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1.
Appl Environ Microbiol ; 84(12)2018 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-29654186

RESUMEN

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.


Asunto(s)
Bacillus anthracis/efectos de la radiación , Rayos gamma , Viabilidad Microbiana/efectos de la radiación , Esporas Bacterianas/efectos de la radiación , Esterilización/métodos , Bacillus anthracis/fisiología , Técnicas Microbiológicas/métodos , Estudios Retrospectivos , Esporas Bacterianas/fisiología
2.
Anal Biochem ; 447: 64-73, 2014 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-24184358

RESUMEN

We constructed a genetic fusion of a single domain antibody (sdAb) with the thermal stable maltose binding protein from the thermophile Pyrococcus furiosus (PfuMBP). Produced in the Escherichia coli cytoplasm with high yield, it proved to be a rugged and effective immunoreagent. The sdAb-A5 binds BclA, a Bacillus anthracis spore protein, with high affinity (K(D) ∼ 50 pM). MBPs, including the thermostable PfuMBP, have been demonstrated to be excellent folding chaperones, improving production of many recombinant proteins. A three-step purification of E. coli shake flask cultures of PfuMBP-sdAb gave a yield of approximately 100mg/L highly purified product. The PfuMBP remained stable up to 120 °C, whereas the sdAb-A5 portion unfolded at approximately 68 to 70 °C but could refold to regain activity. This fusion construct was stable to heating at 1mg/ml for 1h at 70 °C, retaining nearly 100% of its binding activity; nearly one-quarter (24%) activity remained after 1h at 90 °C. The PfuMBP-sdAb construct also provides a stable and effective method to coat gold nanoparticles. Most important, the construct was found to provide enhanced detection of B. anthracis Sterne strain (34F2) spores relative to the sdAb-A5 both as a capture reagent and as a detection reagent.


Asunto(s)
Proteínas Arqueales/genética , Inmunoensayo/métodos , Proteínas de Unión a Maltosa/genética , Glicoproteínas de Membrana/análisis , Proteínas Recombinantes de Fusión/química , Anticuerpos de Dominio Único/química , Temperatura , Citoplasma/genética , Microesferas , Estabilidad Proteica , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Esporas Bacterianas , Temperatura de Transición
3.
Chem Biol ; 19(4): 449-55, 2012 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-22520751

RESUMEN

Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.


Asunto(s)
Anticuerpos de Cadena Única/química , Enlace de Hidrógeno , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Terciaria de Proteína , Anticuerpos de Cadena Única/metabolismo , Programas Informáticos , Temperatura
4.
Toxins (Basel) ; 3(11): 1405-19, 2011 11.
Artículo en Inglés | MEDLINE | ID: mdl-22174977

RESUMEN

Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations.


Asunto(s)
Abrina/inmunología , Antígenos/inmunología , Camélidos del Nuevo Mundo/inmunología , Lectinas de Plantas/inmunología , Anticuerpos de Dominio Único/inmunología , Abrina/administración & dosificación , Animales , Antígenos/administración & dosificación , Dicroismo Circular , Inmunización
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