SCREENING ASSAYS AND METHODS
Screening assays and methods of performing such assays are provided. In certain examples, the assays and methods may be designed to determine whether or not two or more species can associate with each other. In some examples, the assays and methods may be used to determine if a known antigen binds to an unknown monoclonal antibody.
1 .- 25 . (canceled)
26 . A method of screening a secreted product comprising:
sealing a moldable slab to a substrate, said moldable slab comprising at least one microwell of less than 100 micrometers in diameter, each of said microwells comprising a single cell that produces a secreted product in a volume of 10 nanoliters or less of fluid in said microwell,
allowing said cell to secrete said product in said volume;
exposing said volume of said product to at least one defined ligand, and
determining if said secreted product binds to said ligand, wherein the steps of the method of screening secreted products are performed in less than one day.
27 . The method of claim 26 , wherein the method of screening is performed in less than 6 hours.
28 . The method of claim 26 , wherein the substrate is pretreated with the defined ligand.
29 . The method of claim 26 , wherein the substrate is pretreated with a secondary antibody.
30 . The method of claim 26 , wherein the moldable slab comprises a microwell array, wherein at least one microwell of the microwell array comprises the cell that produces the secreted product.
31 . The method of claim 30 , wherein the microwell array is incubated for less than 24 hours while contacting the substrate.
32 . The method of claim 30 , wherein the microwell array is incubated for less than 5 hours while contacting the substrate.
33 . The method of claim 30 , wherein the microwell array is incubated for less than 2 hours while contacting the substrate.
34 . The method of claim 23 , wherein the secreted product and the defined ligand associate on the substrate forming a microarray.
35 . The method of claim 34 , wherein the microarray on the substrate is removed from the moldable slab.
36 . The method of claim 35 , wherein cells in the microwells are maintained in the medium and the microwell array on the moldable slab contacts a new substrate and forms at least one new microarray.
37 . The method of claim 36 , wherein between 5 and 100 new microarrays are formed.
38 . The method of claim 26 , wherein the secreted bound to the defined ligand is detected.
39 . The method of claim 26 , wherein said cell is a primary cell.
40 . The method of claim 26 , wherein said cell is a cancer cell.
41 . The method of claim 26 , wherein said cell is of a tissue type selected from the group consisting of heart, brain, liver prostate, breast, kidney, and colon.
42 . The method of claim 26 , wherein said cell is an immune cell.
43 . The method of claim 42 , wherein said immune cell is a T cell or a B cell.
44 . The method of claim 23 , wherein said secreted product comprises a cytokine, chemokine, antibody, or growth factor.
45 . The method of claim 44 , wherein said secreted product is selected from the group consisting of tumor necrosis factor alpha (TNF-α), immunoglobulin E (IgE), interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-2, IL-4, IL-10, and IL-13.
46 . The method of claim 26 , wherein said secreted product is a recombinant polypeptide or a recombinant immunoglobulin.
47 . The method of claim 26 , wherein said secreted product comprises a bacteria, yeast, parasite, or virus.
48 . The method of claim 26 , wherein said volume is 1 picoliter to 1 nanoliter.
49 . The method of claim 26 , wherein said determining step is carried out by fluorescence detection, mass spectrometry, surface plasmon resonance, or a colorimetric assay.
50 . The method of claim 26 , wherein secreted product-ligand binding is detected using a fluorescence microscope, fluorimeter, or camera.
51 . The method of claim 26 , wherein said secreted product-ligand binding is detected using an instrument comprising a charge coupled device, a photomultiplier tube, or a diode array.
52 . The method of claim 26 , wherein the steps of the method of screening are performed in less than 12 hours.
53 . The method of claim 30 , wherein the monoclonal antibody associates with the substrate to form a microarray.
54 . The method of claim 38 , wherein the secreted product bound to the defined ligand is identified using a detectable marker.
55 . The method of claim 54 , wherein the detectable marker comprises a fluorescent label, a colorimetric label, or a radiolabel.
56 . The method of claim 54 , wherein the detectable marker is a radiolabel.
57 . The method of claim 56 , wherein the radiolabel comprises 3 H, 14 C, 32 P, 33 P, 35 S or 125 I.
58 . The method of claim 26 , wherein the cell is retrieved from the moldable slab.
59 . The method of claim 26 , further comprising characterization of said cell after the capture of said secreted product to determine a phenotype of said cell.
60 . The method of claim 59 , wherein said phenotype is determined using immunofluorescence labeling or genetic sequencing.