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Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

Walper SA, Anderson GP, Brozozog Lee PA, Glaven RH, Liu JL, Bernstein RD, Zabetakis D, Johnson L, Czarnecki JM, Goldman ER - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Naval Research Laboratory, Washington D. C., 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.

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Related in: MedlinePlus

Specificity of sdAbs for Bacillus species spores.Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.
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pone-0032801-g004: Specificity of sdAbs for Bacillus species spores.Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.

Mentions: To assess specificity of the isolated sdAb a direct binding ELISA with several representatives of the B. cereus group (B. anthracis Sterne 34F2, B. cereus ATCC 4342, B. cereus ATCC 14579, B. cereus ATCC 13061, B. mycoides ATCC 6462, B. thuringiensis 4D9, B. thuringiensis 4Q2, and B. thuringiensis ATCC 33680) and additional non-target bacteria (B. subtilis ATCC 31028, E. coli O157:H7, L. monocytogenes, S. enterica serovar Typhimurium, F. tularensis, and Shigella) was employed. Minimal interaction (absorbance <0.05) with the non-target Bacillus species was observed for all of the sdAbs examined and no cross-reactivity with the non-Bacillus bacteria in the panel (Figure 3). The sdAbs C7, d10x, and G10 demonstrated affinity for the vegetative cell; while the sdAbs A1, B2, and B12 bound the target only minimally. Similar results were observed when the bacterial spores were directly immobilized on microtiter plates. Given that all six sdAbs bound the EA1 protein in immunoblots, a second ELISA was performed in which the spores were first broken with a combination of boiling and sonication to fracture and release the exosporium and loosely attached proteins from the spore. The suspension, with no prior centrifugation, was then immobilized to the microtiter plate to allow for a mix of intact spores, exosporium fragments, and proteins on the well surface. In contrast to the results of the intact spore ELISA; the A1, B2, and B12 sdAbs showed a significant improvement in antigen binding suggesting an epitope of the EA1 protein that is not exposed in the intact spore (Figure 4).


Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

Walper SA, Anderson GP, Brozozog Lee PA, Glaven RH, Liu JL, Bernstein RD, Zabetakis D, Johnson L, Czarnecki JM, Goldman ER - PLoS ONE (2012)

Specificity of sdAbs for Bacillus species spores.Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295763&req=5

pone-0032801-g004: Specificity of sdAbs for Bacillus species spores.Intact and broken spores were passively immobilized to microtiter plates at equivalent optical densities to assess specificity of sdAbs.
Mentions: To assess specificity of the isolated sdAb a direct binding ELISA with several representatives of the B. cereus group (B. anthracis Sterne 34F2, B. cereus ATCC 4342, B. cereus ATCC 14579, B. cereus ATCC 13061, B. mycoides ATCC 6462, B. thuringiensis 4D9, B. thuringiensis 4Q2, and B. thuringiensis ATCC 33680) and additional non-target bacteria (B. subtilis ATCC 31028, E. coli O157:H7, L. monocytogenes, S. enterica serovar Typhimurium, F. tularensis, and Shigella) was employed. Minimal interaction (absorbance <0.05) with the non-target Bacillus species was observed for all of the sdAbs examined and no cross-reactivity with the non-Bacillus bacteria in the panel (Figure 3). The sdAbs C7, d10x, and G10 demonstrated affinity for the vegetative cell; while the sdAbs A1, B2, and B12 bound the target only minimally. Similar results were observed when the bacterial spores were directly immobilized on microtiter plates. Given that all six sdAbs bound the EA1 protein in immunoblots, a second ELISA was performed in which the spores were first broken with a combination of boiling and sonication to fracture and release the exosporium and loosely attached proteins from the spore. The suspension, with no prior centrifugation, was then immobilized to the microtiter plate to allow for a mix of intact spores, exosporium fragments, and proteins on the well surface. In contrast to the results of the intact spore ELISA; the A1, B2, and B12 sdAbs showed a significant improvement in antigen binding suggesting an epitope of the EA1 protein that is not exposed in the intact spore (Figure 4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Naval Research Laboratory, Washington D. C., 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.

Show MeSH
Related in: MedlinePlus