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PubMed1992

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.

Animals↗
PubMed1984

Topographical mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain JHM): correlation with biological activities.

Monoclonal hybridoma antibodies (MAb) of defined polypeptide specificity and biological activity were used in a competition binding assay to identify antibody binding sites (epitopes) on the glycoproteins of murine hepatitis virus-4 strain JHM (MHV-4). Individual MAb were labeled with horseradish peroxidase (HRP) and used as probes in a competition enzyme immunoassay (EIA). Four topographically distinct antigenic sites were detected on the E2 glycoprotein of MHV-4. Antibodies reacting with these four determinants provisionally designated A(E2), B(E2), C(E2), and D(E2) had corresponding biological activities (M. J. Buchmeier, H. A. Lewicki, P. J. Talbot, and R. L. Knobler (1984) Virology 132, 261-270). Antibodies to sites A(E2) and B(E2) mediated virus neutralization in vitro and passively protected mice against lethal virus challenge in vivo. Antibody to site C(E2) neutralized virus efficiently in vitro but did not alter disease in vivo, while antibody to site D(E2) neither neutralized nor protected. Two major nonoverlapping antigenic sites were defined on the E1 glycoprotein. Overlapping epitopes A(E1) and B(E1) constituted one site and epitope C(E1) the other.

Animals↗
PubMed1991

Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein.

Six sequential epitopes (A, B, B', D, D', E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142-161 (MCF4), B and B' in peptide 195-205, D and D' in peptide 218-237 (MCF6), and A in peptide 249-268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51.

Amino Acid Sequence↗
PubMed1990

Mapping of epitopes on the 86 kDa subunit of the Ku autoantigen.

Patients with rheumatic disorders develop autoantibodies to a nuclear protein antigen termed Ku. The Ku antigen has been identified as a non-histone DNA-binding protein complex composed of two polypeptides: 86 kDa and 70 kDa. Initial competition experiments with four monoclonal antibodies specific for the 86 kDa subunit of Ku protein suggested there was more than one epitope on this polypeptide. To determine the region for these epitopes, cDNA deletions were made from both 5' and 3' ends. The immunoreactivity of the expressed proteins was assayed by immunoblotting. A 40 amino acids region located near the C-terminus of the polypeptide between amino acids 667-708 was essential for binding the monoclonal and autoimmune antibodies. Within this region, we differentiated three epitopes for the monoclonal antibodies. The sequence critical for binding of the autoimmune antibodies was in the same region, between amino acids 689-708. None of the four monoclonal antibodies blocked the binding of the autoantibodies. The implications of these results for the etiology of the anti-Ku autoantibody response are discussed.

Amino Acid Sequence↗
PubMed1986

An antigen-binding assay to determine the specificity of monoclonal antibodies against influenza virus and mapping of epitopes.

A simple antigen-binding assay is described for determining the specificity of monoclonal antibodies against A/Victoria/3/75 influenza virus. Monoclonal antibodies were bound to polyvinylchloride microtitre wells via protein A and anti-immunoglobulin serum. Radiolabelled cell extracts were then added and allowed to adsorb to the antigen. The bound material was eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. The method can also be used to study competitive binding of antibodies to a given antigen. In this case, one antibody was adsorbed to the wells as above and the competitor antibody was added in high excess with the radioactive antigen. The results obtained with this method are comparable to those obtained by competitive RIA or ELISA.

Animals↗
PubMed1986

Mapping functional epitopes of the human LFA-1 glycoprotein: monoclonal antibody inhibition of NK and CTL effectors.

Anti-LFA-1 monoclonal antibody (MoAb) was originally identified by screening antibodies for their ability to inhibit cytolysis in the absence of complement. Anti-LFA-1 MoAb has been shown to inhibit both natural killer (NK) and cytolytic T lymphocyte (CTL) mediated cytolysis. To further define the utilization of this molecule in cell-mediated cytolysis, we used a panel of MoAb to functional epitopes on both the alpha and beta chains of the LFA-1 heterodimer. The panel was used to compare OKT3- NK effectors and OKT3+ CTL clones. As expected, function-associated MoAb to CTL antigens (T3, T8, LFA-2) and target cell antigens (HLA, LFA-3) blocked only CTL clones and not NK effectors. In contrast, anti-LFA-1 MoAb blocked both NK effectors and CTL clones. In addition, the panel of anti-LFA-1 MoAb demonstrated an identical hierarchy of functionally relevant LFA-1 epitopes. Given the similar utilization of LFA-1 in NK and CTL mediated cytotoxicity assays, we explored the ability of MoAb to different epitopes on LFA-1 to inhibit conjugate formation. Anti-LFA-1 MoAb inhibition of NK-target binding paralleled the inhibition of CTL-target binding. Thus, functional epitopes on the LFA-1 molecule have been defined for NK and CTL effectors. The identical hierarchy of functional epitopes indicates that the LFA-1 molecule is similarly utilized in NK and CTL mediated cytotoxicity and that the relevant epitopes are involved in effector-target conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Identification of assembled epitopes on the alpha-subunit of human follicle stimulating hormone.

In order to characterize further the antigenic structure of human follitropin (hFSH), BALB/c mice were immunized with hFSH and anti-hFSH monoclonal antibodies (mAbs) were generated. The hFSH subunit specificity of the mAbs was assessed by a solid-phase enzyme-linked immunosorbent assay (ELISA) and a solution-phase radioimmunoassay (RIA), each using hFSH, hFSH alpha, and hFSH beta. Five mAbs bound hFSH and hFSH alpha in the ELISA and the RIA. In addition, some mAbs recognized hFSH beta, albeit to a much lower degree, as demonstrated by displacement of [125I]hFSH binding to the mAbs by hFSH beta, in the solution-phase RIAs. Next, synthetic peptides corresponding to the hFSH alpha-subunit sequence were used to identify sequences specific to the epitopes of each of the five mAbs. Using this epitope mapping strategy, two assembled epitopes were identified. mAbs 3A and 4B distinguish one discontinuous epitope comprised minimally of sequences alpha-16-21 and alpha-66-92, whereas mAbs 5F and 2E distinguish a second discontinuous epitope comprised minimally of sequences alpha-40-50 and alpha-66-72.

Amino Acid Sequence↗
PubMed2005

Have we cut ourselves too short in mapping CTL epitopes?

MHC class I molecules generally present peptides of eight to ten amino acids; however, peptides of 11-14 residues can also elicit dominant cytotoxic T lymphocyte responses, sometimes at the expense of overlapping shorter peptides. Although long-bulged epitopes are considered to represent a barrier for T cell receptor recognition, recent structural data reveal how these super-bulged peptides are engaged while simultaneously maintaining MHC restriction. We propose that algorithms widely used to predict class I-binding peptides should now be broadened to include peptides of over ten residues in length.

Animals↗
PubMed2004

Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library.

BACKGROUND: Phl p 5 represents a major allergen of timothy grass pollen (Phleum pratense). Detailed knowledge about the structures responsible for IgE binding would allow the design of a novel generation of allergy vaccines. OBJECTIVE: We aimed to characterize the IgE epitopes of Phl p 5a using phage display combined with a molecular modeling approach. METHODS: Phl p 5a-specific IgE from sera of patients with grass pollen allergy was used for screening of a random peptide phage library displaying constrained decamers. RESULTS: Fifteen phage clones that shared sequence motifs and could be grouped into families were selected by using Phl p 5a-specific IgE. Peptide alignment with the solvent-accessible amino acids of Phl p 5a revealed 3 sequence sections with frequent hits of identical or similar amino acids. On the surface of Phl p 5a, these sections assembled in compact patches, most likely representing conformational IgE epitopes, whereas no matching clusters were found on the back sides of the 2 Phl p 5a halves. In surface plasmon resonance experiments, the high-affinity interaction between IgE and Phl p 5 could be competed by phage-displayed peptides up to 24%, indicating that they represent true epitope mimics (ie, mimotopes). Allergen-specific immunogenicity of the mimotopes was proved in Biozzi mice. CONCLUSION: The selected mimotopes facilitated the localization of conformational IgE epitopes of Phl p 5. We suggest them to be suitable candidates for the development of an epitope-specific immunotherapy.

Amino Acid Sequence↗
PubMed2001

Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display.

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K(d)) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20-51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R(1)), Bombyx mori (Bt-R(175)), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R(1) or Bt-R(175) are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R(1) or Bt-R(175) receptors. Finally, we showed that synthetic peptides homologous to Bt-R(1) and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R(1) and Bt-R(175) involved in Cry1A toxin interaction.

Amino Acid Sequence↗
PubMed2003

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope.

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.

Amino Acid Sequence↗
PubMed1987

Fine mapping of epitopes by intradomain Kd/Dd recombinants.

11 intradomain recombinants between H-2Kd and H-2Dd were produced using an original technique based on in vivo recombination in Escherichia coli. After transfection into mouse L cells, all these recombinants were expressed at high levels on the cell surface. The specificities of 77 mAbs were examined on these cell lines. mAbs could be organized in 12 groups. In each group, a small number of amino acids participating in the recognized epitope(s) were identified. In a few instances, noncontinuous epitopes comprising amino acids belonging to different domains of the antigen were found. The data thus obtained are compatible with those produced in previous exon-shuffling experiments, but permit a much more precise definition of recognized epitope(s).

Animals↗
PubMed1994

Differential immune responsiveness to the immunodominant epitopes of regulatory proteins (tax and rex) in human T cell lymphotropic virus type I-associated myelopathy.

