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PubMed1998

Autoantibodies against human calpastatin in rheumatoid arthritis: epitope mapping and analysis of patient sera.

Autoantibodies against calpastatin have recently been described to be highly prevalent in sera of patients with rheumatoid arthritis (RA). When the sera of 45 patients with RA were analysed for autoantibodies against calpastatin by a newly developed enzyme-linked immunosorbent assay (ELISA), only four sera (8.9%) tested positive, which is not significantly different from the frequency observed in healthy controls. Since the ELISA is based on a synthetic peptide containing the C-terminal 27 amino acids of calpastatin bound to the solid phase, this negative result might be the consequence of the small antigen used. Therefore, a systematic analysis of the epitopes for autoantibodies in calpastatin was performed using sera from RA patients and healthy individuals. Recombinant fusion proteins containing fragments of calpastatin or the complete protein were produced and sera analysed by Western blots. In the N-terminal portion (amino acids 1-369), at least two major epitopes exist, against which 65% of normal sera as well as 76% of RA sera show reactivity in Western blot assays. These epitopes are not useful for clinical diagnostics. Only five out of 45 (11.1%) RA sera reacted against the C-terminal portion (amino acids 363-708) of calpastatin, while four out of 52 (7.7%) control sera showed reactivity. Three of the five RA sera and two out of four control sera had autoantibodies against the C-terminal 27 amino acids of calpastatin. These three patient sera had already been tested positive in the ELISA. The fourth patient positive in the ELISA was Western blot negative. The differences between the group of RA patients and controls are not statistically significant. When the clinical characteristics of the four patients with autoantibodies against the carboxyl end of calpastatin were analysed, it became apparent that all four had significantly elevated C-reactive protein (>50 mg/l). This observation might indicate that calpastatin autoantibodies are found in RA patients with more active disease. Thus, while the majority of RA patients do not have an increased prevalence of calpastatin autoantibodies, it cannot be ruled out definitively that a small subgroup may be characterized by autoantibodies to the C-terminus of calpastatin.

Adult↗
PubMed2004

Epitope mapping of the antigenic protein TsNTxP from Tityus serrulatus scorpion venom using mouse, rabbit and sheep antibodies.

In the present investigation we used native and recombinant TsNTxP to elicit antibodies in three different animal models (mouse, rabbit and sheep). Differences among anti-TsNTxP antibodies were analyzed using sets of overlapping pentadecapeptides of the TsNTxP amino acid sequence and also modified peptides to reveal key residues in antibody-peptide binding. Despite the identification of similar peptides by the antibodies in the C and N-terminal, peculiarities of each system were observed including the level of reactivity and also the number and type of key residues in the continuous epitopes of TsNTxP. In addition, in vitro neutralization assays indicated that sheep are an alternative and efficient model for the production of anti-Tityus serrulatus venom.

Amino Acid Sequence↗
PubMed2003

Epitope mapping of antigenic MUC1 peptides to breast cancer antibody fragment B27.29: a heteronuclear NMR study.

MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant "reverse templates" of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], (1)H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including (15)N and (13)C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)(2). (15)N and (13)C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The (13)C(alpha) T(1) values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the (15)N- and (13)C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast cancer vaccine design.

Amino Acid Sequence↗
PubMed2001

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

Amino Acid Sequence↗
PubMed2000

Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

Amino Acid Sequence↗
PubMed2001

Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha.

The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibalpha (GPIbalpha) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbalpha were identified through the characterization of 5 new inhibitory anti-GPIb monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb mAbs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 area by the use of canine-human chimeras. These antibodies, however, had little or no effect (approximately 40% inhibition) on the binding of vWF induced by either botrocetin or ristocetin. On the other hand, the anti-GPIb mAbs 24G10 and 6B4, which blocked GPIb-vWF binding under all conditions examined, bound to 2 different regions of GPIbalpha, aa 1-81 and aa 201-268, respectively. The epitope for 6B4 was further narrowed by phage display revealing 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIbalpha, the gain-of-function platelet-type von Willebrand disease (PT-vWD) mutations have been identified. Alignment was partially confirmed because the binding of 6B4 to recombinant GPIbalpha fragments carrying either one of the PT-vWD mutations was considerably impaired but not completely abolished. In contrast, mAb 24G10 bound more strongly to mutant PT-vWD GPIbalpha. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbalpha. In conclusion, 2 functionally important areas within GPIbalpha were identified: one localized within the leucine-rich repeat N-terminal aa 1-59 area and one composed of residues aa 1-81 in close contact with aa 201-268. Moreover, further support is provided for the existence of an intramolecular interaction between the N-terminal flanking (aa 1-81) and C-terminal flanking (aa 201-268) regions. (Blood. 2001;98:652-660)

Animals↗
PubMed2002

Use of epitope mapping to identify a PCR template for protein amplification and detection by enzyme-linked immunosorbent assay of bovine herpesvirus type 1 glycoprotein D.

