Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance.

Differences in the affinity of a monoclonal antibody raised against the protein of tobacco mosaic virus for 15 related peptides (residues 134-146) carrying single-residue modifications were investigated using a novel biosensor technology (Pharmacia BIAcore). Analysis of the peptide-antibody interaction in real time allowed fast and reproducible measurements of both association and dissociation rate constants. Out of 15 mutant peptides analyzed, five were not recognized by the antibody at all, and seven were recognized as well as the wild-type peptide. For three of the peptides, the rate constants were different for the mutant and wild-type peptides. The pattern of residue recognition suggests that the epitope is formed by three residues (140, 143, and 144) in a helical conformation that mimics the structure in the protein. Even a minor modification of these residues totally abolishes recognition by the antibody. Modifications of adjacent residues result in small but significant differences in association and/or dissociation rate constants. One of the recognized residues is totally buried in the three-dimensional structure of TMV protein, suggesting that a structural rearrangement next to the helix occurs during protein-antibody interaction.

Cross-reactive potential of monoclonal antibodies raised against proteolysed tobacco etch virus.

Monoclonal antibodies (mAb) capable of reacting with different potyviruses were obtained by immunizing mice with proteolysed tobacco etch virus. The mAb were not equally effective in all ELISA formats and some were specific for different conformational states of the viral coat protein. The mAb also detected antigenic differences between purified virus particles and viral antigen in infected plant sap. In an ELISA format using antigen-coated plates, 5 different potyviruses (out of 7 viruses tested) could be detected in plant sap by one mAb. Different combinations of mAb and polyclonal antiserum could also be used for detecting several potyviruses by ELISA.

Detection of potyviruses with antisera to synthetic peptides.

Eight peptides corresponding to conserved regions of the coat protein of potyviruses were synthesized. All the peptides were recognized by anti-virus or anti-core-virus. Antisera raised to the synthetic peptides were tested with purified viruses and viral antigens present in plant sap. In many cases, the extent of cross-reactivity between different potyviruses was not correlated with the degree of sequence homology between the peptide used for immunization and the corresponding region in the coat protein of the potyvirus tested. An antiserum raised to a peptide of 18 residues containing a highly conserved region was found to react with all seven potyviruses tested.

Mapping of viral epitopes with conformationally specific monoclonal antibodies using biosensor technology.

An automated biosensor system (BIAcore) designed for measuring molecular interactions in real time and without labelling any of the reactants was used for mapping the epitopes of tobacco mosaic virus protein using conformationally specific monoclonal antibodies (MAbs). Some of the MAbs used as capturing antibody on the sensor chip allowed a conformational change to occur in the viral protein. As a result, MAbs specific for the quaternary structure of polymerized viral protein were able to bind to monomeric viral subunits. Compared with classical solid-phase enzyme immunoassay, the biosensor technology possesses several advantages for epitope mapping of viral proteins.

Interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.

An automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. Using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immobilized on the dextran matrix present on the sensor chip. Compared to conventional solid phase immunoassays, in which immobilized proteins are usually partly denatured, the biosensor system presents several advantages for studying virus-antibody interaction.

Antigenic cross-reactivity potential of synthetic peptides immobilized on polyethylene rods.

A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin.

Odontoglossum ringspot virus coat protein: sequence and antigenic comparisons with other tobamoviruses.

Comparative immunochemical analysis of different tobamoviruses indicated that the previously reported coat protein sequence of Odontoglossum ringspot virus was likely erroneous. This sequence has been determined again by direct sequencing of the genomic RNA and was found to differ from the previously proposed sequence in 31 of the 157 amino acid residues. The extent of antigenic cross-reactivity between ORSV protein and other tobamovirus proteins was measured by ELISA and found to correlate satisfactorily with the degree of sequence homology.