Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

Salicylic acid-induced resistance to Cucumber mosaic virus in squash and Arabidopsis thaliana: contrasting mechanisms of induction and antiviral action.

Salicylic acid (SA)-induced resistance to Cucumber mosaic virus (CMV) in tobacco (Nicotiana tabacum) results from inhibition of systemic virus movement and is induced via a signal transduction pathway that also can be triggered by antimycin A, an inducer of the mitochondrial enzyme alternative oxidase (AOX). In Arabidopsis thaliana, inhibition of CMV systemic movement also is induced by SA and antimycin A. These results indicate that the mechanisms underlying induced resistance to CMV in tobacco and A. thaliana are very similar. In contrast to the situation in tobacco and A. thaliana, in squash (Cucurbita pepo), SA-induced resistance to CMV results from inhibited virus accumulation in directly inoculated tissue, most likely through inhibition of cell-to-cell movement. Furthermore, neither of the AOX inducers antimycin A or KCN induced resistance to CMV in squash. Additionally, AOX inhibitors that compromise SA-induced resistance to CMV in tobacco did not inhibit SA-induced resistance to the virus in squash. The results show that different host species may use significantly different approaches to resist infection by the same virus. These findings also imply that caution is required when attempting to apply findings on plant-virus interactions from model systems to a wider range of host species.

Anti-immunoglobulin responses to IgG, F(ab')2, and Fab botulinum antitoxins in mice.

Side effects to botulinum antitoxins, including anaphylaxis and serum sickness, are common. This is due to the immunogenicity of the antitoxin, which can be measured by the production of anti-immunoglobulin antibodies. An ideal botulinum antitoxin would elicit a minimal production of anti-immunoglobulin antibodies from a patient, aiding its safety. To investigate the immunogenicity of different immunoglobulin fragments, whole IgG, F(ab')2 and Fab botulinum antitoxins were administered to mice by either the intravenous or intramuscular route. The production of anti-immunoglobulin antibodies was measured over time after a single dose of antitoxin, and the anti-immunoglobulin antibodies isotyped. When administered by the intramuscular route, Fab showed significantly lower immunogenicity than IgG, while F(ab')2 had an immunogenicity that was intermediate between the two. When administered by the intravenous route there was no significant difference in immunogenicity between IgG and F(ab')2 antitoxins, although Fab antitoxin had a significantly lower immunogenicity than either IgG or F(ab')2. IgG antitoxin was significantly more immunogenic by the intramuscular route than by the intravenous route. Sheep IgG had a lower immunogenicity than goat IgG in mice. There was no significant difference in immunogenicity between the two dosing routes for either F(ab')2 or Fab antitoxin. The anti-antibodies were predominantly IgG1, suggesting a strong Th2 bias to the anti-antibody response. In all cases, Fab represents the least immunogenic form of antitoxin.