Ultrasensitive Detection of Ricin Toxin
Ricin is one of the most toxic and easily produced plant toxins, and is readily available globally. Currently, there is no way to test for ricin toxin in samples like blood or urine. Since we do not know how much ricin is enough to hurt people, it is important that public health authorities are able to detect even very small amounts. Our test will be able to accurately detect tiny amounts of ricin toxin, providing important information about exposure events.
Project Goal: The overall goal is to develop an ultra-sensitive test for the detection and quantitation of the biothreat toxin ricin in biological and environmental samples.
Rationale: Currently, no widely accepted method exists for detection of ricin in exposed individuals, and methods to detect biothreat toxins in environmental samples possess limited analytical sensitivity. By combining serologic protein capture and nucleic acid amplification, Enzyme Mediated Immuno-PCR (EMI-PCR) offers unparalleled sensitivity for the detection of toxins such as ricin, in both environmental and biological sample types. Sub-clinical exposures and environmental contamination can be confirmed more accurately where ultra-low levels of various toxins can be detected and quantified.
Hypothesis: A real-time EMI-PCR method will be developed to detect ricin toxin in various sample types and at concentrations several thousand-fold lower than that which current methods can detect.
Specific Aim 1: Independently develop and validate EMI-PCR components for ricin detection. First, a reference ELISA to detect ricin will be developed for future comparison to the EMI-PCR method. Differing solid supports, and variable coating, blocking, washing/dilution, incubation, and detection conditions will be evaluated. Preliminary evaluations with titrated reporter DNA, alkaline phosphatase (AP) conjugates, and λ-exonuclease (λ-exo) followed by PCR amplification will be performed. The milestone is the independent optimizations of the ELISA and AP/λ-exo/amplification assay components. Aim 1 will be completed during months 1-18.
Specific Aim 2: Combine and Optimize Complete EMI-PCR method and compare to that of the reference ELISA method. Guided by results from Specific Aim 1, all EMI-PCR reaction conditions will be further optimized in the context of a complete EMI-PCR. The analytical sensitivities of the EMI-PCR and the ELISA will be established and compared. The milestone is to have an optimized EMI-PCR method for ricin detection that exhibits a sensitivity at least 1000-fold greater than that of the corresponding ELISA. Aim 2 will be completed during months 19-26.
Specific Aim 3: Challenge EMI-PCR methods with various sample matrices. EMI-PCR will be assessed using ricin-spiked human serum and urine. Swabs representing drinking water spiked with ricin will also be evaluated. Sensitivity of EMI-PCR when applied to these matrices will be compared to the ELISA method. Standard Operating Procedure will be drafted. The milestone is to show that the EMI-PCR platforms can detect ricin toxin at ultra-low levels in biological media, and in typical environmental samples. Aim 3 will be completed during months 27-36.
Niel T. Constantine, Ph.D.
University of Maryland at Baltimore