High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures

Abstract

Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3–9.9 % coefficient of variation), and accurate (0.1–12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % (n = 27 animal/clinical cases). Diagnostic specificity was 100 % (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.Graphical AbstractMethod schematic for analysis of anthrax lethal toxin activity by ID-MALDI-TOF MS

Advance detection technologies for select biothreat agents

“…The findings can be used as preliminary analytical data. The instrumental techniques to be used for generating fingerprints of toxins and bioagents include GC-MS, LC-MS, and MALDI-TOF (Boyer et al, 2015).…”

Mass spectrometry for the detection of bioterrorism agents: from environmental to clinical applications

“…However, the limit of detection of that EF assay was not precisely reported . As the detection of the lethal toxin is clinically more relevant than the detection of LF protein, a precise and accurate method was recently developed by John Barr's group for LTx‐specific quantification in plasma . This bioassay involves an anti‐PA IgG magnetic immunoprecipitation of PA, a substrate cleavage reaction, and MALDI–TOF MS quantification of LF activity, which co‐purifies with PA.…”

“…[77] As the detection of the lethal toxin is clinically more relevant than the detection of LF protein, a precise and accurate method was recently developed by John Barr's group for LTx-specific quantification in plasma. [82] This bioassay involves an anti-PA IgG magnetic immunoprecipitation of PA, a substrate cleavage reaction, and MALDI-TOF MS quantification of LF activity, which co-purifies with PA. The entire analytical method takes less than 4 h and detects LF levels as low as 0.033 ng/ml in plasma and can reach an LOD of 0.0075 ng/ml with an 18-h incubation time.…”

Anthrax in injecting drug users: the need for increased vigilance in the clinic

“…A number of further diagnostic tests may be employed to confirm IA cases; these fall broadly into assays which detect the presence of the bacteria, such as fluorescence immunoassays and mass spectrometry identifying cell-associated proteins [25][26][27] and assays which detect the presence of antibodies to PA and LF [28,29]. Several caveats must be borne in mind though when considering these methods, as phenotypic characteristics may vary between strains and hemolytic, motile bacilli, which are resistant to phage lysis, have been identified in B. anthracis isolates, closely related Bacillus species may lead to misdiagnosis, and (perhaps most importantly) antimicrobial therapy rapidly decreases the sensitivity of these assays [30].…”