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PubMed1990

Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis.

The gene encoding the major Mycobacterium bovis secreted protein MPB70 was cloned and isolated from a DNA library in lambda EMBL 3, and the restriction map of the area of chromosome containing the gene ascertained. After sub-cloning, the complete DNA sequence and predicted amino acid sequence were determined, and from this information a series of overlapping octapeptides encoding all possible linear epitopes of eight or less amino acids were synthesized. These peptides were probed with monoclonal antibodies specific for M. bovis and with sera from M. bovis-infected cattle. Epitopes defined by this technique were then examined using a substitution analysis that allowed the significance of each amino acid in the putative epitope to be quantified, and the exact specificity of the antibody response for the epitope determined.

Amino Acid Sequence↗
PubMed1990

B cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides.

Envelope glycoproteins, gp 120 and gp41, of the human immunodeficiency virus type 1 (HIV-1) elicit immune responses, including virus-neutralizing antibodies, which are expected to play a role in the defence against HIV-1 infection. Subregions of the gp120/gp41 sequence have immunosuppressive effects or may be implicated in autoimmune responses. Some of the immunodominant epitopes of gp120/gp41 do not contribute to protective immunity and act as immunological decoys. These circumstances emphasize the need to select from gp120/gp41 regions inducing protective responses. Towards this goal, 30 peptides covering approximately 87% of the HIV-1 strain BH10 gp120/gp41 sequence were synthesized. Antibodies in rabbit and human anti-HIV-1 sera recognized 28 and nine of the peptides, respectively, indicating that most of the gp120/gp41 sequence is immunogenic and secondly, that the antibody response to HIV-1 is restricted in infected humans. Most of the peptides, without conjugation to carriers, elicited high levels of anti-peptide (endpoints 1: greater than 10(4] and anti-gp120/gp41 (endpoints 1: greater than or equal to 10(3] antibodies. The highest levels of virus-neutralizing antibodies were elicited by peptide 306 to 338 from a hypervariable loop of gp120. Additional peptides from the full-length hypervariable loop (303 to 338) of HIV-1 BH10 and from 20 additional HIV-1 isolates were recognized differentially by human anti-HIV, suggesting that success of passive immunization may depend on a match between administered antibodies and the challenging HIV-1 strain, and also that active immunization with selected peptides from a hypervariable region of distinct HIV-1 isolates should be explored further as a method for prophylaxis against infection.

Antibody Specificity↗
PubMed1989

Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping.

Monoclonal antibodies (MAbs) to human immunodeficiency virus type 1 were produced. Two antibodies reacted with the 17-kilodalton core protein (p17) of the virus and with its polyprotein precursor. To various degrees, each MAb neutralized infection by the cell-free virus. With a series of sequential overlapping hexapeptides which represent the p17 gene product, the epitopes identified by the MAbs were defined. The epitopes localize to overlapping regions near the amino terminus of the protein. Soluble synthetic peptides which span the antibody-binding sites of interest were demonstrated to competitively inhibit the reactivity of p17 MAbs, thus confirming the location of virus-neutralizing sites within the core protein.

Amino Acid Sequence↗
PubMed1991

Epitope mapping of human thyroglobulin reveals a central immunodominant region.

Thyroglobulin is the thyroid hormone precursor and the major antigen frequently involved in autoimmune diseases. The primary structure of human thyroglobulin is known but the spatial structure remains largely undetermined. By using fusion protein produced in prokaryotic system we have characterized seven short immunoreactive peptides carrying at least one epitope. None of them includes hormonogenic sites, but five are concentrated in the central part of the monomeric molecule, which thus emerges as the major immunogenic region of this protein.

Epitopes↗
PubMed1987

Epitope mapping of the major Epstein-Barr virus outer envelope glycoprotein gp350/220.

