Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.
Anal Bioanal Chem
; 411(12): 2493-2509, 2019 May.
Article
em En
| MEDLINE
| ID: mdl-30911800
ABSTRACT
Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Infecções Respiratórias
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Bacillus anthracis
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Toxinas Bacterianas
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Toxemia
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Cromatografia Líquida de Alta Pressão
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Espectrometria de Massas em Tandem
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Antraz
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Antígenos de Bactérias
Tipo de estudo:
Diagnostic_studies
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Observational_studies
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Screening_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos