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PubMed1992

An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53.

Somatic mutation of the p53 gene is a very frequent event in the development of human neoplasia, and germ line mutations in p53 are responsible for an inherited cancer susceptibility syndrome. Many of the mutations in p53 found in human tumours are point mutations that result in the substitution of a single amino acid in the protein. These point mutant proteins are much more stable than the normal protein and the mutant product accumulates to a high level which permits important information about p53 expression to be obtained by immunochemical analysis. Using bacterial expression systems to produce fragments of human p53 we have isolated and characterized new monoclonal antibodies to p53. These antibodies are suitable for the measurement of p53 in ELISA, immunoblotting and immunoprecipitation analyses. They are especially useful in immunohistochemistry as they are able to react strongly with p53 in conventionally fixed and processed histological sections.

Antibodies, Monoclonal↗
PubMed1990

Epitope mapping of apamin by means of monoclonal antibodies raised against free or carrier-coupled peptide.

A panel of 20 monoclonal antibodies raised against the bee-venom peptide apamin (18 residues, 2 disulfide bridges) was prepared. Nine monoclonal antibodies (mAb) were obtained from a mouse immunized with free apamin and 11 from a mouse immunized with a mixture of free and carrier-coupled peptide. Using a panel of 11 synthetic apamin analogs, we examined the fine antigenic specificity of each antibody. The mAb generated against free apamin preferentially bound to the central part of the peptide and less frequently recognized the N- and C-terminal regions. However, monoclonal antibodies obtained by immunization with carrier-bound apamin showed a broader range of specificities, consistent with the possibility of the entire surface of this small antigen becoming immunogenic upon coupling to the carrier.

Amino Acid Sequence↗
PubMed1986

Epitope mapping of the outer structural protein VP1 of three different serotypes of foot-and-mouth disease virus.

All overlapping hexapeptides of the outer structural protein VP1 of type O1, type A10, and type C1 were reacted with the appropriate anti-virus, anti-viral subunit and anti-VP1 sera. The results suggest that anti-virus sera may contain activities against viral subunit and VP1 as well as against virus. Furthermore the antigenic peptides associated with the intact virion of all three serotypes are found at similar locations on their respective VP1s, and produced neutralizing activities when used for vaccination. The results further offer an explanation for the often observed cross-reactions between serotypes, especially at the level of the viral subunit and VP1. The reliability of predictions of useful peptides from hydrophilicity profiles and secondary structure predictions is questioned. Predictions based on variation profiles appear to be more useful.

Amino Acids↗
PubMed1988

Epitope mapping of alpha-transforming growth factor: evidence of an immunodominant region.

Antisera were produced in rabbits and sheep against both full-length synthetic rat alpha-transforming growth factor and peptides corresponding to the carboxy-terminal 17 amino acids. These antisera were used to develop a peptide based radioimmunoassay of alpha-TGF. All antisera reacted only with a restricted region of the alpha-TGF corresponding to the 8 residues (43-50) at the carboxy-terminus: (formula; see text) A series of synthetic peptides representing deletions or substitutions of amino acids in this carboxy-terminal region were tested for competition with 125I-alpha-TGF. All changes in the above peptide sequence resulted in a marked reduction in competition. All of the polyclonal antisera demonstrated similar specificity whether they were produced against the 50 amino acid, full-length alpha-TGF, against shorter 17 amino acid and 8 amino acid carboxy-terminal sequences.

Amino Acid Sequence↗
PubMed1993

Epitope mapping of the major inner capsid protein of group A rotavirus using peptide synthesis.

Three hundred and ninety-one consecutive heptapeptides derived from the VP6 protein of bovine rotavirus (397 AA) were synthesized using the "pepscan" method and were assayed on the synthesis pins with monoclonal antibodies to VP6. Heptapeptides reactive with MAbs were located in four main regions: regions AA 32-64, AA 155-167, AA 208-274, and a fourth region at the C-terminal, from AA 380 to AA 397. Among these regions, two sequences were also reactive with the MAbs when longer peptides were assayed. The sequence located between AA 58 and AA 62 (NWNFD), recognized by MAbs RV-1026, RV-50, and RV-443, was previously reported. A new site was defined in the region essential for trimerization, between AA 159 and AA 165 (PYSASFT), which was recognized by MAbs RV-133 and RV-138.

