AbstractOne method of laboratory‐ or field‐based testing for anthrax is detection of Bacillus anthracis spores by high‐affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re‐formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross‐reactivity between species. These results were rationalized by determination of the three‐dimensional crystallographic structure of the scFv‐tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter‐spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high‐affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure‐based antibody design for this difficult target. Proteins 2011. © 2011 Wiley‐Liss, Inc.