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PubMed1985

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin.

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.

Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.

Adult↗
PubMed1996

Epitope mapping and reactivity of autoantibodies to the E2 component of 2-oxoglutarate dehydrogenase complex in primary biliary cirrhosis using recombinant 2-oxoglutarate dehydrogenase complex.

Five different target mitochondrial autoantigens recognized by sera from patients with primary biliary cirrhosis (PBC) have been identified as subunits of the following 2-oxo acid dehydrogenase complexes: the pyruvate dehydrogenase complex (PDC), the branched chain 2-oxo acid dehydrogenase complex (BCOADC), and the 2-oxoglutarate dehydrogenase complex (OGDC). Unlike the E2 subunits of PDC (PDC-E2) and BCOADC (BCOADC-E2), the E2 subunits of OGDC (OGDC-E2) reactivity of PBC sera and the reactive epitope of OGDC-D2 have not hitherto been studied in detail. In this report, we took advantage of a recombinant fusion protein for OGDC-E2 to address these issues. Eighty of 268 (29.9%) PBC patient sera but none of the 45 controls reacted with recombinant OGDC-E2. The recombinant OGDC-E2 was judged to express the immunodominant epitope, because when sera from patients with PBC were preabsorbed with the recombinant fusion protein, such sera were depleted of reactivity against 48 kD OGDC-E2 when probed on beef heart mitochondria (BHM) but retained reactivity toward PDC-E2 and/or BCOADC-E2. Furthermore, affinity-purified PBC sera against recombinant OGDC-E2 reacted only with native OGDC-E2 and not with any other enzyme components of the 2-oxo acid dehydrogenase complex. Antimitochondrial autoantibodies (AMA) against OGDC-E2 included immunoglobulin (Ig)G2, IgG3 and IgM and the relative titers were as follows: IgG2 > IgG3 > IgM. Finally, using overlapping recombinant polypeptides, it was determined that a minimum of 81 amino acids (residues 67-147) corresponding to the lipoyl domain of OGDC-E2 are necessary for reactivity, suggesting that a conformational autoepitope is recognized by AMA. These data suggest that each of the 2-oxo acid dehydrogenase enzymes has distinct antigenicity despite their similarities in structure and function. The availability of recombinant OGDC-E2 autoantigen will allow the design of additional studies to further our understanding of the role of mitochondrial autoantigens in the pathogenesis of PBC.

Animals↗
PubMed1992

Epitope mapping of Streptococcus mutans SR protein and human IgG cross-reactive determinants, by using recombinant proteins and synthetic peptides.

alpha-Hemolytic oral streptococci are known to possess a family of cell surface cross-reactive proteins termed Ag I/II, having a molecular mass of approximately 180 to 210 kDa. These proteins are implicated in bacterial adherence to various oral tissues, and we showed recently that the SR protein, an I/II Ag-related protein, from Streptococcus mutans OMZ 175 serogroup f possesses Ag mimicry with human IgG. In this study, regions of the SR protein encoding the cross-reactive epitope(s) were analyzed by expressing selected restriction fragments from the cloned sr gene. The three SR-derived polypeptides reacted in ELISA with anti-SR rabbit IgG, whereas only the two polypeptides located along the carboxyl-terminal two thirds of the SR protein reacted with anti-human IgG rabbit IgG. In order to locate more precisely the human IgG-cross-reactive region, we synthesized six peptides, on the basis of the recently determined complete nucleotide sequence of the sr gene. Among these peptides, peptide 2, corresponding to the alanine-rich repeating amino-terminal region, peptide 3, located in the three tandem proline-rich regions, and peptide 6, located near the cell wall-spanning region, were the most interesting in term of antigenicity and immunogenicity. Anti-peptide 2, 3, and 6 rabbit IgG reacted with free SR and with cell wall-associated SR. Peptide 1, located near the amino terminus, was poorly immunogenic. Peptides 4 and 5, located in the putative human IgG-cross-reactive region, were immunogenic; however, anti-peptide 4 rabbit IgG reacted only weakly with SR or human IgG, whereas anti-peptide 5 rabbit IgG reacted strongly with SR and human IgG, and peptide 5 was recognized by anti-SR and anti-human IgG rabbit IgG. These results confirm the cell surface accessibility of this epitope and its potential participation in eliciting, in rabbits, anti-SR IgG cross-reactive with human IgG.

Amino Acid Sequence↗
PubMed1992

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.

3T3 Cells↗
PubMed1998

Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.

Animals↗
PubMed1994

Fibroblast activation protein: purification, epitope mapping and induction by growth factors.

The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a M(r) 95,000 subunit (FAP alpha) carrying the F19 epitope and a non-covalently bound M(r) 105,000 subunit (FAP beta) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAP alpha and tentatively assign one epitope to FAP beta. Analysis of detergent extracts of a FAP alpha high beta- sarcoma cell line by size exclusion-high performance liquid chromatography (HPLC) revealed that FAP alpha does not elute as a M(r) 95,000 species but as part of a high-molecular weight complex (M(r) > 400,000) that dissociates into M(r) 95,000 subunits in SDS gels. Immunoaffinity purification of FAP alpha followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAP alpha peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAP alpha has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-beta, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

Amino Acid Sequence↗
PubMed1990

Carbohydrate-dependent epitope mapping of human thyrotropin.

