Immunoassay methods utilizing trapping conjugate
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Publication Number
US-9891216-B2
Publication Date
2018-02-13
Expiration Date
Abstract
Methods are provided for determining the presence of a first ligand in a sample. In some embodiments depletion conjugates are used to deplete the ligands different from but related to the first ligands from the sample. In some embodiments, interim binding agents are used to enhance the test signal.
Core Innovation
The disclosed invention relates to dual-path lateral flow immunoassay devices and methods for detecting a first ligand in a sample that contains related ligands. The test device includes a first sorbent strip with a first migration path for carrying a marker conjugate adapted to bind the first ligand, and a second sorbent strip with a second migration path for carrying the sample toward a test site. At the test site, an immobilized ligand-binding mechanism is provided for binding the first ligand.
A second, related-ligand binding functionality is placed in the sample migration path as depletion molecules including an immobilized ligand-binding element adapted to bind second ligands that are different from but related to the first ligand. This depletion structure is positioned so that the sample applied to the second location requires time to migrate to the test site and does not immediately wet the test site. As a result, interfering related ligands are bound and removed before reaching the test line, increasing sensitivity/specificity.
The disclosed embodiments describe the use of particles in the marker conjugate, including white latex particles or white latex beads, that reduce visibility against the background of the test site, and optional design features such as pore size or membrane pore design to prevent conjugate movement. The disclosure further presents example applications for influenza, including differentiating pandemic versus seasonal flu antibody/antigen behavior, and includes kit-related configurations with downstream use that involves filtration and interim binding agent signal enhancement embodiments for HIV p24 antigen detection.
Claims Coverage
The document includes one independent claim that defines a dual-sorbent, dual-path testing method with a depletion function in the sample path, followed by test-site inspection through a window. The independent claim specifies multiple inventive features including the separate migration paths, time-separated wetting of the test site, immobilized ligand-binding depletion elements, and an inspection configuration with openings and a viewable test site.
Dual-sorbent dual-path device with separated migration paths
A test device having a first sorbent strip with a first location defining a first migration path and a marker conjugate adapted to bind a first ligand, and a second sorbent strip distinct from the first having a second location defining a second migration path, with a test site located on or in at least one of the first and second sorbent strips and having an immobilized ligand-binding mechanism for the first ligand.
Sample-path depletion of related ligands before the test site
Depletion molecules located on or in the second migration path, including an immobilized ligand-binding element adapted to bind second ligands that are different from but related to the first ligand, configured so the sample requires time to migrate to the test site and does not immediately wet the test site.
Two-step application sequence for marker migration and test readout
Applying the sample to the second location, then after applying the sample applying a solution to the first location to cause the marker conjugate to migrate along the first migration path, and inspecting the test site to determine an indication the presence or lack thereof of the first ligand in the sample.
Conjugate depletion with particles not readily seen and window-based inspection
The depletion molecules comprise a conjugate including an antigen or antibody for the second ligand and particles that are not readily seen by the human eye against a background of the test site, wherein the test device has a housing defining a first opening adjacent the first location, a second opening adjacent the second location, and a window adjacent the test site through which the test site is viewable, and inspecting comprises inspecting through the window.
Influenza antibody pair with pandemic and non-pandemic ligands
The method wherein the first ligand is a first flu antibody and the second ligand is a second flu antibody different than the first flu antibody.
Overall, the independent claim covers a dual-sorbent lateral flow testing method in which the sample path includes immobilized depletion molecules for related second ligands, timed separation prevents immediate wetting of the test site, and a marker conjugate migrates later along a separate path for inspection through a windowed housing.
Stated Advantages
Increases sensitivity/specificity by binding and removing interfering related antibodies/antigens before they reach the test line.
Documented Applications
Influenza detection using a dual-path lateral flow immunoassay device to support distinguishing behavior associated with pandemic versus seasonal flu antibody/antigen pairs.
HIV p24 antigen detection using interim binding agent signal enhancement embodiments with interim binding agents including biotin/streptavidin and anti-biotin antibody configurations (single and double interim binding).
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