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PubMed1992

Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library.

Many naturally occurring point mutations in the p53 gene lead to a proportion of the encoded protein molecules adopting a distinct, "mutant" conformation characterized by exposure of a normally cryptic epitope recognized by the monoclonal antibody PAb240. Here the PAb240 epitope is defined using a filamentous phage epitope library. The hexapeptides displayed by the PAb240-binding phage isolated from the library were all highly related and allowed both direct localization of the epitope and prediction of a specific interaction between PAb240 and Xenopus TFIIIA. This study demonstrates for the first time the power of phage epitope libraries in the precise definition of previously unmapped epitopes. Identification of the PAb240 epitope precisely defines a region of the p53 molecule structurally altered by the mutation-induced conformational shift.

Animals↗
PubMed1997

Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments.

Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin. Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.

3T3 Cells↗
PubMed1988

A rapid method of epitope mapping. Application to the study of immunogenic domains and to the characterization of various forms of rabbit progesterone receptor.

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action. This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101-110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1-60). The 79-kDa form ('subunit A') of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101-110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.

Animals↗
PubMed1993

Epitope mapping of form-specific and nonspecific antibodies to acetylcholinesterase.

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44-82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp59, an N-linked glycosylation site not present in mouse AChE. A 20-amino-acid synthetic peptide, RFRRPEPKKPWSGVWNASTY, representing residues 44-63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first alpha-helix.

Acetylcholinesterase↗
PubMed2001

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

CD4-Positive T-Lymphocytes↗
PubMed2001

A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes.

We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with freshly isolated splenocytes in a sensitive enzyme-linked immunospot (ELISPOT) assay. This technique was applied for the analysis of splenocytes from Listeria monocytogenes-infected BALB/c and C57BL/6 mice. The screening of peptide spot libraries covering the whole listeriolysin O and p60 of L. monocytogenes confirmed all known CD4 and CD8 T cell epitopes of these proteins and additionally revealed six new H-2(d) and six new H-2(b)-restricted T cell epitopes. New epitopes were categorized into CD4 and CD8 T cell epitopes by ex vivo ELISPOT analysis with separated T cell populations. The quantitative analysis of cells reactive with these CD4 and CD8 T cell epitopes revealed the existence of dominant and subdominant CD4 and CD8 T cell populations during L. monocytogenes infection. As a consequence of these data we suggest that ELISPOT-based screening of peptide spot libraries could be a general approach for the rapid identification and characterization of pathogen-specific T cell populations during various infectious diseases.

Animals↗
PubMed1999

Epitope mapping of mouse monoclonal antibodies to the ppUL83 lower matrix phosphoprotein of human cytomegalovirus.

Of nine mouse monoclonal antibodies (MAbs) directed against the lower matrix protein (pp65; ppUL83) of human cytomegalovirus (HCMV), all immunoprecipitated the 65-kDa protein. Only five were reactive by Western blotting, however, and four of these mapped to linear antigenic epitopes located between amino acids 184-195 (MAb C6), 343-357 (MAb C11), 448-462 (MAb C5), and 448-459 (MAb C13). The epitope specificity of the fifth antibody (MAb C3) and the four that recognised nonlinear sites could not be determined. Competition binding studies using HCMV antigen extracted from productively infected human embryonic lung fibroblasts (HELF), in an enzyme immunoassay (EIA), showed that three of the antibodies reactive with linear epitopes and two of those reactive with conformational epitopes (MAbs C3, C6, C11, C14, and C18), were unique in their binding specificities. MAb C4 competed with MAb C8 and MAb C5 competed with MAb C13 for binding to ppUL83. One of the linear epitopes identified, corresponding to amino acids SAFVFPTKDVAL (MAb C6), was an epitope described previously for CD8+ cytotoxic T lymphocytes.

Amino Acid Sequence↗
PubMed1992

B-cell epitope mapping of the entire SIVagm envelope glycoprotein including fine mapping of immunogenic regions.

