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At least 595 records · Page 33Linked to original sources
PubMed1997

Functional epitope mapping of insulin-like growth factor I (IGF-I) by anti-IGF-I monoclonal antibodies.

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 10(6) to 10(10) M-1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

Animals↗
PubMed1996

Comparative epitope mapping of sera from United States (US) and Japanese patients with bullous pemphigoid (BP) to fusion proteins encoded by BPAG1.

Sera from patients with bullous pemphigoid (BP) from the United States (US), Japan, and Britain demonstrate similar reactivity to the major target antigens BPAG1 and BPAG2. The purpose of the present study was to determine if the epitope specificity of circulating autoantibodies in patients with BP from the US and Japan is similar as mapped by binding to fusion proteins encoded by BPAG1. Sera from patients and controls with BP from the US and Japan were assayed for reactivity to intact BPAG1 and BPAG2 by immunoblot, and to fusion proteins encoded by BPAG1 by immunoblot and enzyme-linked immunosorbant assay (ELISA). Significant reactivity to fusion proteins encoded by the carboxyl region (FP 16-8) and coiled-coil region (FP3) was seen in sera from the US and Japanese patients, but not from normal controls from the US or Japan. Sera from US and Japanese patients differed in their response to FP7; namely, the reactivity of sera from US patients but not from Japanese patients to FP7 was significantly different from the reactivity of their respective control sera. The reasons for this difference in reactivity are unknown but may reflect genetic or environmental factors relevant in the generation of an autoantibody response to these target antigens.

Aged↗
PubMed2003

Displaying and epitope mapping of CD147 on VCSM13 phages: influence of Escherichia coli strains.

The external domain of a human leukocyte surface molecule, CD147 was displayed on the surface of phage. Two Escherichia coli laboratory strains, XL-1 Blue and TG-1, were chosen to separately propagate the recombinant phages. By sandwich enzyme linked immunosorbent assay (ELISA), CD147 on phage particles were individually captured by six CD147 mAbs and subsequently detected by anti-M13 conjugated HRP. All mAbs specifically bound the CD147 on phage particles derived from TG-1. On the contrary, only four of them could recognize the CD147 on phages produced by XL-1 Blue. The results indicate that the environment in the TG-1 periplasm is more appropriate than that of XL-1 Blue for promoting the suitable folding of CD147. This finding emphasizes the importance of selecting the appropriate E. coli host for display of a complex protein. The epitopes of CD147 displayed on the phage were further mapped by competitive inhibition ELISA, which is a reliable and economical method. Certain clusters of mAb recognition areas were identified and will provide valuable information for the discovery of the ligand for CD147.

Antigens, CD↗
PubMed1991

Epitope mapping of apolipoprotein A-I using endoproteinase cleavage and monoclonal antibodies in an enzyme-linked immunosorbent assay.

The epitopes for two monoclonal antibodies (MAbs) directed towards human apolipoprotein A-I (apoA-I), designated AI-1 and AI-3, have been more precisely defined. Previous work in our laboratory demonstrated that AI-1 and AI-3 recognize antigenic determinants located within cyanogen bromide (CNBr) fragments 1 (CF1) and 3 (CF3), respectively. Using peptides generated from endoproteinase cleavage of CF1 and CF3, we now report that both MAbs are specific for two previously unreported epitopes along the apoA-I molecule. The ability of whole endoproteinase digest mixtures to bind the MAbs, as determined by means of a competitive enzyme-linked immunosorbent assay (ELISA), indicated regions of CF1 and CF3 that were likely to form the epitopes. Purified peptides derived from the digests were then used to localize the epitopes recognized by MAbs AI-1 and AI-3 to within residues 28-47 and 140-147 of apoA-I, respectively. We have previously reported that the epitopes for both MAbs are exposed on HDL2, HDL3, and free apoA-I. Thus, the precise mapping of the binding sites recognized by AI-1 and AI-3 has enabled the identification of regions along apoA-I that are exposed on the surface of lipoprotein particles.

Amino Acid Sequence↗
PubMed2000

Epitope mapping of rat cytochrome P450 2B1 using glutathione S-transferase-2B1 fusion constructs.

