Biomedical subjects

B Friguet

Publications and source records attributed to B Friguet.

43 records · Page 3Linked to original sources

PubMed1986

Conformational changes induced by domain assembly within the beta 2 subunit of Escherichia coli tryptophan synthase analysed with monoclonal antibodies.

The effects of domain assembly on the conformation of the F1 (N-terminal) and F2 (C-terminal) domains of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were analysed using six monoclonal antibodies which recognize six different epitopes of the native beta 2 subunit (five carried by the F1 domain and one carried by the F2 domain). For this purpose, the affinity constant of each monoclonal antibody for the isolated domains F1 or F2, the associated domains in the trypsin-nicked apo-beta 2 and in the native apo-beta 2 subunits were determined, both with the intact immunoglobulin and the Fab fragment. It was found that the association of the F1 and F2 domains within beta 2 is accompanied by structural changes of the two domains, as detected by variations of their affinity constants for the monoclonal antibodies.

Antibodies, Monoclonal↗

PubMed1986

Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies.

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.

Antibodies, Monoclonal↗

PubMed1985

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.

Antibodies, Monoclonal↗

PubMed1984

Structural and functional influence of enzyme-antibody interactions: effects of eight different monoclonal antibodies on the enzymatic activity of Escherichia coli tryptophan synthase.

Twelve monoclonal antibodies directed against the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) were produced from hybridoma clones. These monoclonal antibodies are found to recognize at least eight different epitopes on beta 2, and eight classes of monoclonal antibodies are thus defined. The effects of these monoclonal antibodies on the enzymatic activities of beta 2 are studied. The monoclonal antibodies from three classes rapidly inhibit the serine deaminase activity catalyzed by the beta 2 subunit alone; two of them lead to an inhibition plateau under stoichiometric conditions, and their inhibitory effects are cumulative. With the antibodies from two of these three classes, the tryptophan synthase activity of the alpha 2 beta 2 complex is recovered, through a competition between the alpha subunit and the monoclonal antibody. On the contrary, the antibody from the third class is inhibitory even in the presence of an excess of alpha subunit. The antibodies from the five other classes, though binding easily to the coated antigen in the enzyme-linked immunosorbent assay, react only very slowly with beta 2 in solution and, only after a long time of incubation, inhibit the enzymatic activity at different levels.

Animals↗

PubMed1984

Some monoclonal antibodies raised with a native protein bind preferentially to the denatured antigen.

Recent studies with monoclonal antibodies directed against different epitopes of the beta 2-subunit of Escherichia coli tryptophan synthase have shown that some of these antibodies bind rapidly in solution to the native protein; others bind very slowly to native beta 2 in solution while they recognize quite rapidly this antigen adsorbed on a microtitration plate. In the present work, an enzyme-linked immunosorbent assay competition test with either the native or a denatured form of the antigen has been developed. It allowed us to show that the rapidly binding antibodies recognize epitopes present on the native protein while those which react very slowly in solution bind preferentially to the denatured form of the protein. These results prompted us to emphasize how important it is, in the characterization of antibodies, to ascertain that the antigen they recognize remains native in the specificity test.

Antibodies, Monoclonal↗

PubMed1983

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase.

Seven hybridoma clones, producing antibodies directed against the beta 2-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on beta 2, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to beta-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes. Using this test, it is shown that, of the 7 anti-beta 2 monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.

Animals↗

PubMedDate not supplied

Peptide/antibody recognition: synthetic peptides derived from the E. coli tryptophan synthase beta 2 subunit interact with high affinity with an anti-beta 2 monoclonal antibody.

Recent studies have shown that the antigenic determinant recognized by a monoclonal antibody (mAb 164-2) elicited against the beta 2 subunit of E. coli tryptophan synthase is localized between residues 276 and 297 of this protein. In order to delineate more precisely the epitope recognized by this antibody, peptides ranging in length from 11 to 29 amino acids and belonging to this region were synthesized, and their interactions with the antibody are described in this paper. The smallest peptide recognized with a high affinity by antibody 164-2 contains 11 residues (273-283). This peptide is recognized by antibody 164-2 with an affinity (KD = 7.5 x 10(-9) M) very close to that of the native beta 2 subunit, suggesting a high structural similarity of the epitope inside the protein and in the isolated peptide. The corresponding sequence of beta 2 is located in a region protruding from the protein surface that contains a beta-turn as unique element of secondary structure in the crystallographic model. The absence of interaction between antibody 164-2 and the octapeptide lacking the three residues at the C-terminal end of peptide 11 suggests that the beta-turn is important in the recognition by the antibody. Kinetic studies were performed to find out whether or not the binding of the antibody to the peptide involves any conformational adaptation. The dissociation equilibrium constant (KD), the dissociation rate constant (koff) and the association rate constant (kon) were measured for eight peptide/antibody complexes. The values obtained were compatible with a one-step reaction, suggesting that no important conformational adaptation is involved in the formation of the peptide/antibody complexes. Furthermore, it has been shown that differences in affinity of antibody 164-2 for the various peptides were mainly due to differences in the dissociation rate constants (koff) and not in the association rate constants (kon). The exceptional location of the epitope in the native protein and the unusually high affinity of the 11-residue peptide for mAb 164-2, makes this peptide a good model for studying the interaction between an antibody and a continuous epitope of a protein.

Amino Acid Sequence↗