Method of detecting an amount of a cholinesterase inhibitor in a sample
Inventors
Loke, Weng Keong • Tan, Yong Teng • Seow, Josefina
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-8187832-B2
Publication Date
2012-05-29
Expiration Date
Abstract
A method of detecting an amount of a cholinesterase inhibitor in a sample comprising steps of: (a) contacting the sample with an agent capable of recovering the cholinesterase inhibitor from the sample so that the cholinesterase inhibitor is recovered, wherein the agent comprises a reactivity towards phosphyl moieties; (b) isolating the recovered cholinesterase inhibitor from the sample; (c) contacting the isolated cholinesterase inhibitor from step (b) with a test cholinesterase wherein the cholinesterase activity of the test cholinesterase before step (a) is known; and (d) measuring the cholinesterase activity to determine the amount of cholinesterase inhibitor in the sample based on the inhibition of the test cholinesterase activity from the known activity of the test cholinesterase before step (a).
Core Innovation
The patent describes a rapid, field-deployable assay for detecting an amount of a cholinesterase inhibitor in a sample. The assay recovers a cholinesterase inhibitor from the sample using an agent that has reactivity towards phosphyl moieties, thereby recovering phosphyl-bound inhibitors from the sample into an isolated form. The recovered inhibitor is then used to inhibit a test cholinesterase whose activity before contact is known, and the cholinesterase activity is measured to determine the amount of inhibitor based on the inhibition relative to the known activity.
A key element of the invention is reactivation and recovery of phosphyl-bound inhibitors by contacting the sample with a phosphyl-reactive agent, notably fluoride/oxime-type chemistry, at an acidic pH. After recovery, the recovered cholinesterase inhibitor is isolated from the sample and then contacted with a test cholinesterase, and inhibitor amount is determined by measuring cholinesterase activity and calculating inhibition from the known pre-contact activity.
The disclosed workflow supports a chromogenic Ellman-type readout using immobilized test cholinesterase and quantifying inhibition via spectrophotometric measurement, including measurement around a wavelength such as about 412 nm using a DTNB chromogen. The disclosure further includes supporting components and processing such as acid and additives, use of filtration-based separation for agent removal, optional immobilized cholinesterase stability options, and evaluation and confirmation by non-specific inhibition thresholding and analytical confirmation approaches.
Claims Coverage
The only independent claim provided is clm-00001, which covers a method for detecting the amount of a cholinesterase inhibitor by phosphyl-reactive recovery, isolation, contact with a test cholinesterase of known activity, and measuring cholinesterase activity to determine inhibitor amount from inhibition.
Phosphyl-reactive recovery and isolation of cholinesterase inhibitor
Recover the cholinesterase inhibitor from the sample using an agent having reactivity towards phosphyl moieties, and isolate the recovered cholinesterase inhibitor from the sample.
Known-activity test cholinesterase contact and inhibition-based quantification
Contact the isolated recovered cholinesterase inhibitor with a test cholinesterase whose activity before contact is known, and measure the cholinesterase activity to determine the amount of cholinesterase inhibitor based on inhibition of the test cholinesterase activity from the known activity.
The provided independent claim requires recovering and isolating a phosphyl-reactive, recovered cholinesterase inhibitor and determining inhibitor amount by measuring inhibition of a test cholinesterase with known pre-contact activity.
Stated Advantages
Rapid, field-deployable assay for detecting cholinesterase inhibitors.
Determination of inhibitor amount based on measured inhibition of a test cholinesterase activity relative to known activity.
Use of immobilized test cholinesterase with a chromogenic Ellman-type readout enabling spectrophotometric measurement.
Documented Applications
Detection of cholinesterase inhibitors including OP nerve agents such as sarin/VX/Tabun and organophosphate pesticide dichlorvos.
Evaluation of robustness relative to prophylactic/treatment drugs including pyridostigmine and 2-pralidoxime chloride.
Analytical confirmation of detected inhibitors using SPE-GCMS as documented in the disclosure.
Interested in licensing this patent?