Amino Acid Sequence↗
PubMed1987

Epitope mapping of the von Willebrand factor subunit distinguishes fragments present in normal and type IIA von Willebrand disease from those generated by plasmin.

A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF. In contrast, plasmin cleaved a 176 kD fragment from the amino terminus and a 145 kD fragment from the carboxy terminus of the subunit. Species similar to these plasmin-cleaved fragments were demonstrated in plasmas from four patients treated with fibrinolytic agents, but not in IIA vWD.

Antibodies, Monoclonal↗
PubMed1991

Epitope mapping of the alpha-toxin of Clostridium perfringens.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.

Amino Acid Sequence↗
PubMed1994

Characterization of FcR Ig-binding sites and epitope mapping.

The low-affinity receptor for IgG, Fc gamma RII, and the high-affinity receptor for IgE, Fc epsilon RI, are functionally distinct but structurally homologous receptors. These characteristics have been exploited using a chimeric receptor strategy to examine segments of human Fc gamma RII for IgG-binding function. A series of chimeric receptors was generated by exchanging coding regions of the extracellular ligand-binding regions between Fc gamma RII and the Fc epsilon RI alpha chain using splice overlap extension by the polymerase chain reaction. The expression of these chimeric receptors in COS-7 cells and analysis of their IgG/IgE binding capacities have enabled the Ig-binding region of Fc gamma RII to be localized to a subregion of the second extracellular domain. The localization of the Ig-binding region of Fc gamma RII has provided the opportunity of performing site-directed mutagenesis to determine the key amino acids involved in the interaction of the receptor with IgG. These findings demonstrate that the chimeric receptor approach is a powerful technique for the dissection of structure/function relationships of structurally related yet functionally different molecules.

Amino Acid Sequence↗
PubMed1991

Fine specificity of the alloantiserum MSD-51: epitope mapping of HLA-DRw53 determinants.

HLA-DRw53 is a supertypic specificity expressed by HLA-DR4-, HLA-DR7-, and HLA-DR9-positive cells. In the present study, the fine specificity of an HLA-DRw53-specific alloantiserum (MSD-51) was analyzed in serology and by the isoelectric focusing technique. In serology, MSD-51 recognized HLA-DRw53-positive cells with the exception of cells expressing the HLA-DR7/DRw53/DQw9 haplotype. The immunoprecipitation studies and the use of the IgM-reducing agent dithiothreitol revealed that MSD-51 consisted of at least two antibodies: (1) an IgM antibody which reacted with the HLA-DRB4 gene product of HLA-DRw53-positive cells, except HLA-DR7/DRw53/DQw9-expressing cells, and (2) at least one IgG antibody which recognized a linear sequence or conformational structure formed by positions 67 to 70 on the HLA-DRB1 gene product of HLA-DR4- and HLA-DR9-positive cells. These findings demonstrate the complexity of the supertypic HLA-DRw53 antigen analyzed with a serologically well-defined HLA-DRw53-specific alloantiserum.

Amino Acid Sequence↗
PubMed1986

Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry.

