Accurate and selective quantification of anthrax protective antigen in plasma by immunocapture and isotope dilution mass spectrometry

The Analyst
Maria I SolanoJohn R Barr

Abstract

Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 μL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF a...Continue Reading

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Citations

Jul 29, 2020·Microorganisms·Jean-Nicolas Tournier, Clémence Rougeaux

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