Biomedical subjects

D Altschuh

Publications and source records attributed to D Altschuh.

At least 37 records · Page 2Linked to original sources

PubMed1992

Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance.

Differences in the affinity of a monoclonal antibody raised against the protein of tobacco mosaic virus for 15 related peptides (residues 134-146) carrying single-residue modifications were investigated using a novel biosensor technology (Pharmacia BIAcore). Analysis of the peptide-antibody interaction in real time allowed fast and reproducible measurements of both association and dissociation rate constants. Out of 15 mutant peptides analyzed, five were not recognized by the antibody at all, and seven were recognized as well as the wild-type peptide. For three of the peptides, the rate constants were different for the mutant and wild-type peptides. The pattern of residue recognition suggests that the epitope is formed by three residues (140, 143, and 144) in a helical conformation that mimics the structure in the protein. Even a minor modification of these residues totally abolishes recognition by the antibody. Modifications of adjacent residues result in small but significant differences in association and/or dissociation rate constants. One of the recognized residues is totally buried in the three-dimensional structure of TMV protein, suggesting that a structural rearrangement next to the helix occurs during protein-antibody interaction.

Amino Acid Sequence↗

PubMed1992

Mapping of viral epitopes with conformationally specific monoclonal antibodies using biosensor technology.

An automated biosensor system (BIAcore) designed for measuring molecular interactions in real time and without labelling any of the reactants was used for mapping the epitopes of tobacco mosaic virus protein using conformationally specific monoclonal antibodies (MAbs). Some of the MAbs used as capturing antibody on the sensor chip allowed a conformational change to occur in the viral protein. As a result, MAbs specific for the quaternary structure of polymerized viral protein were able to bind to monomeric viral subunits. Compared with classical solid-phase enzyme immunoassay, the biosensor technology possesses several advantages for epitope mapping of viral proteins.

Antibodies, Monoclonal↗

PubMed1992

A conformation of cyclosporin A in aqueous environment revealed by the X-ray structure of a cyclosporin-Fab complex.

The conformation of the immunosuppressive drug cyclosporin A (CsA) in a complex with a Fab molecule has been established by crystallographic analysis to 2.65 angstrom resolution. This conformation of CsA is similar to that recently observed in the complex with the rotamase cyclophilin, its binding protein in vivo, and totally different from its conformation in an isolated form as determined from x-ray and nuclear magnetic resonance analysis. Because the surfaces of CsA interacting with cyclophilin or with the Fab are not identical, these results suggest that the conformation of CsA observed in the bound form preexists in aqueous solution and is not produced by interaction with the proteins.

Amino Acid Isomerases↗

PubMed1992

Correlation of co-ordinated amino acid changes at the two-domain interface of cysteine proteases with protein stability.

The engineering of a protein containing an alternative local residue packing for a set of side-chains has proven to be a major challenge because compositional, volumetric and steric constraints must be respected. Homologous proteins should provide examples of alternative groups of residues leading to a similar functional result. The functional significance of a pair of co-ordinated changes that are observed in the cysteine proteases family has been investigated by comparing the effect of individual or double changes on secretion, stability and activity of papain. The two changes are not independent. Detrimental effects of single mutations at one of the two positions can be partly suppressed by the co-ordinated mutation that reproduces naturally occurring contacts, indicating that these changes are concerted. Single mutations at the other position produce milder effects, suggesting a pathway for evolution.

Amino Acids↗

PubMed1992

Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.

Antibodies, Monoclonal↗

PubMed1989

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin.

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.

Antibodies, Monoclonal↗

PubMed1988

Visualization by electron microscopy of the location of tobacco mosaic virus epitopes reacting with monoclonal antibodies in enzyme immunoassay.

