Sequential lateral flow device
Inventors
Vallejo, Yli Remo • Lawrence, Gregory M. • Lousararian, Adam P. • Shinefeld, Lisa
Assignees
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Publication Number
US-11293922-B2
Publication Date
2022-04-05
Expiration Date
Abstract
The present disclosure provides methods and lateral flow devices for detecting a plurality of target analytes in a liquid sample. In some implementations, the disclosed lateral flow device comprises a housing unit, a capillary flow bed, a sample-receiving zone, a buffer-receiving zone, and a capture zone. The device is configured to control the flow of the sample and reagent buffer in a sequential manner with minimal mixing. In some implementations, the disclosed method is capable of detecting a plurality of target analytes in an assay by applying the binding agents and the signaling agents in separate or sequential steps.
Core Innovation
The invention describes a sequential lateral flow device and a corresponding multiplex immunometric (sandwich) lateral flow assay for detecting multiple target analytes in a liquid sample. The method forms sandwich complexes in which a binding agent, a target analyte, and a capture agent are brought together to provide detection complexes. The target analytes comprise a plurality of bacterial antigens, viral antigens, or fungal antigens.
The binding agents are tagged with one member of a conjugate pair, while a non-enzymatic signaling agent is tagged with the second member of the conjugate pair. The signaling agent is prevented from contacting the plurality of binding agents prior to formation of the sandwich complex, so the signal is generated after detection complexes are formed. The assay uses conjugate-pair labeling to generate a signal indicative of the presence of the target analytes.
The invention also specifies device architecture for sequential fluid delivery and containment, including a housing unit, a capillary flow bed, a sample-receiving zone/pad and a buffer-receiving zone/pad, a conjugate pad, a capture zone with immobilized capture agents, and a reservoir pad/terminal wick, together with inlets and a reading window. Additional hardware features are described for inhibiting backflow and minimizing mixing, including impermeable backing, ribs, pinch points, and control of a capillary flow bed configuration. The approach enables multiplexing while improving sensitivity, including a reported improvement in limits of detection compared with assays where the signaling agent is directly conjugated to the binding agent.
Claims Coverage
The provided independent claim covers a multiplex sandwich lateral flow immunometric detection method using sequential inhibition of signaling-agent contact, a conjugate-pair tagging strategy, and non-enzymatic signal generation. The claim includes 3 main inventive areas: sandwich complex formation with polyclonal antibody binding agents and capture agents, delayed signaling using a non-enzymatic signaling agent tagged with a second conjugate-pair member with the device adapted to inhibit premature signaling-agent contact, and a sensitivity improvement requirement versus direct conjugation.
Non-enzymatic conjugate-pair signaling after inhibited contact
The method contacts the sandwich complex with a non-enzymatic signaling agent tagged with a second member of the conjugate pair and binding the signaling agent to the binding agent of the sandwich complex to form a detection complex which indicates the presence of target analytes, wherein the lateral flow device is adapted to inhibit the signaling agent from contacting the plurality of binding agents prior to formation of the sandwich complex.
Multiplex sandwich complex formation with polyclonal antibodies
The method contacts the liquid sample with a plurality of capture agents disposed on a solid support in the lateral flow device and a plurality of binding agents to form sandwich complexes comprising a binding agent, a target analyte, and a capture agent, wherein the binding agents are tagged with one member of a conjugate pair, the binding agents and the capture agents each comprise polyclonal antibodies, the binding agents comprise a plurality of polyclonal antibody binding agents binding to different target analytes, and the binding agents and/or the capture agents can bind to an antigen common to at least a subset of the target analytes.
Improved assay limit of detection for multiplex bacterial, viral, or fungal antigens
The plurality of target analytes comprise a bacterial antigen, a viral antigen, or a fungal antigen, and wherein the limit of detection of the assay is at least 2-fold lower than in an assay wherein the signaling agent is directly conjugated to the binding agent.
Overall, the claim coverage centers on forming multiplex sandwich complexes using polyclonal antibody binding agents and capture agents, using a conjugate pair with non-enzymatic signal generation, and adapting the lateral flow device to inhibit signaling-agent contact until after sandwich complex formation, yielding at least a 2-fold improved limit of detection versus direct conjugation.
Stated Advantages
Improved sensitivity with a limit of detection at least 2-fold lower than an assay wherein the signaling agent is directly conjugated to the binding agent.
Documented Applications
Multiplex detection of bacterial antigens, viral antigens, or fungal antigens in a liquid sample using a sequential lateral flow device and a multiplex immunometric (sandwich) assay.
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