Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors

Specificity of sdAbs for <i>Bacillus</i> species spores.

<p>Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.</p

Melting temperatures and recovery of native structure.

<p> <b>The recovery of native structure (rounds 1–3) was calculated from the total change in left and right ellipticity for each cycle of heating and cooling compared to the initial total ellipticity.</b></p

Immunoblot analysis of sdAb target protein.

<p><i>B. anthracis</i> Sterne strain cell and spore lysates were separated via SDS-PAGE for immunoblotting using each of the sdAbs.</p

Amino acid alignments of sdAb families.

<p>Families were identified based on amino acid alignments and variability in the complementarity determining regions (CDRs) bracketed in red boxes. Single representatives of each family are shown here. Clones d3x, d9x, G1, H4, and C8x are members of families that are not described in this study.</p

Specificity of sdAbs for whole bacteria.

<p>Bacteria were either grown overnight as live cultures or obtained from either CRP or KPL as killed material. Equivalent optical densities were immobilized to microtiter dishes for analysis of specificity using a direct binding ELISA.</p

Affinity of sdAbs for <i>Bacillus anthracis</i> strains.

<p>Biotinylated sdAbs were tested for their ability to recognize a panel of <i>B. anthracis</i> strains from a number of geographic locations (acquired as irradiated spore preparation from NMRC).</p

Binding kinetics for sdAbs.

a<p>KD values are calculated from kd/ka.</p><p>NA = No binding observed.</p