Methods for screening cells and antibodies

• US 20070172894A1
• Filed: 12/08/2006
• Published: 07/26/2007
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: X-Body Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Sru Biosystems Incorporated
• Inventors: Li, Peter, Smith, Timothy, Madison, Lara, Laing, Lance, Karl, William, Genick, Christine
• Granted Patent: US 7,927,822 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 30/10 by measuring physical properties, e.g. mass
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 21/4788 Diffraction for sizing particles G01N15/0205
• CPC Class Codes: G01N 21/7743 the reagent-coated grating coupling light in or out of the waveguide
• CPC Class Codes: G01N 33/5026 on cell morphology
• CPC Class Codes: G01N 33/5029 on cell motility
• CPC Class Codes: G01N 33/5032 on intercellular interactions
• 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 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G02B 5/1809 with pitch less than or comparable to the wavelength
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

Abstract

The invention provides methods of detecting a change in cell growth patterns, methods of screening many different antibodies in one receptacle, and methods of detecting specific binding of an antibody to a protein or cell, wherein the antibody is in a mixture of many different antibodies.

Claims

1. A method of detecting a change in a cell growth pattern comprising:

a) applying one or more cells to a location on a surface of a calorimetric resonant reflectance optical biosensor;

b) detecting a calorimetric resonant reflectance optical peak wavelength value (PWV) for the location;

c) incubating the one or more cells for a period of time or applying a test reagent to the one or more cells and incubating the one or more cells for a period of time;

d) detecting the colorimetric resonant reflectance optical PWV for the location; and

e) comparing the PWV of step (b) to the PWV of step (d);

wherein a difference between the colorimetric resonant reflectance optical PWV of step (b) in relation to the calorimetric resonant reflectance optical PWV of step (d) indicates a change in the cell growth pattern in the one or more cells.

2. The method of claim 1 wherein the change in cell growth pattern is a change in cell morphology, change in cell adhesion, change in cell migration, change in cell proliferation, change in cell death, change in microtubule structure, change in microfilament structure, granule exocytosis, respiratory burst, cell differentiation, or a combination thereof.

3. The method of claim 1, wherein the PWV is detected using a scanner with a lens having a lower limit pixel size of about 2 micrometers to about 15 micrometers.

4. The method of claim 1, wherein the location on a surface of a calorimetric resonant reflectance optical biosensor is an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.

5. A method of screening about 100 or more different antibodies in one receptacle comprising:

(a) immobilizing the antibodies to a calorimetric resonant reflectance biosensor, wherein the colorimetric resonant reflectance biosensor comprises an inner surface of the receptacle;

(b) determining a first peak wavelength value for the receptacle;

(c) adding one or more natively folded proteins to the receptacle;

(d) determining a second peak wavelength value for the receptacle;

(e) comparing the first and second peak wavelength values

wherein, if the second peak wavelength value is higher than the first peak wavelength value, then one or more immobilized antibodies in the receptacle have specifically bound the one or more natively folded proteins.

6. The method of claim 5, wherein the total antibody concentration in the receptacle is greater than 2 mg/ml.

7. The method of claim 5, wherein the one or more antibodies in the receptacle that have specifically bound the one or more natively folded proteins are present at a concentration of less than 5 ng/ml.

8. The method of claim 5, wherein the natively folded protein is a cell surface protein.

9. The method of claim 5, wherein the natively folded protein is part of a whole cell that is added to the receptacle.

10. The method of claim 9, wherein the cell is pre-treated with one or more antibodies prior to the cell being added to the receptacle.

11. The method of claim 5, wherein the antibodies are produced by about 100 or more different hybridoma cells.

12. The method of claim 5, wherein the antibodies are produce by about 1,000 or more different hybridoma cells.

13. A method of detecting specific binding of a first antibody to a protein, wherein the antibody is in a mixture of more than 100 different antibodies, wherein the first antibody is in the mixture of antibodies at a concentration of less than about 3 ng/ml, and wherein the concentration of the mixture of antibodies is greater than about 3 ug/ml comprising:

(a) immobilizing the mixture of antibodies to a colorimetric resonant reflectance biosensor, wherein the colorimetric resonant reflectance biosensor comprises an inner surface of the receptacle;

(b) determining a first peak wavelength value for the receptacle;

(c) adding one or more proteins to the receptacle, wherein one or more of the proteins may specifically bind to the first antibody;

(d) determining a second peak wavelength value for the receptacle;

(e) comparing the first and second peak wavelength values

wherein, if the second peak wavelength value is higher than the first peak wavelength value, then one or more antibodies in the receptacle have specifically bound the one or more of the proteins.

14. A method of screening about 100 or more different antibodies in one receptacle comprising:

(a) immobilizing the antibodies to a biosensor comprising a substrate having a periodic surface grating structure wherein the periodic grating structure is constructed in a manner designed for both

     1) optical interrogation of the biosensor with light in an evanescent resonance (ER) detection mode, and

     2) optical interrogation of the biosensor with light in a label-free detection mode, wherein the biosensor comprises an inner surface of the receptacle;

(b) adding one or more cells to the receptacle;

(c) illuminating the biosensor in a readout detection instrument with light from at least one light source designed for the ER detection mode and illuminating the sensor with the at least one light source designed for the label-free detection mode; and

analyzing light reflected from the biosensor.

