Femtomolar Detection of the Anthrax Edema Factor in Human and Animal Plasma

Abstract

Edema factor (EF), a calmodulin-activated adenylyl cyclase, is a toxin which contributes to cutaneous and systemic anthrax. As a novel strategy to detect anthrax toxins in humans or animals infected by Bacillus anthracis, we have developed a sensitive enzymatic assay to be able to monitor functional EF in human and animal plasma. Samples containing EF are incubated in the presence of calmodulin and ATP, which is converted to cAMP. After oxidation and derivatization, cAMP is monitored by competitive enzyme immunoassay. Because of the high turnover of EF and the sensitivity of cAMP detection, EF can be detected at concentrations of 1 pg/mL (10 fM) in 4 h in plasma from humans or at 10 pg/mL in the plasma of various animal species using only a blood volume of 5 microL. The assay has good reproducibility with intra- and interday coefficients of variation in the range of 20% and is not subject to significant interindividual matrix effects. In an experimental study performed in mice infected with the Berne strain, we were able to detect EF in serum and ear tissues. This simple and robust combination of enzymatic reaction and enzyme immunoassay for the diagnosis of anthrax toxemia could prove useful in biological threat detection as well in research and clinical practice.