Sequential lateral flow device
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.
1. A method for detecting a plurality of target analytes in a liquid sample using a lateral flow device, the method comprising:
(a) contacting 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;
(b) contacting 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 in the liquid sample, 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; and
(c) detecting a signal generated by the formation of the detection complexes to determine the presence of the plurality of target analytes,
wherein 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.
2. The method of claim 1 , wherein the contacting in step (a) comprises:
contacting the liquid sample with the plurality of binding agents under conditions that permit formation of a first complex between at least some of the target analytes and at least some of the binding agents; and
contacting the first complex with the plurality of capture agents to form the sandwich complex, such that the liquid sample contacts the plurality of capture agents only after formation of the first complex.
3. The method of claim 1 , wherein the contacting in step (a) comprises:
contacting the liquid sample with the plurality of capture agents under conditions that permit formation of a complex between at least some of the target analytes and at least some of the capture agents; and
contacting the complex with the plurality of binding agents to form the sandwich complex, such that the binding agents contact the target analytes only after formation of the complex.
4. The method of claim 1 , wherein the lateral flow device comprises a substantially impermeable backing disposed between a sample-receiving pad in a sample-receiving zone for introducing the liquid sample into the lateral flow device and a capillary flow bed in the solid support that facilitates a flow of the liquid sample, thereby reducing backflow of the liquid sample in a proximal direction of the lateral flow device.
5. The method of claim 1 , wherein the plurality of capture agents comprise more than one type of capture agents, each adapted to specifically bind to a common antigen of at least the subset of target analytes in the liquid sample.
6. The method of claim 1 , wherein the plurality of capture agents bind a Gram-positive and/or Gram-negative bacterial antigen.
7. The method of claim 1 , wherein the plurality of capture agents comprise one or more capture agent groupings on the solid support, the groupings spatially separated from each other, each grouping comprising an antibody that binds to a different target analyte.
8. The method of claim 1 , wherein the binding agents comprise more than one type of binding agents, each adapted to specifically bind to a common antigen of at least the subset of target analytes in the liquid sample.
9. The method of claim 1 , wherein the binding agents bind to a Gram-positive and/or Gram-negative bacterial antigen.
10. The method of claim 1 wherein at least some of the binding agents are different types of antibodies from at least some of the capture agents.
11. The method of claim 4 , wherein the lateral flow device comprises a housing unit, wherein the upper inner surface of the housing unit comprises a series of ribs to contain liquid in excess of the capacity of the sample-receiving pad, thereby inhibiting the excess fluid from overflowing the sample-receiving pad.
12. The method of claim 1 , wherein the conjugate pair comprises biotin and a biotin-binding protein, and either each of the binding agents or each of the signaling agents is labeled with biotin.
13. The method of claim 12 , wherein the biotin-binding protein is selected from the group consisting of avidin, neutravidin, anti-biotin antibody, streptavidin, and other biotin binding proteins.
14. The method of claim 1 , wherein the signaling agent is selected from of the group consisting of metallic particles, fluorescent dyes, and latex particles.
15. The method of claim 1 , wherein the sample is pre-treated using a base digestion followed by neutralization.
16. The method of claim 15 , wherein the pre-treated sample is mixed with the binding agents.