Matrix of antibody-coated spots for determination of antigens

• US 4,591,570 A
• Filed: 02/02/1983
• Issued: 05/27/1986
• Est. Priority Date: 02/02/1983
• Status: Expired due to Term

• Current Assignee: Tanox Biosystems Inc. (Roche Holding AG)
• Original Assignee: Centocor Incorporated (Johnson & Johnson)
• Inventors: Chang, Tse-Wen
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): Wieder, Stephen C.
• Application Number:
  US06/463,188
• Time in Patent Office: 1,210 Days
• Field of Search: 435/7, 435/300, 436/519, 436/532, 436/531, 436/527, 436/809, 436/815, 436/518
• US Class Current: 435/7.24
• CPC Class Codes: B01J 2219/00367 capillary
• 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 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C40B 60/14 for creating libraries
• 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 33/545 Synthetic resin
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56977 HLA or MHC typing
• CPC Class Codes: Y10S 435/965 involving idiotype or anti-idiotype antibody
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

This invention relates to a novel immunoassay device and method for the determination of antigenic substances. The device essentially comprises a pattern or array of minute antibody-coated spots on the surface of a support. The array of antibody-coated spots is preferably in the form of a rectangular matrix. Each antibody-coated spot is made up of antibodies of a different and distinct specificity. A large number of different antibody-coated spots can be assembled on a very small portion of the surface of the support. The spots serve as tiny, specific immunoadsorbents of cells. The expression of particular surface antigen by cells may be detected by determining to which antibody-coated spot the cells bind.

Claims

1. An immunoassay device comprising a support having a substantially planar surface and having an array of small, discrete, closely spaced antibody-coated areas on the planar surface, the antibodies in the discrete, antibody-coated surface areas being absorbed to the planar surface at a density and uniformity such that when cells bearing a surface antigen are brought into contact in sufficient concentration with a discrete, antibody-coated area containing antibodies which bind the antigen, the cells adhere tightly to the discrete, antibody-coated area and form a layer of bound cells which is substantially microscopically uniform over essentially all of the discrete antibody-coated area.

2. An immunoassay device according to claim 1, wherein all of the discrete, antibody-coated areas have substantially the same size and shape.

3. An immunoassay device according to claim 1, wherein the antibodies in the discrete, antibody-coated areas are covalently bound to the surface of said support.

4. An immunoassay device according to claim 1, wherein the antibody in each of the discrete, antibody-coated surface areas, or in identifiable sets of said antibody-coated surface areas, is antibody of different and distinct specificity.

5. An immunoassay device according to claim 1, wherein the antibodies in the discrete, antibody-coated areas are monoclonal antibodies.

6. An immunoassay device according to claim 1, wherein the antibodies in the discrete, antibody-coated areas are monoclonal antibodies.

7. An immunoassay device according to claim 1, wherein the support is a glass or plastic coverslip.

8. An immunoassay device according to claim 1, wherein the array of discrete, antibody-coated areas is a rectangular matrix.

9. An immunoassay device according to claim 1, wherein discrete, antibody-coated surface areas are dots having diameters of about 0.25 mm to 1.0 mm.

10. An immunoassay device according to claim 1, wherein said antibody-coated dots on the surface of the support are arranged in a compact 5-20 dot by 5-20 dot rectangular matrix.

11. An immunoassay device according to claim 9, wherein the entire rectangular matrix is contained within an area of about 0.5-1.0 cm² of the surface of the support.

20. An immunoassay device according to claim 1, for tissue typing, wherein the antibody in each of the discrete, antibody-coated areas, or in identifiable sets of the antibody-coated areas, is specific for a different and distinct transplantation antigen.

12. A method of producing an immunoassay device which comprises the steps of:

(a) applying droplets of substantially enriched aqueous solutions of antibodies of distinct specifications to a substantially planar surface of a support, locating said droplets so that they cover small, discrete, closely spaced areas of the planar surface of the support and form an array;

(b) allowing said aqueous solutions of antibodies to remain on said areas of the surface for a period of time long enough for the antibodies in solution to be adsorbed to said areas of the surface to form discrete, antibody-coated areas;

(c) swiftly flooding the entire surface of the solid support with an aqueous solution of protein in order to dilute the unadsorbed antibodies in the various solutions of antibodies and in order to block non-specific binding sites in areas of the surface of the solid support which surround the antibody-coated areas; and

(d) washing and drying the device.

