Danièle Altschuh

Comparative sensitivity of different ELISA procedures for detecting monoclonal antibodies

Abstract Seven different ELISA procedures using alkaline phosphatase as a label were compared for their sensitivity in detecting binding activity of 9 monoclonal antibodies raised against tobacco mosaic virus. Two procedures, by which the microtitre plates were coated with antigen, were found to be unreliable for testing monoclonal antibodies. The 2 most sensitive assays were of the double sandwich type and involved the use of viral antibody obtained from the egg yolk of immunized chickens. The use of avian antibody in these multilayered sandwich procedures was essential to avoid high background readings caused by cross-reactivity between different mammalian antibodies. In the less sensitive ELISA procedures, the 9 monoclonal antibodies raised against the virus did not react with the monomeric subunit of the viral coat protein; however, in the 2 most sensitive assays, 4 of these antibodies were clearly able to recognize the protein subunits.

Protein-protein recognition and interaction hot spots in an antigen-antibody complex: free energy decomposition identifies "efficient amino acids".

The molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) method was applied to the study of the protein–protein complex between a camelid single chain variable domain (cAb‐Lys3) and hen egg white lysozyme (HEL), and between cAb‐Lys3 and turkey egg white lysozyme (TEL). The electrostatic energy was estimated by solving the linear Poisson–Boltzmann equation. A free energy decomposition scheme was developed to determine binding energy hot spots of each complex. The calculations identified amino acids of the antibody that make important contributions to the interaction with lysozyme. They further showed the influence of small structural variations on the energetics of binding and they showed that the antibody amino acids that make up the hot spots are organized in such a way as to mimic the lysozyme substrate. Through further analysis of the results, we define the concept of “efficient amino acids,” which can provide an assessment of the binding potential of a particular hot spot interaction. This information, in turn, can be useful in the rational design of small molecules that mimic the antibody. The implications of using free energy decomposition to identify regions of a protein–protein complex that could be targeted by small molecules inhibitors are discussed. Proteins 2007.

Visualization by electron microscopy of the location of tobacco mosaic virus epitopes reacting with monoclonal antibodies in enzyme immunoassay

The binding of monoclonal antibodies obtained after immunization with tobacco mosaic protein was analyzed by electron microscopy. A method was developed for visualizing the viral antigen reacting in different ELISA procedures. It was found that the use of a pH 9.6 buffer during the coating of ELISA plates led to the dissociation of virions into subunits which bound preferentially to the solid phase. MAbs that reacted with both virions and subunits in ELISA were found to bind to one of the two extremities of viral rods. These MAbs also reacted with viral protein aggregated in the form of disks. In contrast, MAbs reacting only with virions in ELISA were found to bind over the entire surface of the virus.

Immunochemical studies of tobacco mosaic virus—VI. Attempts to localize viral epitopes with monoclonal antibodies

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV. By means of computer-generated images of the surface residues of the viral subunit, it was possible to identify certain clusters of residues involved in binding to some of the monoclonal antibodies. The results clearly illustrate the operational limitations encountered when monoclonal antibodies are used for elucidating the antigenic structure of 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.

Cutting Edge: Monovalency of CD28 Maintains the Antigen Dependence of T Cell Costimulatory Responses

CD28 and CTLA-4 are the major costimulatory receptors on naive T cells. But it is not clear why CD28 is monovalent whereas CTLA-4 is bivalent for their shared ligands CD80/86. We generated bivalent CD28 constructs by fusing the extracellular domains of CTLA-4 or CD80 with the intracellular domains of CD28. Bivalent or monovalent CD28 constructs were ligated with recombinant ligands with or without TCR coligation. Monovalent CD28 ligation did not induce responses unless the TCR was coligated. By contrast, bivalent CD28 ligation induced responses in the absence of TCR engagement. To extend these findings to primary cells, we used novel superagonistic and conventional CD28 Abs. Superagonistic Ab D665, but not conventional Ab E18, predominantly ligates CD28 bivalently at low CD28/Ab ratios and induces Ag-independent T cell proliferation. Monovalency of CD28 for its natural ligands is thus essential to provide costimulation without inducing responses in the absence of TCR engagement.

A Peptide-Based, Ratiometric Biosensor Construct for Direct Fluorescence Detection of a Protein Analyte

We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore ( I(N*)/I(T*)) upon binding of Fab57P. When the peptide was labeled in the C terminus, the I(N*)/I(T*) ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats.

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context.

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi‐conservative amino acid replacements at the VH–VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE® instrument. The substitutions introduced at the VH–VL interface had no significant effects on ka but showed small, significant effects on kd. Mutations in the VH module affected kd not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation FL37L–QL38L, alone or in combination with other mutations, consistently decreased kd about two‐fold in Fab 145P. Other mutations in the VL module had no effect on kd in 145P, but always decreased kd in 1F1h. Moreover, in both systems, small‐magnitude non‐additive effects on kd were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes. Copyright

Monoclonal antipeptide antibodies: Affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

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.