Project Goal: The overall goal is to develop an ultra-sensitive test for the detection and quantification of the biothreat toxin ricin in biological and environmental samples. This is a continuation of a previously fundedgrant from MARGE.Rationale: Currently, no widely accepted method exists for detection of ricin in exposed individuals, andmethods to detect biothreat toxins in environmental samples possess limited analytical sensitivity. Bycombining serologic protein capture and nucleic acid amplification, Enzyme Mediated Immuno-PCREMI-PCR) offers unparalleled sensitivity for the detection of toxins such as ricin, in both environmental andbiological sample types. Sub-clinical exposures and environmental contamination can be confirmed moreaccurately 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 andat 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. A'roiect Goal: The overall goal is to develop an ultra-sensitive test for the detection and quantification of thereference ELISA to detect ricin has been developed and will be finalized with maximum sensitivity using an APconjugate. Remaining items include use of a newly identified blocking reagent and two additional detectorantibodies; these will be evaluated as described previously. For EMI-PCR, preliminary evaluations usingtitrated reporter DMA, alkaline phosphatase (AP) conjugates, and A-exonuclease (A-exo) followed by PCRamplification have shown that the components of the system work. Further titrations of these reagents will beperformed to maximize reactivity at concentrations that minimize false positives and false negatives. Themilestone is the finalization of both the ELISA and the components of the EMI-PCR components.
Aim 1 will becompleted by the end of month 1 (March 2008); final experiments are in progress currently.
Specific Aim 2 : Combine and Optimize Complete EMI-PCR method and compare to that of thereference ELISA method. Guided by results from Specific Aim 1, all EMI-PCR reaction conditions will befurther optimized in the context of a complete EMI-PCR. The analytical sensitivities of the EMI-PCR and theELISA will be established and compared. The milestone is to have an optimized EMI-PCR method for ricindetection that exhibits a sensitivity at least 1000-fold greater than that of the corresponding ELISA. Should theEMI-PCR method not attain the desired sensitivity, Immuno-PCR, a similar ultrasensitive method that can bemodified for ricin detection, will be evaluated for its analytical sensitivity.
Aim 2 will be completed duringmonths 2-8.
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 waterspiked with ricin will also be evaluated. Sensitivity of EMI-PCR when applied to these matrices will becompared to the ELISA method. Standard Operating Procedure will be drafted. The milestone is to show thathe EMI-PCR method (or Immuno-PCR if necessary) can detect ricin toxin at ultra-low levels in biologicalmedia, and in typical environmental samples.
Aim 3 will be completed during months 8-12.Ricin is one of the most toxic and easily produced plant toxins, and is readily available globally.
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