Methods for Label Free Testing of Cells

• US 20120107840A1
• Filed: 11/08/2011
• Published: 05/03/2012
• Est. Priority Date: 04/09/2008
• Status: Abandoned Application

• Current Assignee: X-Body Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Sru Biosystems Incorporated
• Inventors: Wagner, Richard W., Shamah, Steven, Yuzhakov, Alexander, Laing, Lance G., Certel, Kaan
• Application Number:
  US13/291,741
• Publication Number:
  US 20120107840A1
• Time in Patent Office: Days
• US Class Current: 435/7.24
• CPC Class Codes: G01N 21/7743 the reagent-coated grating coupling light in or out of the waveguide
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• 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 33/5052 involving B-cells
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

The invention provides methods of detecting activation of an immune cell, methods for detecting blocking or enhancing properties of a test reagent or stimuli on activation of one or more immune cells or platelets, methods for selecting hybridomas producing antibodies to an antigen for quality of strength of binding to the antigen, methods for determining if a subject has had an immune response to an immunogen, methods of isolating neutralizing antibodies for an immunogen, methods of classifying a B cell lymphoma, and methods for selecting activated B-cells expressing an antibody to one or more antigens.

Claims

1. A method for detecting activation of immune cells or platelets comprising:

(a) immobilizing one or more adhesion proteins or extracellular matrix molecules on the surface of a colorimetric resonance reflectance biosensor;

(b) adding one or more types of immune cells or platelets to the surface of the biosensor;

(c) adding a potential activator of the immune cells or the platelets to the surface of the biosensor;

(d) detecting adhesion of the immune cells to the adhesion proteins or the adhesion of the platelets to the extracellular matrix molecules on the surface of the biosensor by illuminating the biosensor and detecting changes in peak wavelength values over time;

wherein an increase in peak wavelength values over time indicates that the potential activator of the immune cells has activated the immune cells or that the potential activator of the platelets has activated the platelets.

2. The method of claim 1, wherein the immune cells are lymphocytes or granulocytes.

3. The method of claim 1, wherein the adhesion protein is ICAM-1 or VCAM-1.

4. A method for detecting blocking or enhancing properties of a test reagent or stimuli on activation of one or more immune cells or platelets comprising:

(a) immobilizing one or more adhesion proteins or extracellular matrix molecules on the surface of a colorimetric resonance reflectance biosensor;

(b) adding one or more types of immune cells or platelets to the surface of the biosensor;

(c) adding an activator of the immune cells or platelets to the surface of the biosensor;

(d) adding a test reagent or stimuli to the surface of the biosensor;

wherein the one or more types of immune cells, platelets, activator, and test reagent or stimuli can be added to the biosensor surface sequentially in any order, or at the same time;

(e) detecting adhesion of the immune cells to the adhesion proteins or adhesion of the platelets to the extracellular matrix molecules on the surface of the biosensor by illuminating the biosensor and detecting changes in peak wavelength values over time for each immune cell or platelet;

(f) comparing the changes in peak wavelength values for each immune cell or platelet to a control that does not comprise the test reagent or stimuli, wherein an increase in peak wavelength values over time indicates that the test reagent or stimuli enhances immune cell or platelet activation, and wherein a decrease in peak wavelength values over time indicates that the test reagent or stimuli blocks activation of the immune cells.

5. The method of claim 4, wherein the adhesion protein is ICAM-1 or VCAM-1.

6. A method of selecting hybridomas producing antibodies to an antigen for highest strength of binding to the antigen comprising:

(a) adding one or more hybridomas to the surface of colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;

(b) adding the antigen to which the hybridomas are specific to the surface of the biosensor;

(c) illuminating the biosensor and detecting changes in peak wavelength value over time for each hybridoma; and

(d) selecting and isolating the hybridomas with the largest increases in peak wavelength value over time;

wherein hybridomas are selected that produce antibodies to the antigen with the highest strength of binding to the antigen.

7. The method of claim 6, further comprising:

(e) adding the individual selected hybridomas to a colorimetric resonant reflectance biosensor, wherein capture molecules for the antibodies produced by the hybridoma are immobilized on the biosensor surface and allowing the hybridoma to multiply to form a hybridoma population;

(f) illuminating the biosensor and detecting changes in peak wavelength value for each hybridoma;

(g) selecting hybridomas having the greatest increase in peak wavelength value;

whereby hybridomas are selected that produce the greatest quantity of antibodies.

8. The method of claim 6, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.

9. A method of determining if a subject has had an immune response to an immunogen comprising:

(a) obtaining test B cells from a subject;

(b) adding the test B cells to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;

(c) adding the immunogen to the surface of the biosensor;

(d) illuminating the biosensor and detecting changes in peak wavelength values over time for the test B cells; and

(e) comparing the changes in peak wavelength values for the test B cells to a control B cell population that does not react with the immunogen;

wherein an increase in peak wavelength values over time for the test B cells as compared to the control B cells indicates that the subject has had an immune response to the immunogen.

10. The method of claim 9, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.

11. A method of isolating neutralizing antibodies for an immunogen comprising:

(a) obtaining test B cells from a subject that has had an immune response to the immunogen;

(b) adding the test B cells to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;

(c) adding the immunogen to the surface of the biosensor;

(d) illuminating the biosensor and detecting changes in peak wavelength values over time for each test B cell;

(e) comparing the changes in peak wavelength values for each test B cell to a control B cell that does not react with the immunogen or that is not exposed to the immunogen;

(f) isolating the test B cells having peak wavelength values higher than the control B cell; and

(g) isolating antibodies produced by the isolated test B cells;

wherein neutralizing antibodies for the immunogen are isolated.

12. The method of claim 11, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.

13. A method of classifying a B cell lymphoma comprising:

(a) obtaining a B cell sample from a patient with an unclassified B cell lymphoma;

(b) adding the B cell sample to the surface of a colorimetric resonance reflectance biosensor having one or more adhesion proteins immobilized to the biosensor surface;

(c) adding one or more chemokines to the biosensor;

(c) optionally adding one or more specific inhibitors of GTPases to the biosensor;

(d) illuminating the biosensor and detecting changes in peak wavelength values over time for each B cell sample; and

(e) comparing the responses of the B cell samples to known responses of B cell samples from patients with classified B cell lymphoma,

wherein the B cell lymphoma is classified.

14. The method of claim 13, wherein the one or more adhesion proteins are VCAM-1 or ICAM-1.

15. A method for selecting activated B-cells expressing an antibody to one or more antigens comprising:

(a) adding B-cells expressing antibody libraries on their cell surfaces to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;

(b) adding the one or more antigens to the surface of the colorimetric resonance reflectance biosensor;

(c) determining the amount of activation of each B-cell on the biosensor surface by illuminating the biosensor and determining the changes in peak wavelength values over time for each B cell, wherein an increase in peak wavelength values over time indicates B cell activation, and wherein a decrease or no change in peak wavelength values over time indicates no B cell activation; and

(d) isolating activated B cells;

wherein a B-cell that expresses an antibody to the one or more antigens is selected.

16. The method of claim 15, wherein polynucleotide sequences corresponding to the expressed antibodies are amplified.

17. The method of claim 15, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.