J. Hoebeke

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity‐purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty‐four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor‐bearing membranes. The M‐35b antibodies interacted only with native digitonin‐solubilized receptors, and not with denatured receptors. The M‐23c antibodies did not react with active digitonin‐solubilized receptors but recognized the denatured form. The M‐23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M‐35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.

The human carcinoma cell line A431 possesses large numbers of functional β-adrenergic receptors

The existence of β‐adrenergic receptors was demonstrated on whole A431 cells as well as A431 membrane preparations by means of binding assays using the hydrophobic 1‐[3H]dihydroalprenolol and the hydrophilic antagonist [3H]CGP‐12,177 as β‐adrenergic ligands. Binding was stereospecific. The receptors, as shown by competition studies, proved to be of the β2‐subtype and appeared functional in the stimulation of adenylate cyclase. The number of receptors per cell and the yield of receptor sites/mg membrane protein render the A431 cell a useful tool for the study of human β‐adrenergic receptors.

Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N‐terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C‐terminal end of the molecules. While the V8 protease produced a fragment with a similar N‐terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11–15 kDa) with hydrophobic N‐terminal sequences. Our results suggest that MP26 is characterised by an N‐terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.

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.

Recognition of Physiological Receptors by Anti-Idiotypic Antibodies: Molecular Mimicry of the Ligand or Cross-Reactivity?

The specific binding and physiological modulation of physiological membrane receptors by anti-idiotypic (anti-Id) antibodies has now been described in several systems (reviewed in Strosberg and Schreiber 1984). Two types of explanations have been given for these unexpected interactions. The anti-Id antibody either acts as an internal image of the ligand recognized by both the physiological receptor and the anti-ligand antibody (Jerne 1974, 1984) or, alternatively, there is recognition, by the anti-Id antibodies, of structural homologies shared by the Id-bearing antiligand antibodies and the receptor (Strosberg 1984).

Isolation, purification and partial analysis of the lipopolysaccharide antigenic determinant recognized by a monoclonal antibody to legionella pneumophila serogroup 1

Monoclonal antibody II-6-18 recognizes a serogroup-1-specific Legionella pneumophila antigenic determinant which has been shown to be virulence-associated. We previously reported the physicochemical characterization by means of a quantitative fluorometric assay of monoclonal antibody II-6-18 binding to L. pneumophila, and its implications concerning the nature of the antigen. We describe here the isolation and the purification of the antigen by chemical and immunological methods, followed by its partial chemical analysis. The results demonstrate that the epitope--an immunodominant carbohydrate which includes a fucosamine-like residue--is part of the cell wall lipopolysaccharide (LPS). It is localized in the polysaccharide moiety of the LPS which contains KDO, rhamnose, mannose, glucosamine and an unidentified aminodideoxyhexose X1, but no heptose. The aminodideoxyhexose X1 could be fucosamine and is probably the immunodominant residue in the epitope, localized, at least partially, at the end of the polysaccharide chain.

Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells.

Abstract The association and dissociation mechanisms of lectin membrane receptor microclustering on HeLa cells have been studied by measuring resonance energy transfer between fluoresceinated and rhodaminated lentil lectin. Compounds known to affect membrane receptor mobility, such as Ca 2+ ions, methylamine, cytochalasin D and nocodazole, did not modify the association kinetics nor the maximal energy transfer values at 4 and 37 °C. Dissociation of the membrane receptor microclusters was followed by measuring the temporal decrease in energy transfer values at 4 °C after preincubation for different time intervals at 37 °C. The rate of dissociation of the lectin receptors decreased in the presence of Ca 2+ ions (10 −3 M) and after cross-linking with anti-lectin antibodies. An increase was observed in the presence of cytochalasin D (10 −6 M) and, to a lesser extent, of methylamine (10 −2 M). When cytochalasin D and methylamine were combined at subliminal concentrations, a partial synergistic effect was observed. Nocodazole (10 −6 M) had no effect. The results suggest that the association of lectin membrane receptors in microclusters is mediated only by physicochemical parameters. Ca 2+ ions, cytochalasin D (microfilaments) and methylamine (transglutaminase)-sensitive components appear, however, to play an important role in the stabilization of the receptor microclusters.

Altered binding properties of β-adrenergic receptors and lack of coupling to adenylate cyclase in P815 mastocytoma cells

P815, a murine mastocytoma cell line, possesses beta-adrenergic binding sites as assessed by using [3H]dihydroalprenolol (antagonist) and [3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by 1-isoproterenol. The intracellular cAMP level of intact cells was not modulated by 1-isoproterenol or by 1-epinephrine, but was increased by forskolin. These results suggest that the beta-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.

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.

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.