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PubMed1996

Antibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis

The multipin method for peptide synthesis was developed for antibody epitope mapping. From its initial invention as a method for testing many short peptides on the solid surface used for their synthesis, it has evolved into a method for producing solutions of peptides of a wide range of lengths in many "formats," such as tagged (biotinylated) peptides for binding studies or affinity chromatography. The power of the multipin technique lies in the large number and variety of peptides that can be rapidly made and efficiently tested.

Journal Article↗
PubMed2001

DiSSiMiL: Diverse Small Size Mini-Libraries applied to simple and rapid epitope mapping of a monoclonal antibody.

Methods for screening protein-protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein-protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH2-X1X2GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide 'hits' were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide-mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4-mAb interactions. Alignment of the three 'hit' pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH2-VYPWMK. Synthesis of NH2-VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor-ligand or other protein-protein interactions, or with enzymatic activity.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.

Animals↗
PubMed1991

CD45 epitope mapping of human CD1a+ dendritic cells and peripheral blood dendritic cells.

The authors studied the pattern of leukocyte common antigen (CD45) epitope expression on dendritic cells in sections of human epidermis, tonsillar epithelium, dermatopathic lymph nodes, and in isolates from blood. The monoclonal antibodies (MAb) used were specific for all known CD45 epitopes, including the seven different CD45 common epitopes as well as the four known CD45R epitopes (two CD45RA, one CD45RB, and one CD45RO). Dendritic cells in all sites were uniformly reactive for the CD45 common epitopes tested except 2B11, which may recognize a CD45R rather than CD45 epitope. By single-label immunoperoxidase and double-label immunofluorescence epitope mapping of CD1a+ dendritic cells in tissue sections, it was generally difficult or impossible to detect expression of CD45RA, CD45RB, CD45RO, or 2B11. In blood dendritic cells, however, low levels of these CD45R epitopes were detected consistently using single-label immunoperoxidase staining of cytocentrifuge preparations. Monocytes were similar to blood dendritic cells except that the staining with MAb to CD45RO and 2B11 was slightly stronger. The authors conclude that dendritic cells differ from most subpopulations of lymphocytes in that CD45 common epitopes are readily detectable but the existing RA, RB, and RO epitopes are either undetectable or expressed at relatively low levels. These studies raise the possibility that CD1a+ dendritic cells may express a novel dominant CD45 isoform.

Antigens, CD↗
PubMed2001

Epitope mapping of neutralizing botulinum neurotoxin A antibodies by phage display.

Single-chain antibodies neutralize activity and bind nonoverlapping epitopes of botulinum A neurotoxin. Two phage display epitope libraries were constructed from the 1.3 kb of binding domain cDNA. The minimal epitopes selected against the single-chain Fv-Fc antibodies correspond to conformational epitopes with amino acid residues 1115 to 1223 (S25), 1131 to 1264 (3D12), and 889 to 1294 (C25).

Animals↗
PubMed1992

Characterization of antibodies against human N-terminal parathyroid hormone by epitope mapping.

Two polyclonal antisera from goat and mouse and two monoclonal antibodies against human parathyroid hormone (1-34) were characterised by epitope mapping. Hexapeptides were synthesized on polystyrene pins, the sequences of which overlapped and represented the entire sequence of hPTH(1-34). Binding of antibodies to these hexapeptides was determined and antigenic determinants thus characterized. At least one predominant binding sequence was detected in the region of hPTH(7-14).

Amino Acid Sequence↗
PubMed1998

Anti-enolase-alpha autoantibodies in cancer-associated retinopathy: epitope mapping and cytotoxicity on retinal cells.

Patients with cancer-associated retinopathy syndrome (CAR), a progressive blinding disease related to retinal degeneration and systemic tumor outside the eye, develop autoantibodies against alpha-enolase. A small percentage of healthy subjects without evident tumor or visual symptoms also possess autoantibody against enolase. In these studies we examined the fine specificity of anti-enolase antibodies derived from patients with CAR and healthy individuals, using synthetic peptides covering the entire sequence of human alpha-enolase. Epitope mapping revealed that three binding regions of enolase within the residues 31-38 (FRAAVPSG), 176-183 (ANFREAMR), and 421-428 (AKFAGRNF) were common for all autoantibodies tested. However, pathogenic sera recognized an additional unique region, the sequence 56-63 (RYMGKGVS). There were also differences in in vitro cytotoxic activities on E1A.NR3 retinal cells and cell-death promoting activities between anti-enolase antibodies of healthy and CAR affected individuals. These studies showed that anti-enolase antibodies from patients with CAR were able to induce apoptotic cell death in E1A.NR3 retinal cells and provided a potential mechanism for retinal degeneration in humans.