Human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome, is cytopathic for CD4+ T cells and binds to these cells via a complex of the 110,000 m.w. viral-envelope glycoprotein, gp110, and the CD4 molecule. We treated virus with several physical, chemical, and enzymic agents to determine their effect on the capacity of HIV to bind to the CD4+ T cell line, CEM. Reduction and alkylation (but not alkylation alone) and trypsin digestion (but not glycolytic enzyme digestions) of HIV destroyed its capacity to bind. If the tertiary protein structure conferred by disulfide bonding is not disrupted, the tertiary and secondary conformations dependent on noncovalent forces appear to be thermodynamically favored, because treatment with denaturants such as sodium dodecyl sulfate, 8 M urea, alcohol, or heat (56 degrees C or 65 degrees C for 30 min) followed by removal of the denaturants did not affect binding. Irreversible denaturation and loss of binding occurred after heating at 100 degrees C for 10 min. HIV binding to CD4+ T cells was inhibited either by murine monoclonal antibodies to the CD4 molecule or by human polyclonal or murine monoclonal antibodies to the gp110 molecule. On the basis of results of binding inhibition obtained with a panel of alpha-CD4 monoclonal antibodies, the receptor site for virus on the CD4 molecule was mapped to the amino-terminal portion of the molecule. Four candidate alpha-CD4 monoclonal antibodies that were potent inhibitors of virus binding (OKT4A, OKT4D, OKT4F, and Leu-3a) were examined for the possibility that their binding sites (idiotopes) might share structural and conformational similarity with the CD4-binding site on gp110. Polyclonal human or rabbit anti-HIV sera (that reacted with gp110 and inhibited virus binding) did not react with or inhibit the binding of these four alpha-CD4 monoclonal antibodies. Conversely, rabbit anti-idiotypic sera raised against each of the four candidate CD4 monoclonal antibodies did not react with virus or inhibit virus binding to CD4+ T cells. Further search or different approaches may yet yield an idiotype that is a structural and conformational "internal image" of the CD4-binding site of virus.

Antibodies, Monoclonal↗
PubMed1989

High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis.

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.

Alanine↗
PubMed1991

Epitope mapping employing immobilized synthetic peptides. How specific is the reactivity of these peptides with antiserum raised against the parent protein?

Peptides synthesized onto polyethylene pins can be directly used to map the antigenic structure of proteins by ELISA (Geysen et al., 1987, J. Immunol. Methods 102, 259-274). The specificity of the reaction between anti-cytochrome c antibodies (IgG) and pin-bound hexapeptides of cytochrome c was tested using a competitive ELISA in which free and pin-bound peptides competed for the antibody. Competition was specific for many of the pin-bound peptides: only free peptides having the same sequence as the pin-bound peptides were able to compete for antibody-binding. However, non-specific reactivity was observed with pin-bound peptides whose sequence corresponded to the N terminal segment of cytochrome c. This segment is predicted to be particularly antigenic because of its high mobility and the nature of its amino acid sequence. In one case no competition by the free peptide could be observed even though the pin-bound peptide reacted strongly with anti-cytochrome c antibodies.

Animals↗
PubMed1985

Variable major proteins of Borrelia hermsii. Epitope mapping and partial sequence analysis of CNBr peptides.

The variable major proteins (VMP) of serotypes 7 and 21 of the relapsing fever agent Borrelia hermsii were isolated by detergent extraction and high performance liquid chromatography. Cyanogen bromide (CNBr) digestion of the isolated VMP yielded two peptides of apparent molecular weights 20,000 (20 K) and 16 K from VMP7, and three peptides of 14.5, 14, and 7 K mol wt from VMP21. Serotype-specific monoclonal antibodies bound in Western blots to one of each of the two or three CNBr fragments from the homologous VMP. A single monoclonal antibody bound to the whole cells, the isolated VMP, and a CNBr fragment of both serotype 7 and serotype 21. (This crossreactive antibody did not, however, bind to any of four other serotypes examined.) Regional conservation of structure between VMP7 and VMP21 was also shown by amino acid sequence analysis of the N-termini of the five CNBr fragments. One pair of aligned fragments from VMP7 and VMP21 had 80% amino acid homology in sequence; a second pair had 40% homology. The partial amino acid homologies between two VMP suggest that these proteins are products of members of a polygene family.

Amino Acid Sequence↗
PubMed1989

Epitope mapping of an HLA-DR-specific monoclonal antibody produced by using human-mouse transfectant cells.

