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Publication Number
US-12339284-B2
Publication Date
2025-06-24
Expiration Date
Abstract
Disclosed herein are assay methods, lateral flow assay test strips, and devices for improved analyte detection. Analyte binding to target is performed both in solution phase and with a target immobilized on a surface, resulting in improved analyte detection.
Core Innovation
The invention provides an assay method for detecting an analyte in a liquid biological sample by combining ligand-target conjugates and receptor-label conjugates with a surface that has immobilized capture agents. In a liquid phase, ligand-target conjugates bind at least a fraction of analytes to form ligand-target-analyte complexes, while a fraction of the analytes remain unbound. The unbound analyte and the ligand-target-analyte complexes are then contacted with a receptor-label conjugate to form analyte-receptor-label complexes and ligand-target-analyte-receptor-label complexes.
The captured complexes are generated on a surface that comprises immobilized capture agents having two fractions with different pre-configurations. A fraction of the immobilized capture agents are pre-complexed with ligand-target conjugates so that the pre-complexed ligand-target conjugates bind the analyte-receptor-label complexes to form captured ligand-target-analyte-receptor-label complexes on the surface. Another fraction of immobilized capture agents are not complexed with ligand-target conjugates and bind the ligand-target-analyte-receptor-label complexes to form captured ligand-target-analyte-receptor-label complexes on the surface.
The method is implemented in a lateral flow assay strip architecture with a sample receiving area, a blocker area downstream comprising ligand-target conjugates, and a conjugate area downstream comprising receptor-label conjugates. At least one test zone downstream includes immobilized capture agents where some capture agents are pre-complexed with ligand-target conjugates and capture analyte-receptor-label complexes, while other capture agents remain uncomplexed and capture ligand-target-analyte-receptor-label complexes. Detection is performed by detecting the ligand-target-analyte-receptor-label complexes captured on the surface, including configurations used for analytes such as anti-HIV antibodies with HIV-1/HIV-2 peptide antigens and recombinant antigens.
Claims Coverage
The provided material includes two independent claims: one assay method claim and one lateral flow assay strip claim. Across these, the central coverage is organized into two key inventive components: (1) forming ligand-target-analyte complexes in a liquid phase and then converting them into analyte-receptor-label and ligand-target-analyte-receptor-label complexes, and (2) capturing both complex types on immobilized capture agents using a surface where one fraction is pre-complexed with ligand-target conjugates and another fraction is not pre-complexed. Overall, the independent claims define the main inventive features in a way that supports both a general assay method and a specific lateral flow strip format.
Liquid phase formation of ligand-target-analyte complexes with unbound fraction
Contacting the liquid biological sample suspected of containing analytes with ligand-target conjugates in a liquid phase, wherein the ligand-target conjugates bind to at least a fraction of the analytes to form ligand-target-analyte complexes, and wherein a fraction of the analytes remain unbound to the ligand-target conjugate
Conversion to analyte-receptor-label and ligand-target-analyte-receptor-label complexes
Contacting the unbound analyte and the ligand-target-analyte complexes with a receptor-label conjugate, to form analyte-receptor-label complexes and ligand-target-analyte-receptor-label complexes
Dual-fraction capture on immobilized capture agents with pre-complexed ligand-target conjugates
Capturing the analyte-receptor-label complexes and ligand-target-analyte-receptor-label complexes on a surface comprising immobilized capture agents, wherein (i) a fraction of the immobilized capture agents are pre-complexed with ligand-target conjugates, wherein the pre-complexed ligand-target conjugates bind to the analyte-receptor-label complexes to form captured ligand-target-analyte-receptor-label complexes on the surface; and (ii) another fraction of the immobilized capture agents are not complexed with ligand-target conjugates and bind the ligand-target-analyte-receptor-label complexes to form captured ligand-target-analyte-receptor-label complexes on the surface
Lateral flow strip with blocker, conjugate area, and test zone using pre-complexed and uncomplexed capture fractions
A lateral flow assay strip for detecting an analyte in a liquid biological sample comprising a sample receiving area; a blocker area downstream from the sample receiving area comprising ligand-target conjugates for forming ligand-target-analyte complexes, wherein the ligand-target conjugates are specific for the analyte; a conjugate area downstream from the blocker area, comprising receptor-label conjugates for forming ligand-target-analyte-receptor-label complexes and analyte-receptor-label complexes, wherein the receptor-label conjugates are specific for any unbound analyte and the ligand-target-analyte complexes; and at least one test zone downstream from the conjugate area comprising immobilized capture agents, wherein (i) a fraction of the immobilized capture agents are pre-complexed with ligand-target conjugates and capture analyte-receptor-label complexes; and (ii) another fraction of the immobilized capture agents are not complexed with ligand-target conjugates and capture ligand-target-analyte-receptor-label-complexes
The independent claims cover an assay in which analyte binding is performed in a liquid phase to form ligand-target-analyte complexes with an unbound fraction, followed by formation of analyte-receptor-label and ligand-target-analyte-receptor-label complexes. Both complex types are captured on immobilized capture agents using a surface or test zone that has two fractions: one fraction pre-complexed with ligand-target conjugates and another fraction not pre-complexed. A corresponding lateral flow strip implementation is claimed with blocker and conjugate areas and a test zone that captures each complex type via the pre-complexed versus uncomplexed capture agent fractions.
Stated Advantages
Solution-phase binding enables significantly lower peptide amounts with similar/maintained reactivity (more than 5× lower).
Recombinant antigen (rAg) shows improved solution-phase contribution versus solid-phase-only striping.
Increasing the fraction of peptides pre-stripped on the nitrocellulose test line yields increased reactivity in HIV EIA positive samples while preserving negativity in HIV-negative samples.
Documented Applications
Detection assays involving anti-HIV antibodies, including configurations using HIV-1/HIV-2 peptides and recombinant antigen (rAg) (including examples related to OraQuick ADVANCE Rapid HIV-1/2).
Use of lateral flow assay strip formats with ligand-target conjugates and receptor-label conjugates to detect an analyte in a liquid biological sample, including examples and performance discussion for HIV EIA positive and HIV-negative samples.
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