Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells Scott A. Walper 1 , George P. Anderson 2 , P. Audrey Brozozog Lee 3 , Richard H. Glaven 3 , Jinny L. Liu 2 , Rachel D. Bernstein 3 , Dan Zabetakis 2 , Linwood Johnson 4 , Jill M. Czarnecki 4 , Ellen R. Goldman 2 * 1 Naval Research Laboratory, Washington D. C., United States of America, 2 Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington D. C., United States of America, 3 Nova Research Inc, Alexandria, Virginia, United States of America, 4 Biological Defense Research Directorate, Naval Medical Research Center, Silver Spring, Maryland, United States of America Abstract 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. Citation: Walper SA, Anderson GP, Brozozog Lee PA, Glaven RH, Liu JL, et al. (2012) Rugged Single Domain Antibody Detection Elements for Bacillus anthracis Spores and Vegetative Cells. PLoS ONE 7(3): e32801. doi:10.1371/journal.pone.0032801 Editor: Adam Driks, Loyola University Medical Center, United States of America Received September 16, 2011; Accepted February 4, 2012; Published March 6, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Defense Threat Reduction Agency, project # CBM.DIAGB.01.10.NRL.011 and the Office of Naval Research NRL base funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: While several authors are employed by Nova Research Inc, this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: ellen.goldman@nrl.navy.mil Introduction Bacillus anthracis, the etiological agent of anthrax, is capable of lethality in both animals and humans, and is a biothreat of great concern [1]. This is due in-part to its persistence and ease of production of the bacterial spore which can be disseminated as an aerosolized bioagent [2,3,4]. The threat posed by Bacillus anthracis is not new; research into its use as a bioweapon has been investigated since the early 1930s by a number of countries around the world. The accidental release of spores from a facility in the former Soviet Union which resulted in 42 cases of anthrax [3] and the Amerithrax incident of 2001 illustrate the devastating potential of this organism as a bioweapon. The Bacillus genus is comprised of a diverse group of Gram- positive, aerobic bacteria, capable of forming an endospore. Bacillus anthracis, is a member of the B. cereus group which also contains the B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstaphenesis species. Within this group, the B. anthracis, B. cereus, and B. thuringiensis are highly similar morphologically and therefore difficult to differentiate using many laboratory tech- niques [5,6,7,8]. The development of scientific tools such as PCR and RT-PCR have allowed for not only the differentiation of closely related Bacillus species, but also the identification of pathogenic versus non-pathogenic strains [9,10,11,12,13]. Addi- tionally, novel and elegant techniques such as mass spectroscopic analysis of small acid soluble proteins [12,14] and optical chromatography [15] continue to be developed. While these methods have all demonstrated their potential for success, each is limited in its ability to be incorporated into a biosensor that satisfies the requirements of ruggedness, portability, and simplicity. Traditional antibodies have served as invaluable tools in medical and scientific assays for many years. Their high specificity and nanomolar binding affinities ensure that assays utilizing antibodies will not soon be replaced. However, while the traditional immunoglobulin is well-established, difficulties such as the cost of production and protein stability in austere environ- ments can be encountered. To circumvent these complications, researchers have explored several recombinant forms including single chain Fv (scFv) antibodies derived from the variable regions of conventional antibodies [16,17], and single domain antibodies (sdAbs) consisting of a single variable domain derived from heavy- chain only antibodies [18,19,20,21]. The sdAb is derived from novel immunoglobulins found in sharks and members of the Camelidae family (camels, llamas, alpacas) that is comprised of only a heavy chain subunit, lacking the light chain found in the more common antibody structure [19,22]. The isolated variable domain of these heavy-chain only antibodies is capable of folding to form the antigen-binding domain, exhibiting binding affinities equiva- lent to the progenitor antibody. Additionally, sdAbs possess the characteristic of thermal stability. This quality can manifest as an PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e32801