Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry

摘要

Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and anti-bioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrate was improved through optimization of the substrate, reaction conditions and sample preparation. In this method, the ricin is captured by an specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than two orders of magnitude enhancement in assay sensitivity. Improving assay’s ruggeddness or reporducibility also made it possible to quantitatively detect active ricin with a three order of magnitude dynamic range.