A polymorphic HLA-DR mAb, TAL15.1, was produced against L cells transfected with DR alpha- and beta-chain cDNA from a cell line homozygous for HLA-DRw8. This antibody reacted with DRw8 plus all other DR types except DR3 and DRw52. DR3 and DRw52 differ uniquely from other other DR antigens at position 77 in the beta 1-domain of their beta-chains where there is asparagine instead of threonine. In Western blots the antibody reacted with DR alpha/beta-dimer but not with free alpha- or beta-chains. Two-dimensional gel analysis of a DRw11, DRw52 cell line showed that TAL15.1 immunoprecipitated the DR products. Although it also coprecipitated the DQ beta chain products, flow microfluorimetric analysis with various transfectant cell lines showed that TAL15.1 failed to bind the DQ or DP products tested. We conclude that TAL15.1 is a DR-specific polymorphic antibody whose activity correlates with a specific residue. It has already proved to be a valuable reagent for distinguishing DR3 homozygotes from DR3, DRw6 heterozygotes, which have in the past been difficult to separate.

Amino Acid Sequence↗
PubMed1991

Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane.

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.

Amino Acid Sequence↗
PubMed1994

Epitope mapping of six monoclonal antibodies recognizing the Shigella dysenteriae type 1 O-antigenic repeating unit expressed in Escherichia coli K-12.

Mouse and rat monoclonal antibodies were generated against lipopolysaccharides expressed in Escherichia coli K-12/Shigella dysenteriae type 1 hybrids and native Shigella dysenteriae type 1 bacteria. Six monoclonal antibodies, all IgM and reacting with the E. coli K-12/Shigella dysenteriae type 1 hybrids, were selected and characterized. The specificities were studied in inhibition experiments using purified native and synthetic saccharides, representing different parts of the S. dysenteriae type 1 tetrasaccharide repeating unit. Three patterns of specificities were seen: (i) MAEC-SD-1 and MAEC-SD-2 recognizing the terminal alpha-D-Galp-(1-->3)-beta-D-GlcpNAc disaccharide as present in an E. coli K-12 class I hybrid strain, (ii) MAEC-SD-11; a class IV specific mAb, recognizing the alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->tris accharide, and (iii) MAEC-SD-21, MASD-1, MASD-2; all mAbs with class V specificity, recognizing the alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1--> trisaccharide. Furthermore, the data strongly suggest that the native repeating unit for S. dysenteriae type 1 is the alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1--> 3)-alpha-D-GlcpNAc tetrasaccharide.

Antibodies, Bacterial↗
PubMed1993

Epitope mapping of the site(s) of binding of Fc epsilon RII/CD23 within human IgE. Determination of the B lymphocyte-binding sites by use of synthetic peptides and anti-peptide antibodies representative of linear Fc sequences.

The work undertaken has investigated the structure-function relationship between IgE and its low affinity receptor on B lymphocytes. To identify sites on the IgE molecule which interact with the low affinity receptor (Fc epsilon RII/CD23), 10 different peptide sequences within the CH2, CH3 and CH4 domains of human IgE were selected according to charge, overall hydrophobicity and possible accessibility on native IgE sequences. Peptides representative of these were synthesized by the solid phase procedure; and their cytophilic activities were examined by determining their capacity to inhibit the binding of radiolabelled or erythrocyte-bound IgE to a Fc epsilon RII/CD23 positive B cell line (RPMI-8866). Moreover, these linear sequences were rendered immunogenic by conjugation to a protein carrier (KLH) and used to produced polyclonal antibodies in rabbits. The reactivity of the anti-peptide antibodies with both free peptides and native IgE bound to a solid phase, as well as their capacity to inhibit binding of IgE to a Fc epsilon RII/CD23 positive cell line, were investigated. Results from such use of peptides and anti-peptide antibodies indicate that two sequences, representative of residues 364-383 and 401-415, could be involved in the binding of IgE to both membrane-bound and soluble form Fc epsilon RII/CD23; indicating that the B lymphocyte-binding site on human IgE may be restricted to the CH3 domain.

Animals↗
PubMed1994

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.

Amino Acid Sequence↗