The use of Endopep–MS for the detection of botulinum toxins A, B, E, and F in serum and stool samples

Abstract

Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Previous work in our laboratory focused on developing Endopep–MS, a mass spectrometric-based endopeptidase method for the detection and differentiation of BoNT serotypes. We have expanded this effort to include an antibody capture method to partially purify and concentrate BoNT from serum and stool extract samples for the Endopep–MS assay. Because complex matrices such as serum and stool contain abundant endogenous proteases, this technique was needed to remove most proteases from the sample while concentrating BoNT from a sample size of 100 to 500 μl to 20 μl. When this antibody capture method is combined with the Endopep–MS reaction, limits of detection in 500 μl of spiked human serum are 10 mouse LD 50 (20 mouse LD 50/ml) for BoNT A, 0.5 mouse LD 50 (1 mouse LD 50/ml) for BoNT B, 0.1 mouse LD 50 (0.2 mouse LD 50/ml) for BoNT E, and 0.5 mouse LD 50 (1 mouse LD 50/ml) for BoNT F. The limits of detection in spiked stool extracts are somewhat higher due to the high-protease environment of stool extract that also requires use of protease inhibitors. The entire method can be performed in as short a time as 4 h.