Jean-Gérard Guillet

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-β-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.

Localisation of three epitopes of the ENV protein of feline immunodeficiency virus

The envelope protein of the feline immunodeficiency virus (FIV) was analyzed using several epitope prediction programs based on profiles of hydrophilicity, antigenicity, and probability of residues to lie on the protein surface. Tentative homologies with the immunodominant epitope sites in simian virus (SIV) or human immunodeficiency virus (HIV) such as the V3 loop, the site of cleavage between surface envelope protein (SU) and transmembrane envelope protein (TM), and sites of N-glycosylation were thus identified. Five peptides corresponding to potential epitopes were synthesized. Four out of five peptides (P99, P100, P101, P103) were from the FIV surface envelope protein (SU). The last one (P102) was from the FIV transmembrane envelope protein TM. Three of these peptides (P99, P100, and P102) were recognized in ELISA by almost all the sera from infected cats. The peptide from TM (102) was recognized by sera from both naturally infected and inoculated cats, whereas peptides P99 and P100 (from SU) were recognized mainly by sera from naturally infected cats. On the basis of these results we propose that peptides P99, and P100 from SU and P102 from TM constitute epitopes on the FIV env protein.

Induction of a pharmacologically active clonotypic B cell response directed to an immunogenic region of the human β2‐adrenergic receptor

It has been reported that autoantibodies against the β2‐adrenergic receptors are involved in the pathology of allergic disorders and of Chagas disease. Therefore, the immune response against a peptide (H26Q) corresponding to the putative second extracellular loop of the human β2‐adrenergic receptor, which could be a target for autoantibody attack, was analysed in view of its possible immunogenicity. The free peptide induced a T cell‐mediated humoral response in the context of three different murine MHC haplotypes. The T cell epitope was found to be localized in the N‐terminal region of the peptide. Highly specific T helper cells were capable of stimulating B cells with the potential to generate a large antibody repertoire reactive with the loop peptide. MoAbs were screened to analyse this B cell response for antibodies potentially interfering with receptor function and a MoAb was found that impaired ligand binding to the receptor.

Physicochemical studies on the antigen-antibody complexes of two monoclonal antibodies against rabbit thymocytes

We have characterized two monoclonal antibodies directed against rabbit thymocyte antigens. Using internally labelled MD3 cells (a transformed T-cell line), ART-F immunoprecipitated a membrane glycoprotein of 95,000 mol. wt, while ART-A immunoprecipitated two major glycoproteins, one of 95,000 mol. wt, the other of 105,000 mol. wt as analysed on reduced SDS-PAGE. ART-A recognizes 650,000 sites both on rabbit thymocytes and MD3 cells with an association constant of 1.5 X 10(5)/M. ART-F shows a biphasic binding curve with a high affinity constant of 3.3 and 0.6 X 10(8)/M and a low affinity constant of 2.2 and 6.9 X 10(5)/M for thymocytes and MD3 cells respectively. The numbers of high affinity and low affinity antigens are 200,000 for the former and 800,000 for the latter. On the basis of the kinetic data, the difference between low and high affinity is attributed to the difference in association-rate constants. The affinity constants calculated from the reaction-rate constants are in good agreement with those obtained from equilibrium measurements. While ART-F prevents the binding of ART-A, ART-A does not inhibit the affinity binding of ART-F. The analysis of the equilibrium, inhibition and kinetic data allow us to present a model for the structural relation of the epitopes recognized on the T-cells by the two monoclonal antibodies.

In situ detection of antigen-specific tumor-infiltrating lymphocytes using newly designed tetramers

We described a new process for the design of HLA tetramers using soluble MHC class I molecules purified from Epstein Barr Virus-transformed B cells. This method does not rely on genetic engineering and presents a significant advantage in view of the polymorphism of MHC class I molecules because tetramers can be produced with any HLA molecule. Here, we showed that our HLA-A*0201 tetramers provided experimental results similar to those obtained with tetramers made with recombinant MHC molecules. Moreover, they can be used to efficiently identify peptide-specific T cells from ex vivo PBMCs as well as from lymphocytes infiltrating human tumor. This innovative and simple method could be widely adopted, specially in diagnostic procedures for monitoring peptide-based immunotherapy.