To allow the precise definition of the anti-lentiviral immune response in the natural host and to facilitate the development of an alternative animal model for vaccine development, we are identifying the immunogenic domains of SIVagm proteins. First, a total of 173 synthetic 15-mer peptides with an overlap of 10 amino acids were produced spanning the entire envelope glycoprotein of the molecular clone SIVagm3. These peptides were used as antigen in enzyme-linked immunosorbent assays for identifying regions recognized by antibodies from naturally infected African green monkeys and monkeys infected with a molecular clone. Regions corresponding to the HIV-1 V3, the transmembrane protein (TMP) "Gnann peptide", and the C-terminal area of the outer envelope protein were shown to be immunodominant. These regions were re-synthesized as 15-mer peptides with an overlap of 14 amino acids and used to precisely map the epitopes recognized. Sera from 93 captive and 61 wild animals were tested by SIVagm-specific Western blot (WB) and for ELISA reactivity against the immunodominant TMP peptide. One hundred percent (76/76) of the WB-positive captive animals and 98% (41/42) wild WB-positive animals also reacted against the peptide. In contrast, only 62% of the WB-positive sera reacted with the "V3" epitope and 46% with the gp130 C-terminal epitope.

Amino Acid Sequence↗
PubMed1990

Epitope mapping and use of anti-idiotypic antibodies to the L6 monoclonal anticarcinoma antibody.

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were raised against L6, a murine IgG2a monoclonal antibody specific for a cell surface antigen expressed by many human carcinomas. Ten distinct anti-ids were generated. Eight anti-ids were shown to inhibit the binding of L6 to its target antigen and were characterized in detail. The heavy and light chain variable region gene segments of the monoclonal antibody L6 linked to human constant regions (chimeric L6) were expressed separately or together, to map the epitopes recognized by the anti-ids. Individual anti-ids were shown to recognize heavy chain, light chain, or combinatorial variable region determinants. Defining these specificities enabled us to select particular anti-ids for assays to monitor the pharmacokinetics of either murine or chimeric L6 antibodies in the circulation of human patients. A quantitative enzyme-linked immunosorbent assay developed with two anti-ids accurately detects less than 5 ng/ml. Anti-ids specific for light chain variable region-encoded determinants were capable of recognizing L6 antigen-binding fragments bound to the surface of human carcinoma cells. These anti-ids can be used to study the binding of chimeric L6 antibody at the surface of tumor cells in histological sections of tumor biopsies.

Animals↗
PubMed1987

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system.

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.

Antibodies, Monoclonal↗
PubMed1994

Epitope mapping for monoclonal antibodies identifies functional domains of pulmonary surfactant protein A that interact with lipids.

Pulmonary surfactant protein A (SP-A) contains 4 domains: a disulfide forming amino terminus, a collagen-like region, a neck region, and a carbohydrate recognition region. The protein binds the lipids dipalmitoylphosphatidylcholine and galactosylceramide and induces aggregation of phospholipid vesicles. SP-A also inhibits lipid secretion and enhances the uptake of phospholipid by alveolar type II cells. Previously described monoclonal antibody 1D6 blocks the inhibitory effect of SP-A on lipid secretion by type II cells, but antibody 6E3 has no effect. In the present study we mapped the epitopes for monoclonal antibodies 1D6 and 6E3 by enzyme-linked immunoassay of recombinant proteins expressed using the baculovirus system, and investigated the domain that is responsible for the SP-A interactions with lipid. Monoclonal antibody 1D6 bound to mutant SP-As in which the neck portion of the molecule was deleted or substituted with that of mannose-binding protein A, but 6E3 failed to bind to these mutants. In contrast, 1D6 did not bind to a chimera in which the carbohydrate recognition domain (CRD) was substituted with that of surfactant protein D (SP-D). In addition, 1D6 failed to recognize antigen in cells infected with the recombinant virus directing the synthesis of a Cys204-Cys218 (small disulfide loop) deletion within the CRD. Antibody 1D6 completely blocked the binding of SP-A to dipalmitoylphosphatidylcholine and galactosylceramide and liposome aggregation. By comparison, 6E3 failed to completely attenuate the interactions of SP-A with lipids. However, both 6E3 and 1D6 blocked the uptake of lipid by type II cells that is caused by SP-A. From these data, we conclude that: 1) the epitope for antibody 6E3 is located at the neck domain of SP-A and that for antibody 1D6 is at the small loop region in the CRD; 2) the CRD is essential for the SP-A functions of lipid binding, liposome aggregation, the inhibitory effect on lipid secretion, and the augmentation of lipid uptake by type II cells, and these activities are largely attributable to amino acid residues within the steric inhibitory footprint of 1D6 bound to the small disulfide loop region; and 3) the neck domain of SP-A may also be involved in the process of SP-A-mediated uptake of phospholipids by alveolar type II cells.

Amino Acid Sequence↗
PubMed2006

MIMOX: a web tool for phage display based epitope mapping.

