Uses of biosensors in the study of viral antigens.

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has greatly simplified the measurement of binding interactions in biology. This new technology known as biomolecular interaction analysis makes it possible to visualize the binding process as a function of time by following the increase in refractive index that occurs when one of the interacting partners binds to its ligand immobilized on the surface of a sensor chip. None of the reactants needs to be labelled, which avoids the artefactual changes in binding properties that often result when the molecules are labelled. Biosensor instruments are well-suited for the rapid mapping of viral epitopes and for identifying which combinations of capturing and detector Mabs will give the best results in sandwich assays. Biosensor binding data are also useful for selecting peptides to be used in diagnostic solid-phase immunoassays. Very small changes in binding affinity can be measured with considerable precision which is a prerequisite for analyzing the functional effect and thermodynamic implications of limited structural changes in interacting molecules. On-rate (ka) and off-rate (kd) kinetic constants of the interaction between virus and antibody can be readily measured and the equilibrium affinity constant K can be calculated from the ratio ka/kd = K.

Kinetic and functional mapping of viral epitopes using biosensor technology.

Some monoclonal antibodies (Mabs) that react with the extremity of the tobacco mosaic virus (TMV) particle containing the 5' end of the RNA are able to block the disassembly of TMV by ribosomes while others are totally devoid of such activity. No correlation could be established between the binding kinetics and affinity of the Mabs and their inhibitory capacity. An epitope map of the Mab binding sites was constructed on the basis of kinetic two-site binding assays with the viral monomeric protein (TMVP) performed using biosensor technology (BlAcore). Mabs possessing inhibitory activity were found to bind to the part of the TMVP surface closest to the central axis in the polymerized particle. As this part of the subunit is known to interact with the viral RNA, it seems that inhibitory Mabs act by sterically preventing the interaction between virus and ribosomes. This study illustrates the advantages of the biosensor technology for locating conformational epitopes in viral proteins.

Mapping of viral conformational epitopes using biosensor measurements.

Earlier electron microscopy studies of the location of various antigenic sites in tobacco mosaic virus indicated that epitopes specific for the quaternary structure and absent in dissociated viral subunits (so-called neotopes) were present along the entire length of the virus particle. In contrast, epitopes expressed in both intact particles and dissociated subunits (so-called metatopes) were found only at the one extremity of the particle containing the 5' end of the RNA. In the present study, the binding properties of antibodies to neotopes and metatopes were studied with the BIAcore. From the results of capture assays with viral subunits and on the basis of binding stoichiometry calculations, it was possible to demonstrate the presence of neotope and metatope specificities on additional parts of the viral surface where they had not been identified before by classical immunoassays. In two site binding assays it was also found that a neotope specificity could be induced in dissociated viral subunits by the binding of a first antimetatope antibody. The results clearly demonstrated the superiority of the biosensor technology for mapping conformational epitopes in viral proteins.

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility.

The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1-14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

Inhibition of in vitro cotranslational disassembly of tobacco mosaic virus by monoclonal antibodies to the viral coat protein.

It has been shown by others that translation of tobacco mosaic virus (TMV) RNA may begin before uncoating of particles is complete. We provide evidence that this cotranslational disassembly can be inhibited by incubating TMV treated at pH 8 with certain monoclonal antibodies (MAbs) specific for TMV coat protein. The most efficient inhibition was achieved by incubation with some anti-metatope MAbs known to bind to the TMV extremity that becomes disassembled first and contains the 5' end of the RNA, as well as with some anti-cryptotope MAbs that bind only to dissociated coat protein subunits.

Stabilisation of emulsions: influence of hydrophilic colloids.

Synopsis The influence of the following hydrophilic polymers on the stability of emulsions has been studied: sodium carboxymethyl cellulose, hydroxyethyl cellulose, agar-agar, water dispersible clay, and a neutralized polyethylene acid. The oil phases used were paraffin oil, olive oil and a synthetic C(8)-C(12) triglyceride. The other emulsifiers were non ionic-types. After 12 months storage at room temperature, olive oil proved to be the most difficult material to emulsify. The hydrocolloids did not give uniformly better stability to the emulsions and the polyacrylic acid gave the best stability of the colloids tested.

Major isomers of the active ingredient of the sunscreen 5(3,3-dimethyl-2-norbornylidene)-3 pentene-2-one.

Synopsis With high-performance liquid chromatography the three major isomers present in the sunscreen agent, 5(3, 3-dimethyl-2-norbornylidene)-3 pentene-2-one, have been isolated and quantified. Their structures have been determined using mass, infra-red, and NMR spectrometry. Isomères dans le filtre solaire 5(3, 3-dimethyl-2-norbornylidene)-3 pentene-2-one.

Influence of homogenization on emulsion stability.

Summary Three manufacturing procedures using a slow propeller mixer, a labyrinth and a valve homogenizer were used for the preparation of emulsions. Three oil phases and fifteen pairs of emulsifiers were used giving a range of HLB values. These homogenizers gave more stable emulsions than the propeller mixer in the majority of cases although several times the reverse was the case. To interpret these reversals several mechanisms are suggested: the mechanical engergy applied to the system modifies the equilibrium of the interaction oil/emulsifier; homogenization gives a considerable increase in the contact to the point where the interfacial film is deficient in surface agents and is unable to stablilise completely the system or the emulsifier is no longer in sufficient concentration in the aqueous phase to play its role in viscosity modification which appears to be the case in non homogenized emulsions.