Single Molecule Assays
The present invention provides single molecule analyses of species of use in analytical, diagnostic or prognostic assays. In exemplary embodiments, the assays utilize samples prepared by novel methods, affording assays of unexpected sensitivity and robustness. The method is described in a non-limiting manner by reference to cytokine assays.
1 - 8 . (canceled)
9 . A method for determining a disease state in an individual comprising measuring the concentration of one or more cytokines and comparing the measured concentration to the concentration range of the cytokine in a healthy individual wherein the cytokine and normal range are selected from the group consisting of:
Cytokine
Normal Range
Il-17A
about 0.05 pg/ml to about 0.7 pg/ml
TNF-α
about 0.6 pg/ml to about 2.7 pg/ml
Il-1β
about 0.03 pg/ml to about 0.3 pg/ml
Il-1α
about 0.05 pg/ml to about 0.6 pg/ml
Il-12
about 0.05 pg/ml to about 1.2 pg/ml
Il-4
about 0.1 pg/ml to about 1 pg/ml
Il-2
about 0.1 pg/ml to about 2 pg/ml
Il-6
about 0.5 pg/ml to about 9 pg/ml
Il-7
about 0.4 pg/ml to about 3 pg/ml
Il-5
about 1.5 pg/ml to about 19.5 pg/ml
Il-10
about 8 pg/ml to about 40 pg/ml
Il-15
about 1 pg/ml to about 4 pg/ml
IL-21
about 2 pg/ml to about 10 pg/ml
Il-22
about 3 pg/ml to about 40 pg/ml
MIP-1alpha
about 22 pg/ml to about 65 pg/ml
G-CSF
about 25 pg/ml to about 110 pg/ml
GM-CSF
about 0.2 pg/ml to about 1.3 pg/ml
IFN-γ
about 2 pg/ml to about 7 pg/ml.
10 . The method of claim 9 wherein the disease state is rheumatoid arthritis, and the cytokine molecule detected is selected from the group consisting of IFN-γ, IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-5, IL-7 and combinations thereof.
11 . The method of claim 9 wherein the disease state is neuroinflammation, and the molecule of cytokine detected is selected from the group consisting of IL-12, GM-CSF, G-CSF, IL-6, IL-17 and combinations thereof.
12 . The method of claim 9 wherein the disease state is Crohn's Disease (CD), and the cytokine detected is selected from the group consisting of TNF-α, IFN-γ, IL-1β, IL-6, IL-7, IL-2, IL-4, GM-CSF, G-CSF and combinations thereof.
13 . The method of claim 9 wherein the disease state is Irritable Bowel Syndrome (IBS), and the molecule of cytokine detected is selected from the group consisting of TNF-α, IFN-γ, IL-1β, IL-6, IL-7, GM-CSF, G-CSF, and combinations thereof.
14 . The method of claim 9 wherein the disease is psoriatic arthritis and the cytokines measured are selected from the group consisting of GM-CSF, IL-17a, IL-2, IL-10, IFN-gamma, IL-6 and combinations thereof.
15 . The method of claim 9 wherein the disease state is ankylosing spondylitis, and the cytokines detected are selected from the group consisting of IL-10, IL-2, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, TNF-α, IFNγ, GM-CSF, G-CSF and combinations thereof.
16 . The method of claim 9 wherein the disease state is reactive arthritis, and the cytokine detected is selected from the group consisting of IL-12, IFN-γ, IL-10, IL-17, TNF-α, IL-4, GM-CSF, IL-6 and combinations thereof.
17 . The method of claim 9 wherein the disease state is enteropathic arthritis, and the of cytokine detected is selected from the group consisting of IL-10, IL-4, G-CSF, IFN-γ, TNF-α and combinations thereof.
18 . The method of claim 9 wherein the disease state is ulcerative colitis (UC), and the cytokine detected is selected from the group consisting of IL-7, IFN-γ, TNF-α, IL-1β and combinations thereof.
19 . The method of claim 9 wherein the disease state is systemic lupus erythematosus, the molecule of cytokine detected is selected from the group consisting of IL-10, IL-2, IL-4, IL-6, IFN-γ, IL-17 and combinations thereof.
20 . The method of claim 9 wherein the disease state is Familial Mediterranean Fever (FMF), and the molecule of cytokine detected is selected from the group consisting of G-CSF, IL-2, IFN-γ, TNF-α, IL-1β, and combinations thereof.
21 . The method of claim 9 wherein the disease state is amyotrophic lateral sclerosis (ALS), and the molecule of cytokine detected is selected from the group consisting of IL-12 and IL-1β, and combinations thereof.
22 . The method of claim 9 wherein the disease state is Juvenile Rheumatoid Arthritis (JRA), and the molecule of cytokine detected is selected from the group consisting of IFN-γ, IL-1β, TNF-α, G-CSF, GM-CSF, IL-6, IL-4, IL-10, IL-5, IL-7 and combinations thereof.
23 . The method of claim 9 wherein the disease state is Sjogren's Syndrome, and the molecule of cytokine detected is selected from the group consisting of IL-12, TNFα, IL-2, IL-15, IL-17, IL-1α, IL-1β, IL-6, GM-CSF and combinations thereof.
24 . The method of claim 9 wherein the disease state is early arthritis, and the molecule of cytokine detected is selected from the group consisting of IL-2, IL-12, IL-17, TNFα, IL-4, IL-5, IL-10 and combinations thereof.
25 . A method for determining a disease state in an individual comprising measuring the concentration of one or more cytokines and comparing the measured concentration to the concentration range of the cytokine in a healthy individual wherein the cytokine and normal range are selected from the group consisting of:
Biomarker
Normal Range
Il-17A
about 0.06 pg/ml to about 0.58 pg/ml
TNF-α
about 0.63 pg/ml to about 2.68 pg/ml
Il-1β
about 0.05 pg/ml to about 0.22 pg/ml
Il-1α
about 0.07 pg/ml to about 0.48 pg/ml
Il-12
about 0.08 pg/ml to about 0.96 pg/ml
Il-4
about 0.19 pg/ml to about 0.77 pg/ml
Il-2
about 0.16 pg/ml to about 1.51 pg/ml
Il-5
about 2.05 pg/ml to about 18.82 pg/ml
Il-6
about 0.48 pg/ml to about 8.90 pg/ml
Il-7
about 0.42 pg/ml to about 2.7 pg/ml
Il-10
about 10.16 pg/ml to about 35.29 pg/ml
Il-15
about 1.24 pg/ml to about 3.88 pg/ml
IL-21
about 1.85 pg/ml to about 9.26 pg/ml
Il-22
about 3.83 pg/ml to about 38.74 pg/ml
MIP-1alpha
about 22.26 pg/ml to about 57.71 pg/ml
G-CSF
about 24.91 pg/ml to about 96.85 pg/ml
GM-CSF
about 0.33 pg/ml to about 1.10 pg/ml
IFN-γ
about 2.66 pg/ml to about 6.65 pg/ml.