Infection with bovine herpesvirus type 1 (BHV-1) occurs worldwide and causes serious economic losses due to the deaths of animals, abortions, decreased milk production, and loss of body weight. BHV-1 is frequently found in bovine semen and is transmitted through natural service and artificial insemination. The detection of BHV-1 in bovine semen is a long-standing problem in veterinary virology which is important in disease control schemes. In the present study, ordered deletions of the full-length BHV-1 glycoprotein open reading frame were used to identify an epitope recognized by a specific monoclonal antibody (MAb). A glycoprotein D fragment containing this epitope was then amplified using an in vitro protein amplification assay developed previously (J. Zhou, J. Lyaku, R. A. Fredrickson, and F. S. Kibenge, J. Virol. Methods 79:181-189, 1999), and the resulting peptide was detected by indirect enzyme-linked immunosorbent assay (ELISA) with the specific MAb. This method detected 0.0395 50% tissue culture infective dose of BHV-1 in raw bovine semen, which was 1,000-fold more sensitive than traditional PCR. We therefore conclude that this in vitro protein amplification assay combined with ELISA has superior sensitivity for direct virus detection in clinical samples.

Animals↗
PubMed1995

Epitope mapping of beta-lactam antibiotics with the use of monoclonal antibodies.

In order to evaluate the antigenic contribution of different regions of the penicillin molecule, monoclonal antibodies were raised against amoxicillin-protein conjugates and their specificities analysed in detail. A random sample of the clones produced was analysed by a quantitative inhibition-ELISA, using, as inhibitors, monomeric conjugates of the following antibiotics to butylamine (BA), amoxicillin (AX), ampicillin (AMP), benzylpenicillin (BP) and the nuclear part of these, 6-aminopenicillanic acid (6-APA); and different parts of the following molecules: N-(p-hydroxyphenyl)-glycine (PHPG), N-phenylglycine (NPG), phenylacetic acid (PA) and thiazolidine (TIAZ). The results showed that 92% of the antibodies recognized an epitope in which the side chain was a major constituent, although with variable contributions from other regions of the molecule. There was a high degree of crossreactivity with aminopenicillins, but low or absent crossreactivity with BP. None of the antibodies recognized the thiazolidine ring or the conjugated nuclear region of the penicillins. Finally, one antibody seemed to recognize, equally, all the different structures tested. The possible relevance of these results to penicillin allergy is discussed.

Amoxicillin↗
PubMed1996

Epitope mapping of C1 inhibitor autoantibodies from patients with acquired C1 inhibitor deficiency.

We report six patients with acquired C1 inhibitor (C1-inh) deficiency associated with serum C1-inh autoantibodies and circulating cleaved (96 kDa), functionally inactive C1-inh. In three patients, all of whom had IgG-kappa paraproteins in their sera, the Abs were IgG-kappa. In the remaining three patients, the Abs were IgM (2 kappa, 1 lambda). These data suggest that all the Abs were monoclonal. The autoantibodies recognized two synthetic peptides (peptides 2 and 3), which spanned the reactive center of C1-inh. Binding to peptide 3 (residues 448-459) was greater than to peptide 2 (residues 438-449), suggesting that the epitope recognized by the autoantibodies was expressed principally by peptide 3. Both peptides inhibited the binding of the autoantibodies to C1-inh. None of the autoantibodies recognized peptide 1 (residues 428-440), and this peptide did not inhibit the binding of the autoantibodies to C1-inh. The use of substituted peptides suggested that residues Q452 and Q453 made significant contributions to the epitope, and computer modeling studies showed their side chains to be surface exposed in the intact molecule. However, computer modeling also showed that none of the side chains of the polar residues in peptide 2 were sufficiently close to Q452 and Q453 to be able to contribute to a shared epitope. As peptide 2 could inhibit the binding of C1-inh autoantibodies to peptide 3 and vice versa, we conclude that an autoepitope also exists in peptide 2. Computer modeling and the use of substituted peptides suggested that the sequence LLVF (residues 446-449) in peptide 2 is structurally similar to the sequence QQPF (residues 452-455) in peptide 3. We therefore conclude that there are two potential epitopes in the intact C1-inh molecule that are capable of binding to C1-inh autoantibodies.