To understand the complete immunochemical structure of the Epstein-Barr virus (EBV) major membrane glycoprotein gp350/220, monoclonal antibodies (MAbs) reacting with this important viral antigen were isolated. Through competitive inhibition binding studies, it was determined that a group of 18 IgG MAbs recognized seven distinct epitopes on the gp350/220 molecule. Eight of these MAbs fell into a single epitope group with four of those MAbs, as well as a single MAb from another epitope group, being capable of neutralizing EBV strain B95-8 transformation of umbilical cord lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

Epitope mapping of an HLA-B27 monoclonal antibody that also reacts with a 35-kD bacterial outer-membrane protein.

Ye-3 is an HLA-B27-specific murine monoclonal antibody recognizing the heat-modifiable protein of Enterobacteriaceae. Here we used recombinant hybrid molecules between the HLA-B7 and HLA-B27 antigens to delineate the epitope recognized by Ye-3. Results of these experiments indicated that the segment of HLA-B*2705 spanning residues 77-81 was critical to the reactive epitope. It is known to be a major serological and functional recognition site of HLA-B*2705 and our data give support for its involvement also in the serological cross-reactivity with bacterial antigens.

Alleles↗
PubMed1994

Epitope mapping of Escherichia coli cell division protein FtsZ with monoclonal antibodies.

A fusion between lacZ and ftsZ of Escherichia coli was constructed to obtain a beta-galactosidase-FtsZ fusion protein. This fusion protein was used to raise antibodies against cell division protein FtsZ. Six monoclonal antibodies were obtained, and they reacted with FtsZ from cytoplasm and membrane fractions. The epitopes in FtsZ were localized by studying the reactions of the monoclonal antibodies with fusion proteins truncated at the carboxy terminus and with fragments that were obtained by CNBr cleavage of purified FtsZ. Five different epitopes were defined. Epitopes I and III reacted with the same monoclonal antibody, without showing apparent amino acid homology. Epitope II was defined by monoclonal antibodies that cross-reacted with an unknown cytoplasmic 50-kDa protein not related to FtsZ. Epitopes IV and V were recognized by different monoclonal antibodies. All monoclonal antibodies reacted strongly under native conditions, so it is likely that the five epitopes are situated on the surface of native FtsZ. By using these data and computer analysis, a provisional model of FtsZ is proposed. The FtsZ protein is considered to be globular, with a hydrophobic pocket containing GTP-binding elements. Epitopes I and II are situated on each side of the hydrophobic pocket. Because the carboxy terminus contains epitope V, the carboxy terminus of FtsZ is likely oriented toward the protein's surface.

Animals↗
PubMed1992

Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer.

Amino Acid Sequence↗
PubMed1989

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies.

Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd, specific for the viral nucleoprotein (p 60). Three domains (A, B, C) were identified on the surface of the Lassa virus nucleoprotein using an ELISA-inhibition test. All domains carried different Lassa virus specific epitopes. In addition, the A-domain carried a group specific epitope present within the arenavirus family as a whole as shown by cross-reaction in immunofluorescence tests. The B-domain only carries Lassa virus specific epitopes, whereas the C-domain has a type specific and a subgroup specific (Lassa, LCM) epitope.

Animals↗
PubMedDate not supplied

Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.

Antibodies, Monoclonal↗
PubMed1996

B-cell epitope mapping within the MA16 antigenic sequence found in Eimeria acervulina merozoites and sporozoites.

Overlapping heptapeptides derived from the MA16 Eimeria acervulina antigenic sequence (Castle et al., 1991) were synthesised on polypropylene pins ('pepskan' technique, Cambridge Research Biochemicals, UK). Binding of antibodies from chickens and rabbits infected and immunised respectively with various species of Eimeria oocysts (E. acervulina, E. tenella, E praecox, E. necatrix and E. maxima), was examined using the coated pins as the solid phase of an enzyme immunoassay (EIA). Antigenicity of the overlapping synthetic heptapeptides was then analysed using a number of algorithms based on the amino acid sequence to predict secondary protein structure, hydrophilicity, acrophilicity and chain flexibility profiles. The antigenicity of this sequence appears to be quite different from that found for the E. tenella GX3264 antigenic sequence (Bhogal et al., 1992) whose profile was similarly examined (Talebi and Mulcahy, 1994) using the same rabbit and chicken anti-Eimeria oocyst sera.

