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PubMed1995

A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. Competitive epitope mapping studies.

Presented here are competitive epitope mapping studies on a monoclonal antibody library to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC1.2.4.1) subunit (E1). Several of the monoclonal antibodies had been found to inhibit PDHc from 0 to 98%. Of the 10 monoclonal antibodies that showed the greatest inhibition of PDHc, 4 were elicited by PDHc and 6 by E1. Surface plasmon resonance was used for competitive epitope mapping and revealed that these 10 monoclonal antibodies had at least 6 separate binding regions on the PDHc. The three monoclonal antibodies that demonstrated the strongest inhibition appeared to bind the same region on the PDHc. Mapping studies with the E1 antigen using an additional five monoclonal antibodies demonstrated that the two strongest inhibitory monoclonal antibodies (18A9 and 21C3) shared the same binding region on E1, whereas the third strongest inhibitor (15A9) displayed an epitope region that overlapped the previous two on the E1 subunit. Antibody 15A9 had been shown to counteract GTP regulation of PDHc. Simultaneous multiple site binding experiments confirmed that the defined epitope regions were indeed independent. Limited competitive epitope binding experiments using radiolabeled E1 confirmed the surface plasmon resonance results.

Animals↗
PubMed1994

Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb.

In the mammalian olfactory system, information from approximately 1000 different odorant receptor types is organized in the nose into four spatial zones. Each zone is a mosaic of randomly distributed neurons expressing different receptor types. In these studies, we have obtained evidence that information highly distributed in the nose is transformed in the olfactory bulb of the brain into a highly organized spatial map. We find that specific odorant receptor gene probes hybridize in situ to small, and distinct, subsets of olfactory bulb glomeruli. The spatial and numerical characteristics of the patterns of hybridization that we observe with different receptor probes indicate that, in the olfactory bulb, olfactory information undergoes a remarkable organization into a fine, and perhaps stereotyped, spatial map. In our view, this map is in essence an epitope map, whose approximately 1000 distinct components are used in a multitude of different combinations to discriminate a vast array of different odors.

Animals↗
PubMed1998

Why certain antibodies cross-react with HLA-A and HLA-G: epitope mapping of two common MHC class I reagents.

Antigen presentation at the maternal-fetal interface has been characterized by a reported lack of classical MHC class I products and the presence of a tissue-restricted, non-classical class I product with limited polymorphism, HLA-G. The lack of HLA-A, -B, and -C products at this interface would allow escape from T-cell mediated attack, while the presence of HLA-G may enable evasion of NK cell-mediated destruction. We provide evidence that in addition to HLA-G, the classical class I product HLA-C is also present in trophoblast. Specifically, cDNA from the trophoblast-derived JEG 3 cell line encodes the HLA-C-locus product, HLA-Cw*0401. This protein, obtained by in vitro transcription/translation, has biochemical characteristics identical to MHC class I products immunoprecipitated directly from the same cells. These findings are in agreement with RNA analysis and immunohistochemistry on both cell lines and primary trophoblast tissues. We report here the preferential reactivity in JEG 3 cells of two widely used monoclonal anti-MHC class I heavy chain antibodies, HC10 and HCA2, with HLA-C and HLA-G, respectively. We have mapped the epitopes recognized by these reagents to distinct areas of the alpha1 domain of the MHC class I heavy chain. HCA2 recognizes the motif xLxTLRGx spanning amino acids 77 84 present in both HLA-A and HLA-G. In contrast, HC10 may recognize a discontinuous epitope, with essential elements of the recognized motif surrounding residue 60 in the alpha1 domain of the class I heavy chain, as shown by truncation analysis. These results adequately explain the immunochemical cross-reactivity of HLA-A and HLA-G.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function.

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.

Amino Acid Sequence↗
PubMed2005

Epitope mapping of monoclonal antibody 4C8 recognizing the protein huntingtin.

Huntington's disease is a dominantly inherited, devastating neurodegenerative disorder, caused by a polyglutamine expansion (>37) in the N-terminal region of huntingtin, a protein of unknown function. In patients and normal individuals, N-terminal fragments of huntingtin are found, and the N-terminal fragments of mutant huntingtin are cytotoxic. The functions of wild-type huntingtin and the mechanisms underlying the toxic effects of mutant huntingtin are still ill defined. To get more insight into these topics, monoclonal antibodies (MAbs) are indispensable tools. Antibodies raised against the N-terminus are especially important. Among these, the 4C8 mouse MAb has been extensively used in various approaches. In this study, we have mapped the epitope of 4C8 to a 15-amino acid (aa) region spanning from aa 443 to 457 of the human protein, and found that mutation of three consecutive glutamic acids present in this region disrupts the recognition by 4C8. These results allow a more accurate interpretation of the results obtained by usage of the 4C8 antibody and broaden the utility of this antibody.

Amino Acid Sequence↗
PubMed2001

Epitope mapping on Wegener's granulomatosis autoantigen proteinase 3.

