Automated imaging and analysis of the hemagglutination inhibition assay (HAI)
Inventors
Nguyen, Michael • Parkhill, Robert
Assignees
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Publication Number
US-10502730-B2
Publication Date
2019-12-10
Expiration Date
Abstract
A system and method provide for high through put determination of agglutination states. The system includes a rotating table and multiple plate tilting stations. The system also includes one or more optical paths positioned to image entire plate arrays in tilted and/or untitled configurations. The system preferably includes image analysis software to analyze an image of an array of test wells and determine an agglutination state of each well based on the image analysis.
Core Innovation
The invention provides a method for determining functional binding activity of an antibody using an agglutination-based assay in an array of test wells on a plate. A sample is prepared consisting of an agglutinating factor and the antibody in at least one well, and a target object is added under conditions permitting agglutination of the target object by the agglutinating factor. The approach establishes functional binding activity based on whether agglutination of the target object occurs in the wells.
An imaging system is used to prepare an image of the plate sample with the target object added. The system includes an optical path with a camera, a lens, and a mirror, and a rotating table with a plurality of plate holding stations. Each plate holding station includes a plate holder for holding a plate comprising an array of test wells and a tilt motor for adjusting a tilt angle of the plate holder, while a controller controls the table motor, tilt motor, and camera to acquire at least one image of at least one plate with the plate disposed in the plate holder.
A programmed processing device processes the image to determine agglutination of the target object and thereby determine functional binding activity of the antibody. The disclosed implementation performs computer-vision analysis of agglutination and streaming patterns in multiwell plates by defining well masks, segmenting agglutination patterns using L*a*b* color gating, computing an HA-parameter, classifying wells, assigning titers, detecting outliers, and selecting an optimal kinetic read time. The HA-parameter enables continuous dose-response fitting and IC50-style characterization beyond discrete titers, with reported concordance versus manual reading and strain independence of fixed algorithm thresholds.
Claims Coverage
The document contains one independent claim directed to determining functional binding activity of an antibody via an imaging and programmed image-processing workflow. The claim set includes dependent claims that refine imaging configuration, geometry, and automation features.
Imaging agglutination in a multiwell plate to determine functional binding activity
A method that prepares a sample with an agglutinating factor and the antibody in at least one well of a plate comprising an array of test wells, adds a target object under conditions permitting agglutination, prepares an image of the sample with the target object added, and processes the image to determine agglutination of the target object and thereby determine functional binding activity of the antibody.
Rotating table with tiltable plate holding stations controlled for plate imaging
A system with an optical path and a rotating table comprising a plurality of plate holding stations and a table motor to move any plate holding station into alignment with the optical path, where each plate holding station includes a plate holder for holding a plate comprising an array of test wells and a tilt motor for adjusting a tilt angle of the plate holder, with a controller controlling the table motor, tilt motor, and camera to acquire at least one image of at least one plate.
Optical path using camera, lens, and mirror
An optical path comprising a camera, a lens, and a mirror, where the plate disposed in the plate holder is imaged to acquire the at least one image for subsequent image processing to determine agglutination and functional binding activity.
Programmed image processing to determine agglutination from acquired images
A programmed processing device that processes the image of the plate sample to determine agglutination of the target object, and thereby determine functional binding activity of the antibody.
Antibody functional binding determination with a constrained imaging tilt-angle range
The method where the angle is between about 0 degrees and about 70 degrees.
Bottom-up plate imaging geometry
The method where the assay plates are imaged from the bottom up.
Optical path including a telecentric lens
The method where the optical path includes a telecentric lens.
Simultaneous tilting of multiple plates
The method where the assay plates are used with simultaneous tilting of up to six plates.
Automated plate handling using a robotic arm
The method where an automated plate handler is implemented as a robotic arm.
Overall, the inventive coverage centers on imaging a multiwell agglutination assay using a controlled rotating table with tiltable plate holding stations and an optical path with camera, lens, and mirror, followed by programmed image processing to determine agglutination and thereby determine functional binding activity of the antibody, with dependent refinements including a tilt-angle range, bottom-up imaging, telecentric lens optics, simultaneous tilting of multiple plates, and robotic-arm plate handling.
Stated Advantages
Concordance with manual readout.
Strain independence of fixed algorithm thresholds.
Enables continuous dose-response fitting and IC50-style characterization beyond discrete titers.
Documented Applications
Automated, high-throughput hemagglutination inhibition assay imaging and analysis using multiwell plate imaging and computer-vision classification of agglutination and streaming patterns.
Characterization of influenza antigens (H1N1, H3N2, B; ether-treated B) using antibody functional binding activity determination.
Red blood cell species referred to as turkey, chicken, horse, guinea pig, and human type O in the described assay context.
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