Infection by human T cell lymphotropic virus type I (HTLV-I) is etiologically linked with HTLV-I-associated myelopathy (HAM) or adult T cell leukemia (ATL). To evaluate the contribution of the viral regulatory proteins tax and rex during the development of disease, antibody responses to these proteins were analyzed in patients with HAM (n = 28) or ATL (n = 48) and in asymptomatic carriers (n = 69). Epitope mapping analysis identified immunodominant epitopes towards the amino terminus (Tax8(106-125)) and at the carboxyl terminus (Tax22(316-335), Tax23(331-350), and Tax24(336-353)) of tax and the amino terminus (Rex1(1-20), Rex2(16-35), Rex4(46-65), and Rex6(76-95)) of rex. Analysis of antibody reactivity to these immunodominant epitopes demonstrated preferential reactivity to Tax8, Tax22, Tax23, and Tax24 (71%-93%) and to Rex4 and Rex6 (52%) in patients with HAM when compared with reactivities in ATL patients (4%-31% for tax and 19%-24% for rex) or asymptomatic carriers (27%-37% for tax and 7%-23% for rex). In contrast, antibody responses to the immunodominant epitopes of the env proteins of HTLV-I (MTA, Env1, Env5) were similar in all of three clinical groups. Thus, differential immune responsiveness to the immunodominant epitopes of tax and rex in patients with HAM may play a role in disease pathogenesis in HTLV-I-infected persons.

Amino Acid Sequence↗
PubMed1999

Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase.

DNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3G8 and betaG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG(1), k. K(d) values were 7.3 x 10(-8) for 3G8 and 1.1 x 10(-6) for betaG1. Nucleotide sequencing of cDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among alpha-like (family B) DNA polymerases (Region II), and the epitope for betaG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).

Amino Acid Sequence↗
PubMed1989

Mapping of epitopes on the 65k DNA-binding protein of herpes simplex virus type 1.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

Amino Acid Sequence↗
PubMed1991

Mapping of epitopes on U1 snRNP polypeptide A with synthetic peptides and autoimmune sera.

The ability of synthetic peptides encompassing almost the entire sequence of snRNP U1A polypeptide to be recognized in ELISA by sera of autoimmune patients was investigated. Sera from 18 patients with mixed connective tissue disease (MCTD), 145 with systemic lupus erythematosus (SLE) and 120 with other rheumatic autoimmune diseases were tested with 13 overlapping peptides. Among them, peptide 257-282 and, to a lower extent, peptide 1-11 were recognized by MCTD, SLE and Sjögren's syndrome sera. In contrast, peptide 35-58 was recognized by 94% of MCTD and only 19% of SLE sera. It did not react with any of the other patient sera. The ELISA results were compared with the pattern of reactivity observed in immunoblotting. The results indicate that peptide 35-58 probably contains a major epitope recognized by MCTD autoantibodies. It is noteworthy that in snRNP particles, this region of U1A interacts with RNA and presents only limited homology with the corresponding sequence 32-50 of U2B''.

Amino Acid Sequence↗
PubMed1994

Mapping of B cell epitopes on steroid 17 alpha-hydroxylase, an autoantigen in autoimmune polyglandular syndrome type I.

Earlier studies have shown that 17 alpha-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc) are the main autoantigens recognized by sera from patients with Addison's disease associated with autoimmune polyglandular syndrome type I (APS I). In this study we tried to identify the autoantigenic epitopes on P450c17 and compared the identified sequences with corresponding regions in two other adrenal autoantigens, P450scc and P450c21. A series of P450c17 cDNA fragments was expressed in Escherichia coli and the recognition of the corresponding protein fragments by patient sera was tested by immunoblotting. Four distinct epitope regions (ER) were found: ER1 (amino acids 122-148), ER2 (280-304), ER3 (396-432) and ER4 (466-508). B cell epitope prediction analysis showed that the four identified ERs were all located in regions of high predicted antigenicity. Homology search revealed that ER3 and ER4 but not ER1 and ER2 were related to similar sequences on P450c21. No significant homologies with P450scc in these regions were found. Although all three P450 cytochromes are genuine autoantigens this finding suggests that the autoantibody reaction against P450c17 and P450c21 can partly be a result of immunological cross-reactivity.

Addison Disease↗
PubMed2006

Plasminogen and plasmin activity in patients with coronary artery disease.

OBJECTIVE: While coronary artery disease (CAD) is associated with disturbances of the plasma fibrinolytic system, the nature of these disturbances is not fully defined. Fibrinolysis is regulated by plasmin, whose production is mediated by plasminogen activator conversion of plasminogen (Plg) to plasmin. The cascade is modulated by feedback loops that include Plg activator inhibitor 1 (PAI-1). Molecular interactions with Plg kringle domains play an important role in regulating plasmin production and its modulation of fibrinolysis. We hypothesized that interactions of tissue plasminogen activator (tPA) with Plg kringle domains regulates plasmin levels in patients with stable CAD. METHODS: Plasma was collected from patients (n = 33) with an angiographically significant CAD and controls (n = 18) with angiographically established normal or minimally diseased arteries. Plasmin activity, tPA activity, and plasma levels of Plg, PAI-1, uPA, and tPA were determined. RESULTS: CAD patients had 1.7-fold greater plasmin activity (P = 0.02) that correlated with 1.5-fold higher tPA activity when compared to controls. Epitope mapping of Plg domains showed Plg differences in epitope exposure between the two groups. Plasma from CAD patients had 50% less (P < 0.001) detectable kringle 4 and 48% less (P = 0.007) detectable kringles 1-3. CONCLUSIONS: Based on detectable differences in Plg, we conclude that in patients with stable CAD, Plg complexed with tPA exists in a conformation that enables increased tPA activity and Plg conversion to plasmin.

Adolescent↗