Screening assays and methods

• US 7,776,553 B2
• Filed: 09/18/2006
• Issued: 08/17/2010
• Est. Priority Date: 09/16/2005
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Ploegh, Hidde L., Ronan, Jehnna, Love, J. Christopher
• Primary Examiner(s): Jung; Unsu
• Application Number:
  US11/523,124
• Publication Number:
  US 20100152054A1
• Time in Patent Office: 1,429 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00734 Lipids
• CPC Class Codes: B01J 2219/0074 Biological products
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/5052 involving B-cells
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/577 : involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: G01N 33/6854 : Immunoglobulins
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/24 : Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

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.

Claims

1. A method of screening a monoclonal antibody 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 antibody-producing cell that secretes monoclonal antibodies in a volume of 10 nanoliters or less of fluid in said microwell allowing said antibody-producing cell to secrete said monoclonal antibodies in said volume;

exposing said volume of said monoclonal antibodies to at least one defined antigen, and

determining if said monoclonal antibodies binds to said antigen, wherein the steps of the method of screening the monoclonal antibody are performed in less than one day.

2. The method of claim 1, wherein the method of screening the monoclonal antibody is performed in less than 6 hours.

3. The method of claim 1, wherein the substrate is pretreated with the defined antigen.

4. The method of claim 1, wherein the substrate is pretreated with a secondary antibody.

5. The method of claim 1, wherein the moldable slab comprises a microwell array, wherein at least one microwell of the microwell array comprises the antibody-producing cell that secretes the monoclonal antibody.

6. The method of claim 5, wherein the microwell array is incubated for less than 24 hours while contacting the substrate.

7. The method of claim 5, wherein the microwell array is incubated for less than 5 hours while contacting the substrate.

8. The method of claim 5, wherein the microwell array is incubated for less than 2 hours while contacting the substrate.

9. The method of claim 1, wherein the monoclonal antibody and the defined antigen associate on the substrate forming a microarray.

10. The method of claim 9, wherein the microarray on the substrate is removed from the moldable slab and is placed in a reservoir containing a medium.

11. The method of claim 10, wherein the hydridoma in the microwells is maintained in the medium and the microwell array on the moldable slab contacts a new substrate and forms at least one new microarray.

12. The method of claim 11, wherein between 5 and 100 new microarrays are formed.

13. The method of claim 1, wherein the antibody bound to the defined antigen is detected.

14. The method of claim 1, further comprising determining whether the monoclonal antibody binds to a defined secondary antibody on the substrate.

15. The method of claim 1, wherein said antigen comprises an intact pathogen.

16. The method of claim 1, wherein said antigen comprises a cell lysate.

17. The method of claim 15, wherein said intact pathogen is a bacteria, virus, or fungus.

18. The method of claim 1, wherein said antibody-producing cell is a hybridoma cell.

19. The method of claim 1, wherein said antibody-producing cell is a primary B cell.

20. The method of claim 1, wherein said volume is 1 picoliter to 1 nanoliter.

21. The method of claim 1, wherein said determining step is carried out by fluorescence detection, mass spectrometry, surface plasmon resonance, or a colorimetric assay.

22. The method of claim 1, wherein antibody-antigen binding is detected using a fluorescence microscope, fluorimeter, or camera.

23. The method of claim 1, wherein said antibody-antigen binding is detected using an instrument comprising a charge coupled device, a photomultiplier tube, or a diode array.

24. The method of claim 1, wherein the steps of the method of screening the monoclonal antibody are performed in less than 12 hours.

25. The method of claim 5, wherein the monoclonal antibody associates with the substrate to form a microarray.

26. The method of claim 13, wherein antibody-antigen binding is identified using a detectable marker.

27. The method of claim 26, wherein the detectable marker comprises a fluorescent label, a colorimetric label, or a radiolabel.

28. The method of claim 26, wherein the detectable marker is a radiolabel.

29. The method of claim 27, wherein the radiolabel comprises ³H, ¹⁴C, ³²P, ³³P, ³⁵S or ¹²⁵I.

30. The method of claim 1, wherein the cell is retrieved from the moldable slab.