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Patent Issued for Method of and System for Enhanced Dynamic Range Assay Analysis

September 2, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Honkanen, Peter (Concord, MA); Douglas, Scott (Exeter, RI); Jung, Jr., Ralph H. (Centennial, CO), filed on April 4, 2012, was published online on August 19, 2014 (see also Aushon Biosystems, Inc.).

The patent's assignee for patent number 8811704 is Aushon Biosystems, Inc. (Billerica, MA).

News editors obtained the following quote from the background information supplied by the inventors: "The present disclosure relates to assay analysis, and, more specifically, to methods and systems for analyzing assay results using enhanced dynamic ranges.

"Antibody-based immunoassays are used for applications such as biomarker verification or validation. One modern medical diagnostic testing technique is an enzyme-linked immunosorbent assay (ELISA). Generally, in an ELISA, a capture antibody is printed in the bottom of a reaction well in an assay substrate. An assay substrate is a surface upon which various chemical and/or biological analyses can be performed. Examples of an assay substrate include microarray plates, glass slides, and microtiter plates. A microtiter plate is a flat plate that has multiple 'wells' formed in its surface. Each reaction well can be used as a small test tube into which various materials can be placed to perform biochemical analyses.

"In an ELISA, the capture antibody has specificity for a particular antigen for which the assay is being performed. A sample to be analyzed is added to the well containing the capture antibody, and the capture antibody 'captures' or immobilizes the antigen contained in the sample. A detect antibody is then added to the well, which also binds and/or forms a complex with the antigen. Further materials are then added to the well that cause a detectable signal to be produced by the detect antibody. For example, when light of a specific wavelength is shone upon the well, the antigen/antibody complexes will fluoresce. The amount of antigen in the sample can be inferred based on the magnitude of the fluorescence. In another example, a compound can be added to the well that causes the detect antibody to emit light within a predetermined wavelength (e.g., 400-500 nm) when properly energized by a suitable source. This light can be read by an optical detector, such as a charged-coupled device (CCD) camera or CMOS sensors, to measure the optical brightness or intensity of the emitted light. During an ELISA, the absorbency, fluorescence, or electrochemical signal of the well can be measured with suitable detection and processing equipment and compared with a standard to determine the presence and quantity of the sample antigen.

"Assay performance involves the ability of the assay to precisely and accurately detect analytes in the sample. A singleplex immunoassay quantifies one analyte per assay, while a multiplex assay simultaneously measures multiple analytes in a single assay. Multiplex assays are used, for example, in functional genomics experiments that detect the presence of biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample.

"Factors that can influence the accuracy of assay detection include antibody pairs, binding between antibody and capture surface, signal amplification, and range of signal linearity detection. Because more variables are involved with multiplex assays as compared to singleplex assays, multiplex assays can be more prone to error. For example, cross-reactivity can occur in multiplex wells, as multiple pairs of capture antibodies and detectors are mixed in a single reaction well. Both pre-analytical error, for example, sample degradation or matrix heterogeneity, and analytical error can affect the accuracy and reliability of the detection signal."

As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "The disclosed systems and methods allow composite images with enhanced dynamic range to be generated that result in more accurate, reliable, and efficient chemical and/or biological analyses.

"In one aspect, the disclosed methods comprise (a) obtaining multiple images of pixels representing light intensity output from an assay, wherein the light intensity output indicates a presence and an amount of a substance within wells of a test plate, and wherein the multiple images of pixels are generated by using a detector to detect the light intensity of the pixels of the wells using a standard exposure time; and a longer-than-standard exposure time; and (b) generating a composite image using the multiple images of pixels, including: determining an exposure compensation ratio based on the standard exposure time and the longer-than-standard exposure time; determining a presence of out-of-range pixels in the image that used the longer-than-standard exposure time, wherein the out-of-range pixels have light intensities that exceed a threshold based on a maximum capacity of the detector; placing out-of-range pixels in the composite image at a same relative position as the out-of-range pixels occurred in the image that used the longer-than-standard exposure time, wherein the light intensities of the out-of-range pixels are based on the light intensities from the image that used the standard exposure time and the exposure compensation ratio; determining a presence of in-range pixels in the image that used a longer-than-standard exposure time, wherein the in-range pixels have light intensities that are below the threshold; and placing in-range pixels in the composite image at a same relative position as said in-range pixels occurred in the image that used the longer-than-standard exposure time, wherein the light intensities of said in-range pixels are based on the light intensities from the image that used the longer-than-standard exposure time adjusted for light intensity decay due to a passage of time between the image that used the standard exposure time and the image that used the longer-than-standard exposure time.