Aged↗
PubMed2005

Detection of citrate synthase-reacting autoantibodies after heart transplantation: an epitope mapping study.

Autoimmune mechanisms play an important role in the pathogenesis of allograft vasculopathy following heart transplantation, but the autoantigens involved have been only sparsely studied. Citrate synthase (CS) enzyme is a conserved molecule, and, as an important mitochondrial autoantigen, it is protected by the "immunological homunculus". Tissue destruction and alteration of the immune regulatory mechanisms can induce pathological immune response against CS in other autoimmune diseases. In our present study we aimed to detect CS-specific autoantibodies in heart transplant patients, therefore, prospective, randomised clinical tests were conducted on 33 heart transplant patients and compared with 130 healthy blood donors. The level and isotype of CS antibodies were detected by simple binding indirect enzyme-linked immunosorbent assay (ELISA). The epitope specificities of the autoantibodies were measured on synthetic overlapping peptide sequences of CS enzyme by an indirect multi-pin ELISA method. Mainly IgM isotype CS autoantibodies were found in healthy controls, while IgG was found at higher levels and frequency (four-times higher) in heart transplant patients. Autoantibodies of IgG isotype recognise different epitopes than do autoantibodies of IgM isotype, even within the same group and individual. New epitope-specific IgG and IgM isotype autoantibodies appeared in heart transplant patients when compared with the controls. Our findings suggest a possible role of CS-specific autoantibodies in the pathomechanism of allograft vasculopathy.

Antibody Specificity↗
PubMed2004

Preparation of human recombinant kinesin heavy chain and epitope mapping of its structural domains.

The isolation of the cDNA sequence encoding the human neuronal kinesin (a force-generating motor protein which transports various membrane organelles along microtubules in an ATP-dependent manner) heavy chain (nKHC) and the construction of expression vectors to produce the full-length nKHC and its domains in Escherichia coli is described. By tuning up the conditions for the expression of nKHC, a sufficient amount of the soluble protein intragenously tagged with 6xHis tag was obtained and purified by nickel chromatography. The recombinant structural domains of nKHC, including the motor domain (FKHC1--amino acids 1-330), the microtubule binding domain (FKHC2--amino acids 174-315) and the coiled-coil stalk domain (FKHC3--amino acids 331-906) were used to determine the epitope location for monoclonal antibodies KN-01, KN-02, and IB II raised against different kinesin heavy chains. The antibodies were shown to recognize epitopes located in the stalk domain of nKHC and represent thus useful probes for this domain.

Antibodies, Monoclonal↗
PubMed1996

Reactivity and epitope mapping of single-chain T cell receptors with monoclonal antibodies.

To examine further the structure of the T cell receptor (TCR) and the specificity of mAbs generated against the native protein, the TCR was expressed in Escherichia coli as a single chain in which the variable regions of the alpha and beta chains are joined by a 25 amino acid linker. Five single-chain TCR that have different alpha and/or beta variable (V) regions were examined with the anti-V beta 8 region mAbs KJ16 and F23.1 and the anti-V alpha 8 mAbs KT50, KT65 and B21.14. Each of the mAbs reacted with one or more of the single-chain receptors. Western blot analysis demonstrated that the intrachain disulfide bonds were required for proper epitope conformation and recognition of the TCR by the antibodies. KT50, KT65 and B21.14 antibodies distinguished between two related V alpha regions that differed at only six residues. A model of the V regions of the TCR based on immunoglobulin (Ig) structure suggests that three of these six variant residues are in the putative CDR1 of the receptor and possibly accessible to antibody. To test this possibility, site-directed mutagenesis of the unreactive V alpha region demonstrated that the combination of all three residues restored binding by the anti-V alpha 8 antibodies. In addition, these three complimentarity determining regions (CDR) residues are likely to be in close proximity to the putative CDR3 which also influenced binding of the antibodies. The epitopes recognized by the V alpha-specific antibodies are thus predicted to reside closer to the putative binding site than the epitopes previously determined to be recognized by the anti-V beta 8 antibodies, KJ16 and F23.1. Finally, the specificities of KT50 and KT65 as determined with the E. coli expression system suggests an explanation for previous observations about the differences in the T cell populations that are recognized by these antibodies.