Amino Acid Sequence↗
PubMed1990

The membrane domain of the human erythrocyte anion transport protein. Epitope mapping of a monoclonal antibody defines the location of a cytoplasmic loop near the C-terminus of the protein.

We have used synthetic peptides to study the location of the amino acid sequences in the human erythrocyte anion transport protein (band 3) which are recognized by four murine monoclonal antibodies, BRIC 130, 132, 154 and 155. These antibodies are known to react with epitopes in the protein which are on the cytoplasmic side of the membrane. The results suggest that the amino acid residues important for the reaction of BRIC 130 and BRIC 154/155 are located within amino acids 899-908 and 895-901 respectively in the cytoplasmic tail of the protein. The BRIC 132 epitope is located within amino acid residues 813-824. This is part of a surface loop in the protein which probably extends from residue 814 to residue 832 and is located on the cytoplasmic side of the membrane. These results provide direct evidence for the topographical location of a sequence in a poorly understood region of the protein.

Amino Acid Sequence↗
PubMed1990

Epitope mapping of the HIV-1 gag region by analysis of gag gene deletion fragments expressed in Escherichia coli defines eight antigenic determinants.

Immune response to HIV infection is generally characterized by appearance of antibodies to the gag protein p24 early in infection, and by apparent loss of p24 antibodies accompanied by increases in p24 antigen levels with disease progression. Precise definition of the immunodominant epitopes present in gag gene proteins has potential clinical significance. Seventeen anti-gag monoclonal antibodies (MAb) were used in enzyme-linked immunosorbent assays (ELISA) with antigens expressed by nine recombinant clones to define epitopes on HIV gag proteins which elicit an immune response. All of the MAbs tested, except two anti-p17, reacted with a clone which expresses the carboxyl terminal 13 amino acids of p17 and all of p24 and p15. All anti-p24 MAbs reacted with clones containing all of p24. MAbs reacted differentially with clones containing deleted regions depending on the antigenic portion expressed. Of thirteen potential identifiably different genomic regions which could be predicted from the genomic structure of the clones, eight different antigen epitopes were defined: two on p17, five on p24, and one on p15 (in the region corresponding to the carboxyl terminal protein p6). Six regions did not appear to react with any of the monoclonal antibodies available. Identification of the epitopes present in the cloned antigens should allow their use to evaluate sera from HIV-infected donors at different clinical stages of progression to AIDS.

Animals↗
PubMed1994

Epitope mapping by cysteine mutagenesis: identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli.

Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329-342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370-412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328-338 regions. The lamB mutants were also tested for phage lambda receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.

Amino Acid Sequence↗
PubMed1998

Epitope mapping of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

Four monoclonal antibodies (mAbs) against 4-aminobenzoate hydroxylase (EC 1.14.13.27) have been produced (H. Tsuji et al., J. Biol. Chem. 265 (1990) 16064; T. Ogawa et al., Biochim. Biophys. Acta 1115 (1992) 220). Of the mAbs, three mAbs (mAb-A, -B1 and -B2) recognize the FAD-binding domain of the enzyme. In the present study, the epitopes of the mAbs on the enzyme have been examined using pGEX-2T expression systems for DNA fragments encoding various partial amino acid sequences of 4-aminobenzoate hydroxylase. The epitopes for mAb-A, -B1 and -B2 were shown to be on sequences 413-434, 435-460 and 380-413, respectively. These findings suggest that these epitopes for mAb-A, -B1 and -B2 may be close to the isoalloxazine moiety of FAD, which plays a central role in the catalysis of the enzyme.

Agaricus↗