To probe possible effects of carbohydrate chains in the conformation of pituitary glycoprotein hormones, two radiolabeled derivatives of human thyroid-stimulating hormone (hTSH), either partially deglycosylated in the beta-subunit or fully deglycosylated in both the alpha- and beta-subunits, were compared to the native hormone for binding to monoclonal as well as polyclonal antibodies. Monoclonal antibodies were screened for their ability to bind the intact hormone (anti-hTSH), hTSH and its free alpha-subunit (anti-alpha) or its free beta-subunit (anti-beta). A panel of 14 monoclonal antibodies directed against at least eight out of the 12 epitopes known to be present in the hormone was tested in solid-phase assays for their capacity to bind intact and deglycosylated forms of hTSH. All of them displayed identical recognition of native and partially deglycosylated 125I-hTSH. In contrast, binding of fully deglycosylated 125I-hTSH to anti-hTSH and anti-beta antibodies was dramatically lost while that of anti-alpha was preserved. This clearly indicates that most of the epitopes specific for subunit association as well as those present on the beta-subunit are glycosylation dependent. No alteration was found in antibody recognition following deglycosylation of free individual subunits, indicating that the carbohydrate effect can only occur in the combined dimer. Using polyclonal antisera raised against the International Reference Preparations, we found that the deglycosylated hormone could be bound by the anti-beta antiserum although at a much lower dilution than the native antigen, suggesting the presence of at least one glycosylation-independent epitope in the beta-subunit. Competitive binding assays revealed that deglycosylated hTSH is 5 times less immunoreactive toward the anti-beta compared to the anti-alpha antiserum. The current data thus demonstrate the presence of the glycosylation-independent epitopes in the alpha-subunit of hTSH and the localization of most of the glycosylation-dependent domains in the beta-subunit.

Antibodies, Monoclonal↗
PubMed1992

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Epitope mapping of human prostate specific antigen and glandular kallikrein expressed in insect cells.

Two human prostate gland proteases were expressed in insect cells using recombinant baculovirus expression system. Prostate specific antigen (PSA) is an established serum marker of prostate cancer whereas the clinical utility of its close homologue, human glandular kallikrein (hK2) is presently unknown. The production levels using Trichoplusia ni cells were roughly 300 &mgr;g/l and 6 mg/l for hK2 and PSA, respectively. On western-blot we estimated the size for both proteins to be approximately 33 kDa which was consistent with PSA purified from seminal plasma. Nine anti-PSA monoclonal antibodies (Mabs) out of 26 tested, representing five independent epitopes, also reacted with hK2. The results obtained in this study may help in designing more accurate diagnostic assays for detection and monitoring of prostate cancer.

Journal Article↗
PubMed1990

Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies.

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase C gamma (PKC gamma); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKC gamma kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J. Biochem. 182, 401-406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKC gamma in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb- and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKC gamma. For this purpose, several deletions were introduced at the 5' end of the PKC gamma cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKC gamma proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151-163 and 164-197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor-binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKC gamma conserves its ability to interact with the non-inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297-310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC. In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.

Amino Acid Sequence↗
PubMed1997

Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human alpha2-macroglobulin.

The human proteinase inhibitor, alpha2-macroglobulin (a2-M), inhibits a large number of proteinases. Alpha2-M-proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (alpha2-M-R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C-terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab alpha-1) has been described which reacts with the receptor-recognizable form of the inhibitor, the so called transformed alpha2-M (a2-Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab alpha-1. Out of 25 phage clones a heptapeptide sequence (S-x1-x2-D-x3-x4-K) was obtained containing identical amino acids in three positions. A consensus peptide (S-R-S-D-P-P-K) was synthesized and found to displace alpha2-Mt from binding to mab alpha-1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.

Amino Acid Sequence↗
PubMed1987

Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium.

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

Alkaline Phosphatase↗
PubMed1990

Epitope mapping of the major allergen from yellow mustard seeds, Sin a I.

The antigenic sites on the major allergen from yellow mustard (Sinapis alba L.) seeds were studied using murine (BALB/c) monoclonal antibodies (mAb) and human IgE antibodies. Ten IgG1 (K) mAb from two fusions were analyzed. Competition and complementation studies performed with peroxidase labeled mAb reveal the existence of two main antigenic sites in Sin a I. All the described mAb failed to recognize the unordered carboxyamidomethylated polypeptide chains, with the single exception of 2B3, which binds the alkylated large chain. However, this mAB cannot react with the tetranitromethane-modified protein which retains the native conformation. This fact suggests that the only tyrosine of Sin a I, located in the large chain, may be part of a sequential epitope of the allergen. This chemical modification also alters the binding of the mAb 4A11 and 3F3 to the allergen, besides 2B3. The three mAb belong to the same complementation group. Specific IgE binding cannot be inhibited either by the large or small carboxyamidomethylated polypeptide chains, while the nitrated allergen shows a weaker inhibitory activity than the native Sin a I. 4A11, which is a tyrosine-dependent mAb, causes the greatest binding inhibition of the tested mAb on human IgE from atopic individuals, as determined from a reverse enzyme immunoassay, suggesting an important role played by tyrosine in the immunochemical recognition of Sin a I.

Allergens↗
PubMed2001

Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus.

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.

Amino Acid Sequence↗
PubMed1991

Epitope mapping of the laminin molecule in murine skin basement membrane zone: demonstration of spatial differences in ultrastructural localization.

Results of studies performed to date with polyclonal antilaminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1 M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non-random spatial orientation for the laminin molecule within murine skin BMZ.

Animals↗
PubMed1991

Epitope mapping of the d flagellar antigen of Salmonella muenchen.

Contiguous exposed antibody-binding regions of the antigen d flagellin of Salmonella muenchen were identified by using octameric peptides synthesized on polyethylene pins. Identification was confirmed by the serological activity of immunoglobulins recovered from specified pin peptides. Peptides equivalent to four regions of the d flagellin reacted with three different sera tested.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.

An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.

Animals↗