Amino Acid Sequence↗
PubMed1996

T cell subsets, epitope mapping, and HLA-restriction in patients with allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease characterized by Aspergillus fumigatus (Af) colonization, IgE and IgG anti-Af antibodies, pulmonary infiltrates, bronchiectasis, and pulmonary fibrosis. Little is known regarding T cell responses and their role in the pathogenesis of ABPA. To examine T cell reactivity to Af antigens, T cell clones (TCC) specific to the Asp f 1 antigen, an 18-kD protein of Af, were established from the peripheral blood of three ABPA patients. The majority of TCC isolated from ABPA patients, and specific for the Asp f 1 allergen of Af, are IL-4 producing CD4+ cells of the Th2 phenotype. Further analysis in this study revealed that the majority of TCC reacted to mainly two epitopes of Asp f 1, while the remaining TCC reacted to three additional "minor" epitopes. Blocking studies using monoclonal antibodies specific for class II HLA-D region gene products showed that most TCC, 19/21, were restricted by HLA-DR molecules, and the remaining two clones by HLA-DP molecules. The use of a panel of HLA-matched and mismatched EBV-transformed B cells as antigen presenting cells revealed that the HLA-DR restriction was mediated exclusively by either the HLA-DR2 or HLA-DR5 alleles. Genotyping of DRB1 gene products showed that class II presentation for most clones was not restricted to a single allele, representing DRB1 gene products of either HLA-DR2 or DR5. These studies offer insight into the cellular and molecular determinants which contribute to the immunopathophysiology of ABPA.

Amino Acid Sequence↗
PubMed1991

Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

Antibodies, Monoclonal↗
PubMed2006

Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy.

Structure-function studies of antibody-antigen systems include the identification of amino acid residues in the antigen that interact with an antibody and elucidation of their individual contributions to binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteines in positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP alanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributions of amino acid residues included in the continuous antigenic epitopes that are recognized by two different monoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of the advantages of solution phase measurements, including high sensitivity, simplicity of manipulation with reagents, and elimination of solid phase interferences or separation steps. Significant differences in the molecular masses of the free and antibody bound BNP results in a substantial ( approximately 2.5-times) increase in the diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusion rates of the ligand at the single molecule level introduces the ultimate opportunity for researching systems where the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of the ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP and bind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx, and Architect platforms.

Animals↗
PubMed1996

Epitope mapping of new monoclonal antibodies recognizing distinct human FcRII (CD32) isoforms.

The class II Fc gamma receptors are widely distributed on cells of the immune system. Nevertheless, the exact cell type distribution of the FcRII isoforms is still unclear because of the lack of appropriate antibodies that discriminate between the various isoforms. In this study we describe the generation and characterization of three monoclonal antibodies (MAbs) raised against recombinant human FcRIIb2 as well as a synthetic peptide (amino acids 30-39) of this receptor. Analyses of the isoform specificity of these antibodies using ELISA and Western blots revealed that the MAbs II1A5 (mIgG1) and ID2.7 (mIgM) are pan FcRII antibodies recognizing all known FcRII isoforms. In contrast, the MAb II8D2 (mIgG1) specifically reacts with FcRIIb but not with FcRIIa. The observed antibody reactivities could be confirmed by examination of the exact epitopes using overlapping 15-mer peptides spanning the entire FcRIIb2. So far these antibodies are the only ones described that detect FcRII in Western blots. Moreover, they can be used to analyze the cellular FcRII isoform distribution at the protein level, which was otherwise not possible.

Amino Acid Sequence↗
PubMed2000

Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages.

cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn(2+). The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide libraries. Polyclonal antibodies were then used to pan the SGRP libraries for antibody-binding phages. Analysis of the phage isolates revealed that the majority (86%) of them only had a single insert. However, phages displaying composite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isolated. Inserts containing A or C region peptide were found to contain extra sequences from the C region, while the B region epitope was linear and had additional sequence from either upstream or downstream. In addition a dominant and possibly non-neutralising VP7 epitope was identified around amino acids 263-270. One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maintain its antigenicity. The results in this study may have significant implication for recreation of conformational epitopes and vaccine development.

Amino Acid Sequence↗
PubMed1994

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

Amino Acid Sequence↗