Method and apparatus for measuring binding affinity

• US 5,324,633 A
• Filed: 11/22/1991
• Issued: 06/28/1994
• Est. Priority Date: 11/22/1991
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Fodor, Stephen P. A., Mazzola, Laura T.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Spiegel, Carol A.
• Application Number:
  US07/796,947
• Time in Patent Office: 949 Days
• Field of Search: 436/517, 436/518, 436/527, 436/531, 436/89, 436/164, 436/171-172, 436/808-810, 436/823, 435/6, 435/7.1, 435/7.92, 435/968, 435/970, 435/973, 435/975, 435/4, 422/52, 422/82.06, 422/99, 422/131, 422/134, 530/333, 530/334, 935/88
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/808 : Automated or kit
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 436/81 : Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/823 : Immunogenic carrier or carrier per se

Abstract

A method and device for measuring the binding affinity of a receptor to a ligand. According to one aspect of the invention, arrays of polymers are synthesized or immobilized on a substrate (212). The array of polymers is exposed to a fluorescently-labelled receptor in solutions of varying concentration. The fluorescence intensity of the labelled receptor is measured by way of, e.g., a photon counter using a confocal microscope (316). Binding affinity is determined through analysis of the relationship between fluorescence intensity and the solution concentration of the receptor. On-rates are measured as a kinetic increase in surface fluorescence intensity, and the on-rate constant rate extracted from fits to the data.

Claims

1. A method of determining the presence and strength of binding affinity, Kd, of a receptor, R, for a selected polymer attached at a selected region on a surface of a substrate comprising an array of polymers attached at a plurality of selected regions on the surface of the substrate, comprising the steps of:

(a) exposing the polymers to a first concentration solution of said receptor, said receptor being labeled with a fluorescent marker;

(b) irradiating the polymers with a light source;

(c) measuring fluorescence intensity of each selected region with a confocal microscope at a time sufficient for the receptor to reach specific binding equilibrium with the selected polymer;

(d) repeating steps (a)-(c) with a second concentration solution of said receptor, said receptor being labeled with the fluorescent marker, wherein the measuring step occurs while at least one of said solutions is in contact with the substrate;

(e) calculating Kd from the fluorescence intensities and the receptor concentrations for the selected polymer by solving an equation, ##EQU7## wherein I=fluorescence intensity, [R]=receptor concentration, Imax=a saturation fluorescence intensity correction factor, to determine the presence and strength of binding of the receptor for the selected polymer.

2. The method as recited in claim 1 wherein said label is fluorescein.

3. The method as recited in claim 1 using at least two substrates, each having a common array of polymers formed thereon, said at least two substrates exposed to said receptor in different solution concentrations.

4. The method as recited in claim 1 using a substrate having duplicate arrays of polymers formed thereon, wherein each array is exposed to a different solution concentration of the receptor so that the exposing, irradiating and measuring steps at the different concentrations of the receptor occur simultaneously.

5. The method as recited in claim 1 further comprising the step of storing the fluorescence intensity for each selected polymer at each of the receptor solution concentrations in an appropriately programmed digital computer.

6. The method as recited in claim 1 wherein the calculating step further comprises plotting a linear relationship between the fluorescence intensities and the receptor concentrations.

7. The method as recited in claim 1 wherein the calculating step further comprises plotting a non-linear relationship between the fluorescence intensities and the receptor concentrations.

8. The method as recited in claim 1 wherein said second receptor concentration is higher than said first receptor concentration.

9. The method as recited in claim 1, wherein said calculating step comprises the steps of:

plotting the ratio of fluorescence intensity to receptor solution concentration on a y-axis for the selected polymer versus total surface fluorescence intensity on a x-axis in order to obtain a function curve which has a slope of -1/Kd;

determining the Kd of the receptor for the selected polymer from said function curve to determine the presence and strength of the binding affinity of the receptor for the selected polymer.

10. The method as recited in claim 1, wherein said receptor is indirectly labelled with the fluorescent marker by means of a second receptor labelled with the fluorescent marker, wherein the second receptor specifically binds to said receptor.

11. The method as recited in claim 1, further comprising the step of attaching said array of polymers to said plurality of selected regions on the surface of the substrate, wherein said attaching step is performed before said exposing step.

12. The method as recited in claim 11 wherein said attaching step comprises the steps of:

removing a photolytic protective group from first selected regions of said surface;

binding a first monomer comprising said photolytic protective group to said first selected regions;

removing said photolytic protective group from second selected regions of said surface, said second selected regions at least partially overlapping said first selected regions;

binding a second monomer to said second selected regions to form at least first and second polymers attached on said surface.

13. The method as recited in claim 12 further comprising the step of removing said photolytic protective group from a third selected region of said substrate and binding a fully synthesized polymer having the same monomer sequence as said first or second polymer to said third region of said substrate.

14. The method as recited in claim 11 wherein said attaching step comprises the steps of:

removing a photolytic protective group from a first selected region of said substrate;

binding a first polymer at said first selected region;

removing said photolytic protective group from a second selected region of said surface;

binding a second polymer at said second selected region.

15. The method as recited in claim 1 wherein the polymers are polypeptides.

16. The method as recited in claim 15 wherein said receptor is an antibody.

17. The method as recited in claim 15 wherein said receptor is an enzyme.

18. The method as recited in claim 1 wherein said step of measuring fluorescence intensity comprises the step of:

directing light at the polymers on said surface in a first selected region;

measuring an amount of light fluoresced from said surface at said first selected region with said confocal microscope; and

repeating said steps of directing light and measuring an amount of light fluoresced for at least one selected region bearing each of the polymers.

19. The method as recited in claim 18 wherein said confocal microscope has a magnification of about 10×

to 40×

.

20. The method as recited in claim 18 comprising the steps of adjusting said magnification of said microscope to a higher magnification at a higher receptor concentration, and adjusting a magnification of said microscope to a lower magnification at a lower receptor concentration.

21. The method as recited in claim 1 wherein said polymers are oligonucleotides.