BACKGROUND: Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. RESULTS: We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. CONCLUSION: A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

Bacteriophages↗
PubMed2003

The effect of relaxation on the epitope mapping by saturation transfer difference NMR.

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand-target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.

Animals↗
PubMed2002

The major conformational IgE-binding epitopes of hevein (Hev b6.02) are identified by a novel chimera-based allergen epitope mapping strategy.

A novel approach to localize and reconstruct conformational IgE-binding epitope regions of hevein (Hev b6.02), a major natural rubber latex allergen, is described. An antimicrobial protein (AMP) from the amaranth Amaranthus caudatus was used as an immunologically non-IgE-binding adaptor molecule to which terminal or central parts of hevein were fused. Hevein and AMP share a structurally identical core region but have different N-terminal and C-terminal regions. Only 1 of 16 hevein-allergic patients showed weak IgE binding to purified native or recombinant AMP. Chimeric AMP with the hevein N terminus was recognized by IgE from 14 (88%) patients, and chimeric AMP with the hevein C terminus was recognized by IgE from 6 (38%) patients. In contrast, chimeric AMP containing the hevein core region was recognized by IgE from only two patients. When both the N-terminal and C-terminal regions of hevein were fused with the AMP core, IgE from all 16 patients bound to the chimera. This chimera was also able to significantly inhibit (>70%) IgE binding to the native hevein. On the contrary, linear synthetic peptides corresponding to hevein regions in the AMP chimeras showed no significant IgE binding capacity in either enzyme-linked immunosorbent assay or inhibition enzyme-linked immunosorbent assay. These results suggest that the IgE binding ability of hevein is essentially determined by its N-terminal and C-terminal regions and that major IgE-binding epitopes of hevein are conformational. The chimera-based epitope mapping strategy described here provides a valuable tool for defining structural epitopes and creating specific reagents for allergen immunotherapy.

Adenosine Monophosphate↗
PubMed1997

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

Amino Acid Sequence↗
PubMed1997

Epitope mapping Candida albicans proteinase (SAP 2).

The continuous epitopes of Candida albicans proteinase SAP 2 were derived by epitope mapping with sera from patients with oral candidiasis (n = 3), necropsy-proven disseminated candidiasis (n = 5), paired sera from patients who had recovered from blood culture-proven disseminated candidiasis (n = 3) and infection due to Candida parapsilosis (n = 2) and Candida tropicalis (n = 2). In C. albicans infection, IgM identified epitopes in amino acid positions 57-61 (QAVPV), 146-151 (SQGTLY) and 346-351 (PYDKCQ) and IgG at position 386-390 (VKYTS). For C. tropicalis IgM and IgG were positive for the same epitopes whilst IgG also detected epitopes at 78-83 (SNNQKL) and 159-164 (GVSIKN). For C. parapsilosis, IgM was positive for SNNQKL and IgG detected no epitopes. Reactivity of two of the epitopes as peptides KTSKRQAVPVTL and SLAQVKYTSASSI was confirmed in an indirect ELISA. At a cut-off optical density of 0.4, IgM against either peptide was associated with survival but present in only about half of the sera (n = 60) from patients who recovered from disseminated candidiasis whilst IgG levels were disappointing. Human recombinant antibodies from a patient who had recovered from disseminated candidiasis against either of these peptides had no activity in a lethal mouse model of candidal infection.

Animals↗
PubMed1997

A linear region of a monoclonal antibody conformational epitope mapped on p185HER2 oncoprotein.

Analysis of epitopes recognized by therapeutic monoclonal antibodies (mAb) is critical in clinical applications and in structure/function studies of target antigen. mAb MGr6 recognizes the extracellular domain of the p185HER2 oncoprotein and is a promising candidate for cancer immunodiagnosis and immunotherapy. Thus, epitope location and structure on p185HER2 need to be investigated. The use of MGr6-selected phage-displayed peptides for epitope analysis served to dissect the MGr6 epitope into at least two subregions, mimicked by CHSDC- and (L)P-(L)K(L) phage displayed peptides, respectively. Comparison of amino acid sequences of CHSDC peptides with the p185HER2 protein sequence and analysis of MGr6 reactivity with p185HER2 deletion mutants identified the linear subregion CCHEQCAAG of the MGr6 epitope, corresponding to amino acids 235-243 of the p185HER2 protein. This continuous subregion is part of a larger conformational epitope, and other amino acids, including a proline, a lysine and leucine residues contained in (L)P-(L)K(L) phage-displayed peptides appear to contribute to the formation of the MGr6 epitope surface.

3T3 Cells↗