"In one or more embodiments, the method further comprises using the composite image to determine the presence and the amount of the substance within the wells.

"In one or more embodiments, the longer-than-standard exposure time in the disclosed methods is about 2 to about 10 times the standard exposure time.

"In one or more embodiments, the methods further comprise applying an image calibration procedure to the multiple images of pixels generated in step (a), wherein the image calibration procedure includes subtracting dark noise and applying flat field correction.

"In one or more embodiments, the methods further comprise obtaining one or more of the composite images; combining the pixels in one or more of the composite images with pixels at a same relative position in other composite images to generate a template image of the wells, wherein the composite pixels at the same relative positions represent anticipated positions of printed features in a test plate; and using the template image to find a location of a feature within the test plate, wherein the location indicates presence and amount of a substance in the test plate.

"In one aspect, the disclosed methods comprise obtaining multiple pixel images representing light intensity output from one or more assays, wherein the wells include features printed in the wells of a testing substrate; combining the multiple pixel images with pixels at a same relative position in other images to generate a template image of the wells, wherein pixels of the template image at the same relative positions represent anticipated positions of printed features in a test plate; and using the template image to find a location of a feature within the test plate, wherein the location indicates a presence and an amount of a substance in the test plate.

"In one aspect, the disclosed systems comprise an image detector; a timer for tracking exposure time of the image detector; and computer readable medium, including instructions that when executed cause a computer system to generate a composite image using the multiple images of pixels by: (a) obtaining multiple images of pixels representing light intensity output from an assay, wherein the light intensity output indicates a presence and an amount of a substance within wells of a test plate, and wherein the multiple images of pixels are generated by using a detector to detect the light intensity of the pixels of the wells using a standard exposure time; and a longer-than-standard exposure time; and (b) generating a composite image using the multiple images of pixels, including: determining an exposure compensation ratio based on the standard exposure time and the longer-than-standard exposure time; determining a presence of out-of-range pixels in the image that used the longer-than-standard exposure time, wherein the out-of-range pixels have light intensities that exceed a threshold based on a maximum capacity of the detector; placing out-of-range pixels in the composite image at a same relative position as the out-of-range pixels occurred in the image that used the longer-than-standard exposure time, wherein the light intensities of the out-of-range pixels are based on the light intensities from the image that used the standard exposure time and the exposure compensation ratio; determining a presence of in-range pixels in the image that used a longer-than-standard exposure time, wherein the in-range pixels have light intensities that are below the threshold; and placing in-range pixels in the composite image at a same relative position as said in-range pixels occurred in the image that used the longer-than-standard exposure time, wherein the light intensities of said in-range pixels are based on the light intensities from the image that used the longer-than-standard exposure time adjusted for light intensity decay due to a passage of time between the image that used the standard exposure time and the image that used the longer-than-standard exposure time.

"In one or more embodiments, the composite image in the systems is used to determine the presence and amount of the substance within the wells.

"In one or more embodiments, the longer-than-standard exposure time in the systems is about 2 to about 10 times the standard exposure time.

"In one or more embodiments, the multiple images of pixels in the systems are processed using a calibration procedure, including subtracting dark noise and applying flat field correction."

For additional information on this patent, see: Honkanen, Peter; Douglas, Scott; Jung, Jr., Ralph H.. Method of and System for Enhanced Dynamic Range Assay Analysis. U.S. Patent Number 8811704, filed April 4, 2012, and published online on August 19, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8811704.PN.&OS=PN/8811704RS=PN/8811704

Keywords for this news article include: Aushon Biosystems Inc.

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Source: Life Science Weekly


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