Partial characterization of a Legionella pneumophila serogroup 1 immunodominant antigenic determinant recognized by a monoclonal antibody Legionella specific antigenic determinant

A monoclonal antibody was used to characterize a serogroup 1 specific Legionella pneumophila Philadelphia strain 1 antigenic determinant. A quantitative fluorometric assay was developed to quantitate the antibody sites (2.7 +/- 0.4 X 10(5)) on Legionella bacteria and to determine the physico-chemical parameters of the antibody-antigen interaction (at 4 degrees C: delta G = -10.9 Kcal X mol-1, delta H = 1.7 Kcal X mol-1, delta S = 45 cal X K-1 X mol-1). The same method was used to study the modification or the removal of the antigen by chemical and enzymatic means (trypsin, papain, lysozyme, acetone, chloroform-methanol and Tris-EDTA); only Tris-EDTA extraction resulted in a significant decrease in antibody binding sites. Inhibition studies of the fluorescein-labelled antibody binding were performed with different sugars of which only L-fucosylamine was inhibitory, and with other monoclonal antibodies to Legionella pneumophila serogroup 1 in order to compare their fine specificity and affinity. The results indicate that the epitope recognized was an immunodominant carbohydrate including an aminodideoxyhexose and carried by the lipopolysaccharide.

Idiotypy of catecholamine-binding proteins

The idiotypic and antiidiotypic response to alprenolol, a beta-adrenergic antagonist, was studied both in rabbits and in mice. Rabbit polyclonal anti-alprenolol antibodies showed binding properties for catecholamine analogs, agonists as well as antagonists, similar to those of the beta-adrenergic receptors. The variability of the anti-alprenolol response was studied by using mouse monoclonal antibodies specific for alprenolol. While the binding properties showed great variations in affinity, the response seemed restricted to the heavy chain classes gamma 1 and gamma 2a. N-terminal sequencing of the light and heavy chains and restriction maps of the corresponding genes suggest that the antibodies use particular subgroups infrequently found in antibodies specific for other antigens. The cyclical antiidiotypic response in rabbits immunized with polyclonal antibodies and in mice immunized with monoclonal antibodies were compared. The response of the latter was dependent on the choice of the monoclonal antibody used to elicit the antiidiotypic response. Finally, the agonist-like properties of a monoclonal antiidiotypic antibody directed against one of the monoclonal anti-alprenolol antibodies were studied extensively. The ability to recognize beta-adrenergic receptors was documented by Western blot and direct immunoprecipitation and visualized by immunofluorescence. The antiidiotypic antibody stimulated catecholamine-sensitive adenylate cyclase and this effect was blocked by the beta-adrenergic antagonist propranolol.

Binding Characteristics of a Monoclonal Antibody Against Rabbit Thymocytes

Abstract A monoclonal antibody raised against rabbit thymocytes was labelled with fluorescein-isothiocyanate (FITC) and its binding characteristics on rabbit thymocytes and a transformed rabbit T lymphocyte line (MD3) were determined by quantitative fluorometry. Scatchard analysis of the binding isotherm was interpreted by the existence of a single class of receptor sites (600 000 per cell) binding to the antibody with a K A of 10 5 M -1 . Inhibition studies proved the specificity of the binding and the integrity of the labelled antibody. They showed also the cross-reaction of the antigenic determinant with another monoclonal antibody directed against rabbit thymocytes. Our results stress the uselfulness of quantitative fluorometry in the study of membrane antigens and reveal constant features in raising monoclonal antibodies against membrane antigens by immunization against whole cells.

Anti-Idiotype Antibodies for the Study of Membrane Receptors: The Double Monoclonal Antibody Approach

Rabbit polyclonal anti-idiotypic antibodies (anti-Id Ab’s) raised against anti-catecholamine Ab’s bind to β-adrenergic receptors and modulate the hormone-sensitive adenylate cyclase. To gain more insight into the interaction between anti-idiotypes and receptors, the unlimited availability of purified components such as monoclonal antibodies (MAb’s) would be of considerable advantage.