15. The method of claim 14, wherein the label-free detection mode indicates that one or more cells have bound to the antibodies and wherein the ER detection mode indicates a biological activity of the one or more cells.

16. The method of claim 14, wherein the at least one light source comprises a first label-free light source and a second ER light source, and wherein the method further comprising the step of selectively illuminating the sensor with light from the first and second light sources.

17. The method of claim 14, wherein the grating structure comprises a two-dimensional grating structure, and wherein:

the first dimension of the periodic grating structure comprises a grating structure designed for label-free detection, and

the second dimension of the periodic grating structure comprises a grating structure designed for ER detection.

18. The method of claim 14, wherein the grating structure further comprises a substrate, a layer applied to the substrate having a grating structure, an intermediate SiO₂ layer deposited on the layer having the grating structures, and a layer of relatively high index of refraction material deposited on the SiO₂ layer.

19. The method of claim 18, wherein the grating structure further comprises a substrate, a layer applied to the substrate having the grating structures in the first and second dimensions, an intermediate SiO₂ layer deposited on the layer having the grating structures, and a layer of relatively high index of refraction material deposited on the SiO₂ layer.

20. The method of claim 18, wherein the SiO₂ layer has a thickness of between about 500 and 5000 Angstroms.

21. The method of claim 18, wherein the grating structure in the first dimension has a period of between 260 and about 1500 nm and a depth of the grating between about 100 nm and about 3000 nm, and wherein the grating structure in the second dimension is between about 200 nm and about 1000 nm, and wherein the depth of the grating in the second dimension is between about 10 nm and about 300 nm.

22. A method of detecting specific binding of a first antibody to unpurified cells or unpurified antigen comprising:

(a) immobilizing the first antibody to a calorimetric resonant reflectance biosensor;

(b) detecting a first peak wavelength value;

(c) adding the unpurified cells or unpurified antigen to the colorimetric resonant reflectance biosensor;

(d) detecting a second peak wavelength value;

(e) comparing the first and second peak wavelength values, wherein an increase in the second peak wavelength value indicates specific binding of the first antibody to the unpurified cells or unpurified cells.

23. The method of claim 22, wherein a first antigen specific for the first antibody is added to the unpurified cells or unpurified antigen prior to adding the unpurified cells or unpurified antigen to the colorimetric resonant reflectance biosensor, and wherein a lower second peak wavelength value than the second peak wavelength value of claim 22 indicates specific binding of the unpurified cells or unpurified antigen to the first antigen.

24. The method of claim 22, wherein a second antibody having the same specificity as the first antibody is added to the unpurified cells or unpurified antigen prior to adding the unpurified cells or unpurified antigen to the colorimetric resonant reflectance biosensor, and wherein a lower second peak wavelength value than the second peak wavelength value of claim 22 indicates specific binding of the unpurified cells or unpurified antigen to the second antibody.

25. The method of claim 22, wherein the unpurified cells are about 10,000 or fewer cells.

26. The method of claim 22, wherein the concentration of the first antibody is about 3 ng/ml or less.

27. The method of claim 22, wherein the unpurified cells are present in HAT media, hybridoma media, or cell culture media.

28. The method of claim 22, wherein the first antibody, unpurified cells, and unpurified antigen do not comprise detection labels.

29. A method of ranking antibodies according to their affinity for an antigen comprising:

(a) immobilizing a specific amount of one or more types of antibodies to a colorimetric resonant reflectance biosensor such that each type of antibody is present at a separate location;

(b) determining a first peak wavelength value for each separate location;

(c) adding antigens or cells comprising cell surface antigens to the calorimetric resonant reflectance biosensor,

(d) determining a second peak wavelength value for each separate location;

(e) comparing the first and second peak wavelength values to determine the ranking of antibodies.

30. The method of claim 29, wherein the cells comprising cell surface antigens are unpurified cells.

31. The method of claim 29, wherein the cells comprising cell surface antigens are about 10,000 or less cells.

32. The method of claim 29, wherein the one or more types of antibodies are present at a concentration of 3 ng/ml or less.

33. The method of claim 29, wherein the one or more types of antibodies are unpurified antibodies.

34. The method of claim 33, wherein the antibodies are present in hybridoma media, HAT media, or cell culture media.

35. The method of claim 29, wherein the one or more types of antibodies are 96 or more types of antibodies that are present at 96 or more separate locations on the colorimetric resonant reflectance biosensor.

36. A method of determining whether different types of antibodies that are each specific for a first antigen bind to the same region of the first antigen comprising:

(a) immobilizing the first antibody to a colorimetric resonant reflectance biosensor;

(b) adding the first antigen to the colorimetric resonant reflectance biosensor;

(c) determining a first peak wavelength value;

(d) adding the second antibody to the colorimetric resonant reflectance biosensor;

(e) determining a second peak wavelength value;

(f) comparing the first and second peak wavelength values, wherein if the first and second antibodies bind different regions of the first antigen, then the first and second antibodies bind to different regions of the first antigen.

37. The method of claim 36, wherein the first and second antibodies are present at a concentration of 3 ng/ml or less.

38. The method of claim 36, wherein the first and second antibodies are unpurified antibodies.

39. The method of claim 38, wherein the antibodies are present in hybridoma media, HAT media, or cell culture media.