We have previously produced 12 monoclonal antibodies (MAbs) against rat cytochrome P450(CYP)2B1, and 8 of these inhibit CYP2B1 catalytic activity to varying extents. Using competitive binding studies we showed that this collection of 12 MAbs recognize at least 6 spatially distinct epitopes. The complete coding sequence of CYP2B1 DNA in plasmid pSR-P450 was inserted into a glutathione S-transferase (GST) expression vector pGEX-1lambdaT so that it was in frame with the GST gene. Expression of GST-CYP2B1 was detected with most of the MAbs in Western blots except those which were conformation-specific. Fourteen different constructs were then made using PCR with oligonucleotide primers having EcoRI sites at their ends and were introduced into the GST expression vector at the EcoRI site. Each fusion construct was expressed in Escherichia coli, subjected to SDS-PAGE, blotted, and probed individually with each MAb. MAbs, which inhibited catalytic activity and were mutually competitive in binding to CYP2B1 (viz. BEA33, BE44, BE45, and BE28), recognized several fusion constructs and by deduction recognized amino acids 250-261 in CYP2B1. Other antibodies inhibiting catalytic activity recognized amino acids 262-272 (BEF29) and 306-491 (BE46, B50, and BE49) on CYP2B1. Non-inhibitory MAbs BE26 and BE32 were mapped to region 380-398 in CYP2B1. It was interesting to note that MAbs BEA33 and BE26, which also recognize spatially distinct epitopes on human CYP2E1 but not rat CYP2E1, had corresponding regions of high homology in human CYP2E1 but not rat CYP2E1. Identifying the epitopes recognized by this collection of MAbs will add to our understanding the sequences that may be important for producing inhibitory and specific antibodies to closely related antigens.

Amino Acid Sequence↗
PubMed2000

Epitope mapping of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae.

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.

Adhesins, Bacterial↗
PubMed1996

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Sequence analyses and antigenic epitope mapping of the putative RNA-directed RNA polymerase of five U.S. bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L1 double-stranded RNA segments of bluetongue virus (BTV) serotypes 2, 11, 13, and 17, which encode the putative RNA-directed RNA polymerase VP1. Each L1 gene contained 3944 nucleotides and was 10 bases shorter than the previously reported L1 gene of BTV 10. A single open reading frame which could encode the reported VP1 protein, 1302 amino acids in size, began with an initiation codon at nucleotides 12-14 and a termination codon at nucleotides 3918-3920. Analyses of the nucleotides of L1 genes and the deduced amino acid sequences of VP1 proteins of the five U.S. BTV serotypes indicated that the most recently isolated BTV-2 serotype from Florida was more distantly related than BTV-10, 11, 13, and 17, which were isolated primarily in the western U.S.A. The results are consistent with our hypothesis that BTVs-10, -11, -13, and -17 are derived from a single and common gene pool, and that BTV-2 belongs to a second, distinct gene pool. These genetic distinctions also reflected well with the known geographic distribution of the five U.S. BTV serotypes in North America. This putative RNA-directed RNA polymerase (149 KDa) was a basic protein, and the deduced amino acid sequences of the VP1 proteins contained seven highly conserved hydrophobic domains and many other sequence motifs which were also found in other known RNA polymerases. Four immunodominant but linear antigenic epitopes conserved among the VP1 of five U.S. BTVs were also been identified and mapped using monospecific oligoclonal antibodies.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of anti-breast and anti-ovarian mucin monoclonal antibodies.

Anti-breast cancer antibodies (BC2, HMPV and 4B6) and an anti-ovarian cancer antibody (OM1) were found to react with mucins--indeed with the protein core encoded by the MUC1 gene. This gene contains a VNTR (variable number of tandem repeats) encoding a 60 bp (= 20 amino acids) repeat sequence and within this amino acid sequence SAPDTRPAP was predicted, by hydrophilicity analysis, to be the immunogenic peptide sequence. The four antibodies were shown to react with MUC1 VNTR encoded peptides in direct binding and inhibition studies. The precise reactivity of the 4 mAbs was mapped using ELISA in both solid and liquid phase, and demonstrated the epitopes to be: APDTR (BC2 and HMPV), PDTR (4B6) and DTRPA (OM1). By using the pepscan method, the epitopes were shorter (PDTR, DTR and DTRP). However when these short peptides (except DTR) were synthesized they did not react; flanking amino acids are needed for the epitopes. Clearly several different methods should be used to define the reactive epitope. Within (S)APDTR, major amino acid substitutions could be made--even of three to four amino acids without altering antibody binding, provided that P and R were not substituted. It was of interest that an anti-ovarian cancer antibody gave similar anti-peptide reactions to the anti-breast cancer antibodies; apparently MUC1 peptides in ovarian cancer are the same as in breast cancer.