13. A method according to claim 12, wherein in step a the concentration of antibodies in said aqueous solutions of antibodies is about 10 μ

g/ml.

14. A method according to claim 12, wherein in step a each solution of antibodies is applied to the surface of the solid support in the form of a tiny droplet having a volume of about 0.005 μ

l-0.5 μ

l.

15. A method according to claim 12, wherein said small, discrete areas of the surface of the support to which said solutions of antibodies are applied are dots having diameters of about 0.25 mm to 1.0 mm.

16. A method according to claim 12, wherein said dots are located on the surface of the solid support to form a rectangular matrix.

17. A method of determining the presence of particular antigens on the surface of cells, comprising the steps of:

(a) applying a suspension of cells to a substantially planar surface of a support, said support having upon the planar surface an array of small, discrete, antibody-coated spots, each spot, or each of certain sets of spots, coated with antibodies of different and distinct specificity, the antibodies in the discrete, antibody-coated spots being adsorbed to the surface at a density and uniformity such that when cells bearing a surface antigen are brought into contact in sufficient concentration with a discrete, antibody-coated spot containing antibodies which bind the antigen, the cells adhere tightly to the discrete, antibody-coated spot and form a layer of bound cells which is substantially microscopically uniform over essentially all of the discrete, antibody-coated spot;

(b) allowing the cells to settle upon the surface of the support so that the cells come into contact with all of the discrete antibody-coated spots;

(c) allowing the cells to react with the antibodies in the discrete antibody-coated spots;

(d) removing non-adherent cells;

(e) determining to which discrete antibody-coated spots the cells have adhered.

18. A method of determining the presence of particular antigens in a liquid comprising the steps of:

(a) applying a sample of the liquid containing antigens to a substantially planar surface of a support, said support having upon its planar surface an array of small, discrete antibody-coated spots, each spot coated with antibodies of different and distinct specificity, so that the antigen containing liquid comes into contact with all of the discrete, antibody-coated spots;

(b) allowing the antigens to react with the antibodies in the discrete, antibody-coated spots;

(c) washing the device to remove all unbound antigens;

(d) determining to which discrete, antibody-coated spots antigens are bound.

19. A method of determining the proportion of cells bearing specific surface differentiation antigens in a mixed population of cells comprising:

(a) applying a suspension of cells to a substantially planar surface of a support, said support having upon its planar surface an array of small, discrete, antibody-coated spots, each spot coated with antibody of different and distinct specificity for cell surface differentiation antigens, the antibodies in the discrete, antibody-coated spots being adsorbed to the surface at a density and uniformity such that when cells bearing a surface antigen are brought into contact in sufficient concentration with a discrete, antibody-coated spot containing antibodies which bind the antigen, the cells adhere tightly to the antibody-coated spot and form a layer of bound cells which is substantially microscopically uniform over essentially all of the discrete, antibody-coated spot;

(b) allowing the cells to settle upon the surface of the support so that the cells come into contact with all of the discrete, antibody-coated spots;

(c) allowing the cells to react with the antibodies in the discrete, antibody-coated spots;

(d) removing non-adherent cells;

(e) quantitating cells bound to the discrete, antibody-coated spots; and

(f) calculating the relative proportion of cells.

21. An immunoassay device for tissue typing comprising a solid support having a substantially planar surface and an array of small, discrete, closely-spaced anti-HLA antibody-coated spots on the planar surface, the anti-HLA antibody in the discrete, antibody-coated surface area being adsorbed to the planar surface at a density and uniformity such that when cells bearing a surface antigen are brought into contact in sufficient concentration with a discrete, antibody-coated area containing antibodies which bind the antigen, the cells adhere tightly to the discrete, antibody-coated area and form a layer of bound cells which is substantially microscopically uniform over essentially all of the discrete antibody-coated area.