The binding of monoclonal antibodies obtained after immunization with tobacco mosaic protein was analyzed by electron microscopy. A method was developed for visualizing the viral antigen reacting in different ELISA procedures. It was found that the use of a pH 9.6 buffer during the coating of ELISA plates led to the dissociation of virions into subunits which bound preferentially to the solid phase. MAbs that reacted with both virions and subunits in ELISA were found to bind to one of the two extremities of viral rods. These MAbs also reacted with viral protein aggregated in the form of disks. In contrast, MAbs reacting only with virions in ELISA were found to bind over the entire surface of the virus.

Antibodies, Monoclonal↗

PubMed1988

Coordinated amino acid changes in homologous protein families.

In the tobamovirus coat protein family, amino acid residues at some spatially close positions are found to be substituted in a coordinated manner [Altschuh et al. (1987) J. Mol. Biol., 193, 693]. Therefore, these positions show an identical pattern of amino acid substitutions when amino acid sequences of these homologous proteins are aligned. Based on this principle, coordinated substitutions have been searched for in three additional protein families: serine proteases, cysteine proteases and the haemoglobins. Coordinated changes have been found in all three protein families mostly within structurally constrained regions. This method works with a varying degree of success depending on the function of the proteins, the range of sequence similarities and the number of sequences considered. By relaxing the criteria for residue selection, the method was adapted to cover a broader range of protein families and to study regions of the proteins having weaker structural constraints. The information derived by these methods provides a general guide for engineering of a large variety of proteins to analyse structure-function relationships.

Amino Acid Sequence↗

PubMed1987

Correlation of co-ordinated amino acid substitutions with function in viruses related to tobacco mosaic virus.

Sequence data are available for the coat proteins of seven tobamoviruses, with homologies ranging from at least 26% to 82%, and atomic co-ordinates are known for tobacco mosaic virus (TMV) vulgare. A significant spatial relationship has been found between groups of residues with identical amino acid substitution patterns. This strongly suggest that their location is linked to a particular function, at least in viruses identical with the wild-type for these residues. The most conserved feature of TMV is the RNA binding region. Core residues are conserved in all viruses or show mutations complementary in volume. The specificity of inter-subunit contacts is achieved in different ways in the three more distantly related viruses.

Amino Acid Sequence↗

PubMed1987

Immunochemical studies of tobacco mosaic virus--VII. Use of comparative surface accessibility of residues in antigenically related viruses for delineating epitopes recognized by monoclonal antibodies.

The binding properties of 18 monoclonal antibodies (MAbs) directed against the tobacco mosaic virus (TMV) coat protein were studied with five related tobamoviruses and seven mutant viruses, as well as with the dissociated coat proteins of these variants. Ten of the antibodies bound to both TMV and TMV protein, but these were able to discriminate between different mutants only when whole virus particles were compared in the immunoassay. Three MAbs reacted with TMV but not with dissociated viral subunits and these recognized the same residue exchanges. Five MAbs recognized synthetic peptides of 13-28 residues corresponding to parts of the protein. By comparing the common accessible surface between TMV and antigenically related viruses, it was possible to narrow down the region recognized by some of these MAbs. The linear peptide sequence recognized by these antibodies did not represent the entire epitope and residue exchanges around this region were able to affect the binding of MAbs.

Amino Acid Sequence↗

PubMed1986

Influence of local structure on the location of antigenic determinants in tobacco mosaic virus protein.

Early work on protein antigenicity led to the common belief that proteins possess a finite number of antigenic determinants located in accessible regions of the molecule's surface. This view is now changing. As a result of extensive studies, seven continuous epitopes have been located on tobacco mosaic virus (TMV) protein by measuring the antigenic activity of short peptides with anti-protein antibodies. The structure of the viral protein has been refined in Cambridge, enabling the Strasbourg workers to correlate the position of these epitopes with regions of high segmental mobility in the protein. Surface accessibility is not a sufficient condition for antigenicity, because six short peptides corresponding to accessible regions of the protein possess no antigenic activity. Recent work in Strasbourg shows that when longer peptides of TMV protein are used, more antigenic determinants are found and these lie in more structured regions of the protein (e.g. helices). In these cases the longer peptide may be folding up in solution to mimic part of the native structure.