OBJECTIVE: To map the epitopes on Wegener's granulomatosis autoantigen proteinase 3. METHODS: Antigenicity of proteinase 3 was studied with Western blot analysis in which proteinase 3 was prepared under reducing and non-reducing conditions. Two anti-proteinase 3 monoclonal antibodies, HZ1F12 and HZ1H3, were used to inhibit each other and to inhibit 22 anti-proteinase 3 positive sera from patients with Wegener's granulomatosis in competitive inhibition enzyme-linked immunosorbent assays (ELISA) and Western blot analysis. RESULTS: All monoclonal antibodies and patient sera recognized proteinase 3 under non-reducing conditions in Western blot analysis. ZH1F12 was inhibited 74% by HZ1H3. 10/22 (46%) sera were completely or partially inhibited by HZ1F12; 9/22 (41%) sera were partially inhibited by HZ1H3; and 6/22 (27%) were inhibited by both monoclonal antibodies. In inhibition Western blot analysis, the binding of patient sera to proteinase 3 could be inhibited by HZ1F12. CONCLUSIONS: The epitopes of Wegener's granulomatosis autoantigen were conformational. Anti-proteinase 3 monoclonal HZ1F12 and HZ1H3 recognized similar or overlapping epitopes on the proteinase 3 molecule. Epitopes of proteinase 3 recognized by anti-proteinase 3 positive sera were not restricted.

Animals↗
PubMed1998

Epitope mapping of a series of human thymidylate synthase monoclonal antibodies.

We have reported previously the development and application of several monoclonal antibodies to thymidylate synthase (TS). In this study, we used a series of overlapping 17-mer peptides that spanned the entire TS protein to map the epitope recognized by three TS monoclonal antibodies (TS 106, TS 109, and TS 110). Using an ELISA, we identified two peptides (R126-F142 and L131-R147) that bound all three antibodies, which suggests that each antibody recognized a similar epitope on TS. A second set of peptides, representing sequential single-residue truncations from either the amino terminus or the carboxyl terminus starting with a G129-E145 17-mer, was synthesized. A 10-amino acid sequence P133-F142 (PVYGFQWRHF) was identified as the binding epitope for all three antibodies. Further investigation via substitution mutational analysis of each residue within this epitope revealed that residues F137, W139, R140, H141, and F142 were critical for maximal binding of TS 106 and TS 110. TS 109 showed a similar pattern except in regard to R140, with which there was no apparent loss of binding. In addition to the utility of the three antibodies in detecting and measuring TS levels, identification of the binding locus permits the potential application of these antibodies in the investigation of TS enzymatic and regulatory function.

Antibodies, Monoclonal↗
PubMed1997

Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein.

The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found.

Amino Acid Sequence↗
PubMed1996

Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody.

Matrix-assisted laser desorption mass spectrometry in combination with proteolytic protection assays has been used to identify the functional epitope on HIV-1 IIIB p26 recognized by a mAb. In this procedure, the intact protein is affinity bound to an immobilized mAb under physiologic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues were identified by matrix-assisted laser desorption mass spectrometry based on their m.w. With this approach, an 11-residue sequence was identified as the most tightly affinity-bound fragment. in addition, two less tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involved in a wider contact area. The combination of matrix-assisted laser desorption and proteolytic epitope footprinting has been applied to the determination of the epitope on a recombinant protein recognized by a mAb but should be equally applicable to the definition of an epitope on a native protein in its natural folded conformation.

Amino Acid Sequence↗
PubMed2003

Monoclonal antibodies to human cartilage oligomeric matrix protein: epitope mapping and characterization of sandwich ELISA.

BACKGROUND: Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids. METHODS: COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody. RESULTS: Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera. CONCLUSIONS: We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.

Antibodies, Monoclonal↗
PubMed1989

Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus.

In this article we report the first topological mapping of neutralizing epitopes of a hepadnavirus. Duck hepatitis B virus is the only hepadnavirus that can replicate and spread from cell to cell in tissue culture. As a result, it is possible to study hepadnaviral neutralization in vitro with this system. To accomplish this goal, we produced a library of monoclonal antibodies against duck hepatitis B virus and identified 12 neutralizing monoclonal antibodies by using an in vitro neutralization assay. The characteristics of six of the neutralizing monoclonal antibodies were further studied by epitope mapping. From the results of competitive binding studies, three distinct neutralizing epitopes were identified on the pre-S polypeptides and one was identified on the S polypeptide. Our findings suggest that antibodies to both the pre-S and S gene products of duck hepatitis B virus can neutralize viral infection in vitro. The pre-S gene product is at least as important as the S gene product in eliciting neutralizing antibodies.

Animals↗
PubMed1993

Mapping epitopes of human Fc gamma RII (CDw32) with monoclonal antibodies and recombinant receptors.