Animals↗
PubMedDate not supplied

Epitope mapping of monoclonal antibodies specific for a macrophage lectin: a calcium-dependent epitope is in the carbohydrate recognition domain.

Mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (mMGL) has a calcium-dependent conformational epitope which is a ligand-induced binding site. A monoclonal antibody (mAb) specific for this epitope (LOM-11) stabilize lectin activity. We performed mapping for this conformational epitope using trypsin fragments that contain a carbohydrate recognition domain (CRD) and chimeric recombinant proteins between mMGL and a human counterpart of this molecule. Binding site for the mAb LOM-11 was mapped within the C-terminal 59 amino acids of CRD. Binding sites for all four mAbs that block carbohydrate ligand binding were also mapped in the C-terminal half of CRD. These results indicated that the calcium-dependent site potentially involved in protein-protein interaction, regulatory or for coordinated binding, is mapped within CRD in addition to the independent carbohydrate binding site, and that both of the distinct sites may have spatial proximity.

Antibodies, Monoclonal↗
PubMed1990

Epitope mapping of horseradish peroxidase with use of monoclonal antibodies.

Nine mouse monoclonal antibodies (MAbs) to horseradish peroxidase (HRP) were used to develop an epitope map of the enzyme. The results of a competitive binding assay indicated three distinct patterns of reactivity. Two groups of MAbs (I and III) recognized epitopes located in separate antigenic regions on the molecule; another (II) bound to sites that overlapped with epitopes in either region I or III. Further definition of these regions was obtained by analyzing the MAbs for their binding to isolated heme, other peroxidases and heme-containing proteins, and to denatured and apo-HRP. None of the group I MAbs bound heme, suggesting that this region was removed from the active site of the enzyme. All of the group II and III MAbs bound heme as well as the other peroxidases and heme-containing proteins, indicating that they recognized heme-associated epitopes at or near the active site. Only one MAb (2A2) in groups II and III bound to apo-HRP but not to denatured HRP; it was also the only MAb in the entire panel that inhibited the catalytic activity of HRP. This suggests that the epitope recognized by 2A2 involves both the heme moiety and a conformationally dependent protein determinant near the active site.

Animals↗
PubMed1993

Epitope mapping: synthetic approaches to the understanding of molecular recognition in the immune system.

Progress in the field of immunochemistry is rapidly increasing due to very efficient methods of epitope mapping. Experimental results on the allele-specific sequence motifs of MHC-binding peptides allow the exact forecast of T-cell epitopes and, in combination with B-cell prediction methods and synthetic adjuvant systems, fully synthetic vaccines may be constructed. Methods of multiple peptide synthesis are of particular use for such constructs and for the fine mapping of monoclonal antibodies or sera of patients. Peptide libraries, containing hundred thousands of different oligopeptides are made available for novel screening procedures. These techniques and their applications in various fields are summarized and discussed with respect to efficiency and productivity.

Amino Acid Sequence↗
PubMed1993

Structure/function analysis of the integrin beta 1 subunit by epitope mapping.

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.

3T3 Cells↗
PubMed1990

Epitope mapping of human thyroglobulin. Heterogeneous recognition by thyroid pathologic sera.

Thyroglobulin is the major Ag of the thyroid gland involved in autoimmune pathologies. Epitope mapping was carried out with a rabbit polyclonal immune serum against fusion proteins expressed in prokaryotic cells. After screening of an initial human thyroglobulin cDNA library and subcloning of immunoreactive clones, seven epitopes were characterized and localized on the human thyroglobulin monomeric molecule. One was close to each extremity of the molecule, and five others were concentrated in the middle, covering a sixth of this 2748-amino-acid chain. The immunoreactivities of 18 autoimmune sera from different thyroid pathologies were tested against the seven previously characterized epitopes. Those from Hashimoto's thyroiditis were the most immunoreactive. Immune responses were heterogeneous for sera from different pathologies as well as for those from the same pathology. The central epitopes and the near-C-terminal epitope, however, were the epitopes most often recognized by the immune sera. These findings show that some autoepitopes overlap accurately with some heteroepitopes characterized by a polyclonal immune serum directed against the mature protein.

Amino Acid Sequence↗
PubMed1992

Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies.

The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.

Animals↗
PubMed1997

Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.

A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1(22-38), the C-terminal of ET-1, six analogues of ET-1(16-21) each containing a substitution with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, with less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38). Ala substitution in positions 16, 17 and 19 of ET-1(16-21) hardly affected the antibody binding capacity of ET-1(16-21), whereas Ala substitution of Asp18, Ile20 and, in particular, Trp21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(15-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.

Amino Acid Sequence↗
PubMed1997

Epitope mapping of human CYP1A2 in dihydralazine-induced autoimmune hepatitis.

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti-LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

Antibody Specificity↗