Abstract
To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis spores and not to other Bacillus species. Additionally, SODA1 and p5303 binding sdAbs detected spores in sandwich assays serving as both captures and tracers. Used in combination, sdAbs targeting B. anthracis proteins could be integrated into emerging biosensors to improve specificity in multiplex assays.
Highlights
Bacillus anthracis, the causative agent of anthrax, has been considered a bioweapon of grave concern since long before the letter-based attacks of 2001
Centavo received a mix of all six proteins; while Whisper was immunized with a cocktail that did not include the BclA protein in an attempt to allow a stronger immune response toward the other spore proteins that may not be as immunodominant as the BclA
SdAbs targeting the proteins of the B. anthracis Sterne strain spores were isolated from an immune llama library using phage display and characterized in terms of target binding kinetics, thermal stability, specificity, and utility in sandwich assays for the detection of both intact and broken spores
Summary
The causative agent of anthrax, has been considered a bioweapon of grave concern since long before the letter-based attacks of 2001. Though discrimination of the closely related bacteria of the B. cereus group is possible with assays that utilize PCR [4,5,6], these techniques frequently require laboratory based equipment and skilled technicians. Immunoassays, must utilize antibodies that target proteins unique to B. anthracis spores and vegetative cells to be useful in detection assays. Though not a universal characteristic, most sdAbs exhibit the ability to refold into active detection elements following denaturation [14] This is advantageous for their application in assays designed for use in more austere environments where cold chains may be limited or completely absent. There are several reports of recombinantly expressed antibody binding domains for the specific detection of B. anthracis [15,16]; these binding elements target EA1, a vegetative protein that is often found as a contaminant in spore preparations. The sdAbs characterized in this study could be used in combination with one another or antibodies targeting other proteins of the spore or vegetative cell to improve discrimination of target and non-target bacterium while potentially eliminating false negatives that may arise from loss or modification of a single epitope
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