Fc gamma RII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1 gamma and D2 gamma), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human Fc gamma RII mAb (8.2, 8.7, 8.26, and 7.30) are detailed, and the mAb are compared with two defined CDw32 mAb, IV.3 and CIKM5. Reactivity of all mAb with Fc gamma RII was demonstrated by (a) specific binding to Fc gamma RII+ L cells (produced after transfection of L cells with human Fc gamma RIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to Fc gamma RII+ L cells, and (c) immunoprecipitation and SDS-PAGE, which detected a 42-kDa protein on K562 and U937 cells and a single 45-kDa protein on Fc gamma RII+ L cells. The mAb were able to detect different forms of Fc gamma RII, by flow cytometry, on Daudi cells (8.7 and 7.30) and U937 cells (8.2, IV.3, and CIKM5); 8.26 stained Daudi cells with intermediate fluorescence and U937 cells with the highest fluorescence, relative to the remaining mAb. Binding to transiently expressed isoforms of Fc gamma RII (a and b1) and four allelic variants of Fc gamma RIIa in COS-7 cells did not distinguish the mAb epitopes. Further mapping of the mAb epitopes was determined by (a) EA inhibition assays, (b) mAb blocking studies, and (c) the binding of the mAb to segments of human Fc gamma RIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1 gamma linked to domain 2 of Fc epsilon RI or domain 1 of Fc epsilon RI linked to D2 gamma were produced by exchanging homologous, but antigenically different, regions of Fc gamma RIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of Fc gamma RII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1 gamma and D2 gamma distant from the IgG Fc binding site, inasmuch as F(ab')2 fragments of 8.2 and CIKM5 do not inhibit the binding of EA to Fc gamma RII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2 gamma and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to Fc gamma RII. Thus, all mAb that inhibit the binding of EA map totally to D2 gamma; it is likely the IgG Fc binding region is also contained in D2 gamma.

Animals↗
PubMed1997

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization.

The B cell epitope mapping of La/SSB was performed using 20mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes: 147HKAFKGSI154, 291NGNLQLRNKEVT302, 301VTWEVLEGEVEKEALKKI318 and 349GSGKGKVQFQGKKTKF364. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147HKAFKGSI154 presented 83.3% similarity with the 139HKGFKGVD146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147HKAFKGSI154 to 100% to epitope 349GSGKGKVQGKKTKF364. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147HKAFKGSI154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.

Adult↗
PubMed1986

Epitope map and processing scheme for the 195,000-dalton surface glycoprotein of Plasmodium falciparum merozoites deduced from cloned overlapping segments of the gene.

DNA fragments from human malaria parasites were cloned into lambda gt11 to produce a genomic DNA expression library. A pool of monoclonal antibodies (mAbs) recognizing three domains of the 195-kDa major merozoite surface glycoprotein (gp195) reacted with seven clones expressing malaria antigens. mAbs recognizing the 83-kDa product of gp195 reacted with the clones, but mAbs recognizing a glycosylated 45-kDa and a nonglycosylated 45-kDa domain did not. Restriction enzyme mapping revealed that the clones contained overlapping segments encoding about 70% of the gene beginning at the 5' end and ending at an EcoRI restriction enzyme site 3.3 kilobase pairs downstream. The mAbs recognizing the 83-kDa domain reacted differently with the clones, allowing the mapping of three epitopes, one of which was repetitive. Affinity-purified antibodies were selected from immune monkey serum with recombinant expression proteins adsorbed to nitrocellulose filters. When used to probe electrophoretic immunoblots of parasite extracts, these antigen-selected antibodies reacted with specific sets of processed products of gp195, including those associated with the 83- and the nonglycosylated 45-kDa domains. This information, combined with the mAb epitope map, allowed a tentative scheme for processing gp195 from the Camp strain to be proposed.

Animals↗
PubMed1995

Cloning of the murine counterpart of the tumor-associated antigen H-L6: epitope mapping of the human and murine L6 antigens.

The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by mAb 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution of the single H-L6 residue Leu122 with Ser resulted in the H-L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Epitope mapping of antibodies to the C-terminal region of the integrin beta 2 subunit reveals regions that become exposed upon receptor activation.

The cysteine-rich repeats in the stalk region of integrin beta subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the beta(2) subunit, one, KIM127, preferentially bound to alpha(L)beta(2) that was activated by mutations in the cytoplasmic domains, and by Mn(2+). KIM127 also bound preferentially to the free beta(2) subunit compared with resting alpha(L)beta(2). Activating beta(2) mutations also greatly enhanced binding of KIM127 to integrins alpha(M)beta(2) and alpha(X)beta(2). Thus, the KIM127 epitope is shielded by the alpha subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated alpha(L)beta(2) and bound equally well to resting and activated alpha(L)beta(2), differentially recognized resting alpha(M)beta(2) and alpha(X)beta(2), and bound fully to activated alpha(M)beta(2) and alpha(X)beta(2). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344-432 and 432-487, respectively. We thus define five different beta(2) stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.

Amino Acid Sequence↗