Amino Acid Sequence↗
PubMed1987

Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping.

Two mAbs, one specific for cardiac alpha-myosin heavy chains (MHC) and the other specific for cardiac beta-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their alpha-or beta-MHC content. alpha alpha-MHC and beta beta-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the alpha-MHC-specific mAb and the beta-MHC-specific mAb, alpha beta-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of alpha alpha-and beta beta-MHC homodimers and alpha beta-MHC heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of alpha- and beta-MHC in alpha alpha-MHC homodimers, beta beta-MHC homodimers, and alpha beta-MHC heterodimers, as initially proposed by Hoh, J. F. Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).

Animals↗
PubMed1992

Epitope mapping on fragments of beet necrotic yellow vein virus coat protein.

The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the beta-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.

Blotting, Western↗
PubMed1996

Epitope mapping of a C5a neutralizing mAb using a combined approach of phage display, synthetic peptides and site-directed mutagenesis.

BACKGROUND: The anaphylatoxin C5a is a powerful proinflammatory protein generated on activation of the complement system. Recently, we described an anti-hC5a neoepitope specific mAb, mAb 2925, which was raised against the nonapeptide ISHKDMQLG (C5a-(65-73). This mAb is unique in that it recognizes both hC5a and hC5adesArg, even when it is denatured. It inhibits binding of [125I]C5a to its receptor on Bt2-cAMP differentiated U937 cells. OBJECTIVES: To define the epitope of mAb 2925, we used a combined approach of a bacteriophage random octapeptide library, synthetic peptides and site-directed mutagenesis. STUDY DESIGN: First a phage peptide library was screened with the anti C5a mAb 2925. Then synthetic peptides were synthesized with respect to the sequence information yielded from the phage approach, and used for binding studies. Site-directed mutagenesis was performed to confirm the results from the mapping experiments. RESULTS AND CONCLUSION: Most phages selected by biotinylated Fab 2925 displayed sequences on the minor coat protein which correspond to residues within the C-terminus of human C5a. A first consensus motif comprised amino acids His-Lys or His-Arg, which allowed us to define position 67 and 68 as part of the epitope. A second consensus motif was selected, comprising Arg/Lys-Trp-Trp. This motif did not match any residues within the C5a C-terminus. However, when expressed together with the consensus motif His-Arg, as in HRWWXXXX or in HRXKWWXX, binding of these peptides to Fab 2925 increased as compared to peptides expressing the His-Arg motif only. Thus, the Arg/Lys-Trp-Trp motif serves to stabilize the binding of His-Arg to mAb 2925. Synthetic peptide studies revealed further N-terminal residues Ile65 and Ser66 as part of the epitope. A C5a mutant with an exchange Lys68Glu (C5aGlu68) confirmed the participation of Lys68 as a contact residue within the epitope of mAb 2925. Hence, the epitope recognized by mAb 2925 is linear and comprises residues Ile65, Ser66, His67, and Lys68. Thus, we could demonstrate for the first time that a mAb inhibits C5a receptor binding through specific interaction with receptor binding residues of the ligand.

Amino Acid Sequence↗
PubMed1991

Epitope mapping studies of snake venom phospholipase A2 using monoclonal antibodies.

Fifteen different monoclonal antibodies developed against pseudexin, a snake venom phospholipase A2 with presynaptic neurotoxicity, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin were synthesized by using alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies bound to toxin peptides and were detected with an enzyme-linked immunosorbent assay. Three of the bound antibodies recognized a site important in calcium binding and the interlocking of dimeric forms of snake venom phospholipase A2. Analogous regions from other phospholipases were synthesized and probed with the four reactive antibodies. A good correlation was found between the reactivity of whole molecule phospholipases and peptide regions with the antibodies. Monoclonal antibodies neutralizing the lethal or enzymatic effects of pseudexin did not recognize any linear epitopes.

Amino Acid Sequence↗