human CYP2D6 containing histidine tag was expressed in Escherichia coli. Purified CYP2D6 was digested by lysyl endopeptidase. The linker including the histidine tag has a lysine residue in its C-terminal and can be removed by digestion. Digested peptides were separated by reversed-phase HPLC and coated on ELISA plates chemically with glutaraldehyde. The immunoreactivity of two LKM-1-positive sera (HCV-negative) and five HCV-positive sera from Japanese patients was investigated with the plates. These sera recognized peptides 1-146, 181-214, 246-281, 284-391, and 412-429. The peptide 1-146 was recognized by LKM-1-positive sera but not HCV-positive sera and is a new epitope found in this study. Seven short peptides spanning peptide 1-146 were synthesized and ELISAs were conducted with these peptides. However, two sera recognized none of these peptides, suggesting that two LKM-1-positive sera recognize the conformational immunogenic site of peptide 1-146.

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target, serum-sensitive cells

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERα in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERα specific monoclonal antibody also revealed specific, multiple M r mERs (67, 110, and 130 k M r). In addition, Western blot analysis with an ABP antiserum revealed a potential 60 k M r ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60 k M r ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.