Monoclonal antibodies to the calcium-binding region peptide of human C-reactive protein alter its conformation.

Five mouse mAb were generated against a synthetic peptide corresponding to the proposed Ca(2+)-binding region of human C-reactive protein (CRP). The peptide consists of amino acids 134 to 148 and possesses a calmodulin Ca(2+)-binding sequence. The mAb reacted with a surface epitope(s) on native, intact CRP as well as the closely related pentraxin protein, serum amyloid P-component. Three of the 5 mAb inhibited the Ca(2+)-dependent phosphorylcholine-(PC) binding activity of CRP, but did not bind to the PC-binding region itself. Four of the five mAb also inhibited the recognition of an epitope in the PC-binding site of CRP. Four of the mAb partially, or completely, protected CRP from selective cleavage by pronase between residues 146 and 147. The findings suggest that the Ca(2+)-binding region is on the surface of CRP, has substantial flexibility, and is probably responsible for the allosteric effects of Ca2+ ions on CRP.     

Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Epitopes in the interacting regions of beta-dystroglycan (PPxY motif) and dystrophin (WW domain).

The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.     

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.

Methods and Results

To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope ¹⁷³LPAPTS¹⁷⁸ is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.

Conclusions and Significance

We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.     

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.     

Diverse VH and VL genes are used to produce antibodies against a defined protein epitope

mAb secreting hybridomas were produced from mice hyperimmune to the model Ag tobacco mosaic virus protein. Six mAb were selected for their ability to bind synthetic peptides corresponding to amino acid residues 103-112 and 97-107 of tobacco mosaic virus protein. These mAb were analyzed for their fine specificity by measuring binding to synthetic analogs of the decapeptide, and cDNA sequences encoding the mAb V regions were determined. These analyses revealed that a wide range of different V regions are capable of binding with the same decapeptide epitope, and that these antibody sequence differences generally coincided with different binding fine specificities. This diverse antibody response with specificity for the same epitope demonstrates both the breadth of potential of the immune system and the lack of exclusivity in specific protein:protein interactions.     

The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

Binding site on human C-reactive Protein (CRP) recognized by the Leukocyte CRP-receptor

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [¹²⁵l]-CRP for cell binding. A peptide of residues 27–38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab′)₂ of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH₂- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31–36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a β-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes. J. Cell. Biochem. 64:140–151. © 1997 Wiley-Liss, Inc.     

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.     

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.     

Elucidation of discontinuous linear determinants in peptides.

Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.     

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.     

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.     

Mapping the binding areas of human C-reactive protein for phosphorylcholine and polycationic compounds. Relationship between the two types of binding sites.

We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g. poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP.PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g. galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap.     

Identification of an epitope of type 1 streptococcal M protein that is shared with a 43-kDa protein of human myocardium and renal glomeruli.

The localization of opsonic and tissue-cross-reactive epitopes within the amino terminus of type 1 streptococcal M protein was investigated by using murine mAb raised against synthetic peptides of type 1 M protein. Two mAb (IIIA2 and IIIB8) reacted with epitopes located within amino acid residues 1-12 of type 1 M protein. These antibodies opsonized type 1 streptococci and did not cross-react with human kidney and heart tissue. Another mAb (IC7) reacted with mesangial cells of renal glomeruli and human myocardium. The cross-reactive epitope of mAb IC7 was localized to position 13-19, indicating that it is not the same epitope as the previously described vimentin-cross-reactive epitope at position 23-26 of type 1 M protein. In Western blots of mesangial cell and myocardial proteins, mAb IC7 cross-reacted with a 43-kDa protein. Neither vimentin nor actin inhibited the binding of mAb IC7 to the cross-reactive protein, as determined by Western blot or immunofluorescence inhibition tests. These results provide evidence that type 1 M protein contains at least one autoimmune epitope shared with both human glomeruli and myocardium.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium.

The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding p6 mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition.