Dominique Simon

Systematic Exploration of the Antigen Binding Activity of Synthetic Peptides Isolated from the Variable Regions of Immunoglobulins

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 × 10−7 to 6.7 × 10−8 m −1 for the soluble form of 9 lysozyme-binding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.

A solid-phase immobilized epitope immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma

We have developed a new enzyme immunometric assay for angiotensin II (AII) based on SPIE-IA technology (solid-phase immobilized epitope-immunoassay). A monoclonal antibody with optimal properties (mAb3 131) was selected from a series of 19 anti-AII mAbs. The mAb had to be purified from ascitic fluid in a specific manner in order to remove endogenous AII from the antibody-binding sites. We established a sensitive (minimum detectable concentration 0.5 pg/ml) and precise (CV below 15% in the 2-100 pg/ml range) SPIE-IA. Using different AII-related peptides, we observed that this new assay has a specificity profile that compares favourably with the corresponding competitive immunoassay. We have used the assay to measure AII in 42 plasma samples, and demonstrated a good correlation with values obtained using a commercial radioimmunoassay. Assay specificity was supported by HPLC fractionation experiments, confirming the absence of interference induced by endogenous AII-related products.

Laser treatment for corrosion prevention of electrical contact gold coating

Abstract The materials used in electrical contact applications are constituted of a copper alloy (brass or bronze) electroplated with two coatings, a nickel layer (diffusion barrier) and a gold layer (corrosion barrier). There are some pores in the nickel and gold layers leading to corrosion of the underlying layers. To modify the gold coating microstructure, a laser surface treatment has been undertaken. An excimer laser is used firstly because the photon absorption coefficient is larger in UV range and secondly because the laser beam homogeneity is available for a surface treatment. The purpose of this surface treatment is to suppress the porosity of the gold layer, which is responsible of the corrosion pits, and to smooth the surface as the roughness prevents a correct electrical contact. The effects of the laser treatment are studied according to different surface parameters (roughness of the substrate, thickness of the two successive coatings, a nickel layer and a gold layer). A numerical code is used to simulate the influence of the laser beam parameter on the surface melting. Tests of corrosion are carried out in the humid synthetic air containing low contents of pollutants (NO 2 , SO 2 and Cl 2 ). The techniques used to control these effects are optical microscopy and scanning electron microscopy (SEM).

New monoclonal antibodies directed against the propart segment of human prorenin as a tool for the exploration of prorenin conformation

Six monoclonal antibodies (MAbs) directed against human prorenin were produced by immunizing BALB/c mice with a peptide corresponding to the sequence (-17 to +9) of prorenin. The new MAbs were screened for their ability to first bind to the immobilized peptide and then to prorenin previously captured by an anti-total renin MAb. The specificity of the MAbs was confirmed by the total lack of binding to active renin. Using BIAcore technology, equilibrium affinity constants of the MAbs were determined and ranged from 3.2 x 10(8) to 5.7 x 10(9) l/mol. Immunoradiometric assays (IRMA) for prorenin were performed using the anti-total renin MAb and the anti-prorenin MAbs. The best results were obtained when an anti-prorenin MAb was immobilized and the anti-total renin MAb was used as tracer in a one-step procedure. Moreover, the signal was significantly increased by the presence of the renin inhibitor SR 43845 suggesting that the inhibitor-induced conformational change of prorenin could be detected by the MAbs.

Interaction of the octapeptide angiotensin II with a high-affinity single-chain Fv and with peptides derived from the antibody paratope

The amino-acid sequence of the very high-affinity anti-angiotensin II monoclonal antibody 4D8 was predicted from the nucleotide sequence of the heavy and light chain variable genes. The single-chain variable fragment (scFv) was constructed and expressed in Escherichia coli as a soluble protein and at the surface of the filamentous M13 phage and was compared with the full-length antibody (Ab). The scFv showed the same specificity profile and affinity constant as the intact antibody (5.0x10(10) and 8.0x10(10) M(-1), respectively, by Scatchard analysis). Several peptides from the set of overlapping dodecapeptides covering the variable domains of 4D8 mAb were found to specifically bind biotinylated angiotensin II: peptides from the L1, L2, L3 and H1 regions had the strongest capacity to bind the antigen.

Laser treatment for corrosion prevention

The laser surface treatment is applied to a multilayer component (copper alloy plated with two thin coatings, nickel and gold). The aim of the study is to melt the whole gold layer (thick < micrometers ) without damaging the underlying layers. The gold melting must be homogeneous and the process must be fast to avoid heat diffusion in the depths. For these reasons, the laser has been chosen for surface treatment. The application of this laser surface treatment is to improve the corrosion of resistance of electrical contacts due to columnar microstructure of gold deposited by electrolytic process. Tests of corrosion are carried out in the humid synthetic air containing low contents of pollutants (NO2, SO2 and Cl2). An numerical study has been realized to find the best laser conditions to melt the whole gold layer.