Sandwich assays in droplets
The invention generally relates to performing sandwich assays in droplets. In certain embodiments, the invention provides methods for detecting a target analyte that involve forming a compartmentalized portion of fluid including a portion of a sample suspected of containing a target analyte and a sample identifier, a first binding agent having a target identifier, and a second binding agent specific to the target analyte under conditions that produce a complex of the first and second binding agents with the target analyte, separating the complexes, and detecting the complexes, thereby detecting the target analyte.
1. A reagent library of a plurality of stabilized fluidic compartments in a container, each fluidic compartment comprising:
a first binding agent specific for a first binding region on a target analyte, the first binding agent comprising a target identifier; and
a second binding agent specific for a second binding region on the target analyte, wherein the second binding agent comprises a functionalized portion for immobilization to a solid support;
wherein the target identifier in each respective fluidic compartment is unique; and
wherein the fluidic compartments do not contain the solid support.
2. The reagent library of claim 1 , wherein each of the plurality of fluidic compartments is a droplet.
3. The reagent library of claim 2 , wherein the droplet is surrounded by an immiscible carrier fluid.
4. The reagent library of claim 1 , wherein the functionalized portion of the second binding agent is a terminal portion of the second binding agent.
5. The reagent library of claim 1 , wherein the functionalized portion of the second binding agent comprises a biotin capture tag, a desthiobiotin capture tag, or an antigen capture tag.
6. The reagent library of claim 1 , wherein the first binding agent and the second binding agents are each independently selected from the group consisting of oligonucleotides, proteins, and antibodies.
7. The reagent library of claim 1 , wherein the first binding agent and/or the second binding agent is an antibody.
8. The reagent library of claim 1 , wherein the first binding agent and/or the second binding agent is an oligonucleotide.
9. The reagent library of claim 8 , wherein the oligonucleotide comprises a sequence substantially complementary to a target analyte and a universal sequence.
10. The reagent library of claim 9 , wherein the universal sequence is a poly(A) sequence.
11. The reagent library of claim 1 , wherein the target identifier is an optically detectable label.
12. The reagent library of claim 1 , wherein the target identifier is a barcode sequence.
13. The reagent library of claim 3 , wherein the carrier fluid is a fluorinated oil.
14. The reagent library of claim 3 , wherein the carrier fluid comprises one or more surfactants.
15. The reagent library of claim 3 , wherein the carrier fluid comprises at least one fluorosurfactant.
16. The reagent library of claim 14 , wherein the one or more surfactants are at a concentration of about 0.2 to 5% by volume.
17. The reagent library of claim 1 , wherein the container is a vial.
18. The reagent library of claim 1 , wherein the container is a well in a plate.