High throughput cell-based assays, methods of use and kits

• US 7,955,867 B2
• Filed: 01/18/2008
• Issued: 06/07/2011
• Est. Priority Date: 01/31/2007
• Status: Expired due to Fees

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Millipore Corporation
• Inventors: Park, Jun Y.
• Primary Examiner(s): GABEL, GAILENE
• Application Number:
  US12/009,508
• Publication Number:
  US 20090081698A1
• Time in Patent Office: 1,236 Days
• Field of Search: 435/2, 435/7.1, 435/7.2, 435/7.92, 435/374, 435/401, 435/287.2, 435/288.4, 435/288.6, 435/40.5, 435/383, 435/395, 435/398, 436/518, 436/523, 436/528, 436/538, 436/172, 436/175, 436/177, 436/178
• US Class Current: 436/538
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2400/0457 passive flow or gravitation
• CPC Class Codes: B01L 3/50255 Multi-well filtration
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

In the present invention cells are placed in a multiwell plate and grown. When the assay is to be performed, one uses gravity to wash away any unbound ligands rather than vacuum or centrifugation. The cells are then examined to detect the bound ligand. To perform the washing step(s) the plate is placed into a carrier plate having open wells in register with the wells of the filter plate or one may use a wicking device or an underdrain attached to the bottom of the filter plate. Sufficient wash liquid is added to allow for filtration by the effect of gravity to occur. Cells are retained within the wells at a rate of 4 times that of other rapid methods.

Claims

1. A method of detecting a target specific ligand that is bound to a target molecule to indicate the presence or absence of the target molecule comprising the steps of:

a. selecting a cell having a selected target molecule located in a position selected from the group consisting of a cell wall or inside the cell,

b. culturing the cell on a multiwell filter plate;

c. contacting the cell on the multiwell filter plate with a reagent comprising a ligand for the selected target molecule in or on the cell, the ligand being selected from the group consisting of ligands having a detectable marker conjugated thereto and ligands being capable of attaching to a secondary ligand that contains a detectable marker,

d. washing the cell with gravity flow at least one time to remove any reagent which is not bound to the selected target molecule of the cell to provide a washed cell by using sufficient fluid to achieve a head height at which filtration occurs; and

e. detecting the presence of the ligand bound to the target molecule to indicate the presence or absence of the target molecule in or on the washed cell on the multiwall filter plate.

2. The method of claim 1 wherein the plate has a polycarbonate filter.

3. The method of claim 1 wherein the plate has a hydrophilic polycarbonate filter.

4. The method of claim 1 wherein the filter is a porous polycarbonate having a pore size of about 3 microns.

5. The method of claim 1 wherein the filter is a porous, hydrophilic, polycarbonate having a pore size of about 3 microns.

6. The method of claim 1 wherein the ligand has a detectable marker selected from the group consisting of fluorescent materials, radioactive materials, chemiluminescent, light absorbence and optical density measurements.

7. The method of claim 1 wherein the target molecule is a marker protein.

8. The method of claim 1 wherein the target molecule is a marker protein which is a cell specific marker protein.

9. The method of claim 1 wherein the target molecule is a marker protein that is a cell surface protein.

10. The method of claim 1 wherein the target molecule is a marker protein that is an intracellular protein of the cell.

11. The method of claim 1 wherein the detection for the presence of the antibody bound to the target molecule occurs in the multiwall plate by a multiwall plate reader.

12. An immunoassay method of detecting presence or absence of a target protein comprising the steps of:

a. selecting a cell having a selected target protein located in a position selected from the group consisting of being on the cell wall or intracellular,

b. culturing the cell on a multiwell filter plate, wherein the filter is formed of porous, hydrophilic polycarbonate having a nominal pore size of about 3 microns;

c. contacting the cell on the multiwell filter plate with at least one antibody for the selected target protein, the at least one antibody being selected from the group consisting of antibodies having a detectable marker conjugated thereto and antibodies being capable of attaching to a secondary antibody that contains a detectable marker,

d. washing the cell with gravity flow at least one time to remove any antibody which is not bound to the selected target protein to provide a washed cell by using sufficient fluid to achieve a head height at which filtration occurs;

e. transferring the washed cell to a cytometer and

f. detecting for the presence of the antibody bound to the target molecule to indicate the presence or absence of the target protein in or on the washed cell in the cytometer.