CELL-MEDIATED IMMUNE RESPONSE ASSAY AND KITS THEREFOR

• US 20100221712A1
• Filed: 03/14/2008
• Published: 09/02/2010
• Est. Priority Date: 03/16/2007
• Status: Active Grant

• Current Assignee: Cellestis Limited
• Original Assignee: Cellestis Limited
• Inventors: Howard, Jenny L., Jones, Stephen L., Radford, Anthony J.
• Granted Patent: US 8,871,508 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2333/35 from Mycobacteriaceae (F)
• CPC Class Codes: G01N 2333/57 IFN-gamma
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

The present invention provides methods and kits for measuring a cell-mediated immune (CMI) in a small volume of whole undiluted blood collected from a subject. In particular, the methods are for measuring responses in undiluted whole blood samples having a volume of, for example, 50 μl to 500 μl. Thus, capillary sampling and rapid testing of subjects including pediatric, adult or geriatric human subjects are facilitated.

Claims

1. A method for assaying cell-mediated immune (CMI) response capacity in a sample of whole blood from a subject, comprising:

a) incubating, with and without an antigen, a substantially undiluted whole blood sample that is selected from (i) peripheral capillary blood from the subject, and (ii) less than 0.5 mL of arterial or venous blood from the subject, wherein said whole blood sample comprises immune system cells which are capable of producing one or more immune effector molecules following stimulation by the antigen; and

b) detecting a level of at least one immune effector molecule, or of a nucleic acid molecule capable of producing said at least one immune effector molecule, that is present in said substantially undiluted whole blood sample, wherein;

in the whole blood sample that is incubated with antigen, the level of the at least one immune effector molecule, or of the nucleic acid molecule capable of producing said at least one immune effector molecule, compared to said level in the whole blood sample incubated without antigen, is indicative of the capacity of the subject to mount a cell-mediated response.

3. The method of claim 1 wherein the subject has symptoms of, or a propensity for developing, an inflammatory disease condition, a pathogenic infection, an autoimmune disorder, immuno-incompetence, allergy or cancer.

5. The method of claim 1 which comprises a step of collecting that comprises (a) collecting with a capillary sampling device;

or (b) collecting into a container containing at least one of the antigen and an anti-coagulant.

8. The method of claim 1 wherein the incubation step is conducted in the presence of a simple sugar.

9. The method of claim 8 wherein the simple sugar is dextrose.

12. The method of claim 1 wherein the step of incubating comprises incubating for from about 4 hours to about 50 hours.

22. The method of claim 1 wherein the step of incubating takes place in a container that has a height of from at least about 4 mm to 6 mm to a maximum height of about 12 mm to about 20 mm.

23. The method of claim 22 wherein the sample in the container has a height of at least about 6 mm to a maximum of about 12 mm.

26. The method of claim 1 wherein the substantially undiluted whole blood sample that is incubated in the step of incubating is selected from

(a) a substantially undiluted whole blood sample having a total volume that is selected from a volume less than 500 μ

l, a volume less than 400 μ

l, a volume less than 300 μ

l, a volume less than 200 μ

l, a volume less than 100 μ

l, and a volume less than 50 μ

l; and

(b) a substantially undiluted whole capillary blood sample having a total volume that is selected from a volume of about 2000 μ

l, 1500 μ

l, 1400 μ

l, 1300 μ

l, 1200 μ

l, 1100 μ

l, 900 μ

l, 800 μ

l, 700 μ

l, 600 μ

l, 500 μ

l, 400 μ

l, 300 μ

l, 200 μ

l, 100 μ

l, 50 μ

l, 40 μ

l, and an intermediate volume therebetween.

29. The method of claim 1 comprising a step of collecting the whole blood sample into a capillary tube having a diameter of about three to four millimeters.

30. The method of claim 1 wherein the step of incubating takes place in an incubation container that maintains an optimal shape of the sample, and wherein the shape has one or two or more dimensions selected from:

(i) a maximum circular diameter of less than about 6 mm;

(ii) a height of from at least about 4 mm to 6 mm to a maximum height of about 12 mm to 20 mm; and

(iii) a volume that is selected from (i) a volume less than 0.5 mL and (ii) a volume less than 400 μ

l.

2. (canceled)

4. (canceled)

6-7. -7. (canceled)

10-11. -11. (canceled)

13-21. -21. (canceled)

24-25. -25. (canceled)

27-28. -28. (canceled)

31. A kit for measuring a cell-mediated immune (CMI) response in a sample of whole blood collected from a subject wherein said whole blood sample comprises cells of the immune system which produce immune effector molecules following stimulation by an antigen, the kit comprising in multicomponent form:

(a) one or more containers for holding a peripheral capillary whole blood sample from the subject, or a whole blood sample comprising less than 0.5 mL of venous or arterial blood from the subject;

(b) one or more antigens selected from the group consisting of a test antigen and a control antigen;

(c) reagents for detecting an immune effector molecule;

(d) either or both of (1) a capillary tube having a diameter of about 3-4 mm, and (2) a set of instructions that comprises instructions;

(i) to collect and mix the whole blood sample in the container;

(ii) to incubate the whole blood sample in the container with at least one of a test antigen, a control antigen and a mitogen;

(iii) to centrifuge the container of (ii) and collect plasma therefrom; and

(iv) to detect an immune effector molecule in the plasma collected in (iii); and

(e) a capillary sampling device,

wherein the container is capable of forming an optimal shape of the sample wherein the shape has one, two or more dimensions selected from;

(1) a maximum circular diameter of less than about 6 mm;

(2) a height of from at least about 4 mm to 6 mm to a maximum height of about 12 mm to 20 mm; and

(3) a volume that is selected from a volume that is less than 0.5 mL and a volume that is less than 400 μ

l,

and wherein the container, when holding a whole blood sample, forms a sample height of from at least about 4 mm to 6 mm to a maximum height of about 12 mm to 20 mm.

48. The kit of claim 31 wherein the container forms a sample height of from at least about 6 mm to a maximum of about 12 mm.

49. The kit of claim 31 wherein the container, when holding a whole blood sample, forms a sample height of 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm or 20 mm or an intervening height therebetween.

50. The kit of claim 31 wherein the container, when holding a whole blood sample, forms a sample height of 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm or 12 mm or an intermediate height therebetween.

54. The kit of claim 31 wherein the reagents comprise an antibody conjugate for detecting IFN-γ

.

32-47. -47. (canceled)

51-53. -53. (canceled)

55. (canceled)