Antigens, Viral↗

PubMed1985

Immunochemical studies of tobacco mosaic virus--VI. Attempts to localize viral epitopes with monoclonal antibodies.

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV. By means of computer-generated images of the surface residues of the viral subunit, it was possible to identify certain clusters of residues involved in binding to some of the monoclonal antibodies. The results clearly illustrate the operational limitations encountered when monoclonal antibodies are used for elucidating the antigenic structure of proteins.

Amino Acid Sequence↗

PubMed1984

Determination of IgG and IgM levels in serum by rocket immunoelectrophoresis using yolk antibodies from immunized chickens.

A method of rocket immunoelectrophoresis based on the use of avian antiglobulin antibodies is described which permits the quantitation of IgG and IgM in human serum. The electrophoretic mobility of avian yolk immunoglobulin (IgY) and mammalian IgG is sufficiently different to obviate the need to chemically modify the antibody, for example by carbamylation. Rockets are obtained in agarose within 3-6 h in 2-(N-morpholino) ethane sulfonic acid buffer at pH 5.5 or 6.0. Avian globulins specific for human IgG and IgM were obtained easily and in large quantities from the egg yolk of hyperimmunized laying hens. The technique is also applicable to quantitation of immunoglobulins in various animal sera.

Animals↗

PubMed1984

Comparative sensitivity of different ELISA methods for detecting monoclonal antibodies to viruses.

Five different ELISA procedures using alkaline phosphatase conjugates were compared for their sensitivity in detecting binding activity in nine monoclonal antibodies raised against tobacco mosaic virus (TMV). The two procedures in which antigen was used to coat the microtiter plates were found to be unreliable for testing the activity of monoclonal antibodies. The two most sensitive procedures utilized a multilayered sandwich of which one component imperatively had to be an avian antibody devoid of serological cross-reactivity with mammalian globulins. The avian antibody was obtained from the egg yolk of immunized chickens. In the two most sensitive assays, four out of nine monoclonal antibodies raised against TMV were also reactive with the dissociated viral protein subunits.

Antibodies, Monoclonal↗

PubMed1983

Immunochemical studies of tobacco mosaic virus--V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains.

Two antigenic determinants have been located in the vicinity of residues 1-10 and 34-39 of the coat protein of tobacco mosaic virus by means of ELISA and complement fixation inhibition experiments with synthetic peptides. The determinant present in residues 1-10 is also expressed at the outer surface of the subunits when they are assembled into virions. Two other determinants expressed in the dissociated protein subunit have been located in the vicinity of residues 55-61 and 80-90 by means of inhibition tests with tryptic peptides. These results bring to seven the number of antigenic determinants of the protein subunit that have been located.

Amino Acid Sequence↗

PubMed1982

Localization of antigenic determinants of a viral protein by inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides.

A new method is described for localizing antigenic determinants in globular proteins by means of inhibition of enzyme-linked immunosorbent assay (ELISA) with tryptic peptides. The inhibitory activity of 10 tryptic peptides of the coat protein of 2 strains of tobacco mosaic virus (TMV) was demonstrated by inhibition of ELISA and verified by inhibition of complement fixation tests. These results bring to 7 the number of antigenic determinants of TMV protein that have been defined.

Amino Acid Sequence↗

PubMedDate not supplied

Comparative interaction kinetics of two recombinant Fabs and of the corresponding antibodies directed to the coat protein of tobacco mosaic virus.

Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134-146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli. They differ by 15 amino acid changes in the sequence of their variable region. The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcoreTM biosensor instrument. The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P. The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P. The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs. The location of amino acid changes was visualized on a model structure of the Fab. Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains. Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes.

Amino Acid Sequence↗

PubMedDate not supplied

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context.

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.

Amino Acid Sequence↗