Method and kit for cytokine analysis from a human whole blood sample

Abstract

The invention relates to a method for prognostic evaluation of the disease progression of rheumatoid arthritis, in particular prognostic evaluation of the disease progression during treatment, and for the diagnosis and/or activity determination of rheumatoid arthritis by analysing cytokines from a human full blood sample. In the method according to the invention, a volume of a full blood sample of a human is transferred into at least one test tube containing a stimulating agent. As control samples, the same volume of a full blood sample of the human in each case is transferred into an empty test tube as a negative control and a test tube containing lipopolysaccharide as a positive control respectively. After incubation, the concentration of at least one proinflammatory cytokine is determined from the cell-free residue of each test tube. By way of an altered concentration of the at least one cytokine in the at least one test tube comprising the stimulating agent, the prognostic evaluation of the disease progression or the diagnosis is subsequently made. The invention further relates to an associated diagnostic kit and to the use thereof. The invention is applied in medical diagnostics and medical research.

Claims

1. A method for prognostic evaluation of disease progression of rheumatoid arthritis (RA) and/or diagnosis of RA, the method comprising: (a) combining a non-activating concentration of a toll-like receptor ligand with a sample of whole blood of a human and transferring a volume of the whole blood with non-activating concentration of a toll-like receptor ligand into at least one test tube containing TNFR2: Ig as a stimulating agent, and as control samples, the same volume of whole blood from the human with non-activating concentration of a toll-like receptor ligand into an empty test tube as a negative control and a test tube containing an activation concentration of lipopolysaccharide (LPS) as a positive control respectively; (b) incubating the test tubes prepared in (a) at a temperature of 30-40C.; (c) obtaining a cell-free reside from each whole blood sample and determining the concentration of TNF- in the cell-free residue of each sample; wherein an increased concentration of TNF- in the test sample when compared to the negative control indicates an increased risk of RA and that a good response to treatment with TNF inhibitor is likely.

2. Method of claim 1, wherein the toll-like receptor ligand is mixed with the whole blood sample at a maximum concentration of 0.1 ng/ml.

3. A diagnostic kit for prognostic evaluation of disease progression of rheumatoid arthritis, comprising: (i) at least one test tube containing TNFR2: Ig as stimulating agent; (ii) a test tube for a negative control and a test tube containing an activation concentration of lipopolysaccharide (LPS) for the positive control; and (iii) a toll-like receptor ligand for addition at a non-activation concentration to a whole blood sample.

4. A diagnostic kit according to claims 3, wherein the TNFR2: Ig is in a surface-bonded form.

5. A Method for evaluation of disease progression of reheumatoid arthritis (RA) and /or diagnosis of RA, the method comprising: (a)combining a whole blood sample from a human with a non-activating concentration of a toll-like receptor ligand and transferring a volume of said whole blood sample containing a non-activation concentration of a toll-like receptor ligand into at least one test tube containing TNFR2-Ig, and as negative and positive control samples, and equal volume of said whole blood sample with a non-activation concentration of a toll-like receptor ligand into an empty test tube and a test tube containing an activation concentration of lipopolysaccaharide, respectively; (b) incubating the test sample and negative and positive controls at a temperature of 30-40 C. for a time sufficient for cells in the whole blood to be stimulated by the TNFR2-Ig; (c) obtaining a cell-free residue from each test sample and negative and positive controls and determining the concentration of TNF- in said samples; and (d) identifying an increased risk of RA and/or the likelihood of a good response to treatment of the human with TNF inhibitor when there is higher concentration of TNF- in the test sample when compared to the negative control.

6. The method of claim 5, wherein the time sufficient for cells in the whole blood to be stimulated by the stimulating agent is between 4 and 48 hours.

7. The method of claim 6, wherein the time sufficient for cells in the whole blood to be stimulated by the stimulating agent is between 12 and 24 hours.

8. A diagnostic kit according to claim 3 additionally comprising an ELISA plate for determining the concentration of TNF-.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is to be explained in greater detail with reference to the following drawings and embodiments, but is not limited thereto.

(2) FIGS. 1a-c show that the spontaneous monocytic in vitro cytokine production and the spontaneous monocytic secretion of dissolved cytokine receptors before the start of treatment are inversely correlated with the response to a TNF inhibitor treatment, measured by the fall in the DAS28. The DAS28 (disease activity score) is a value for assessing the disease activity of rheumatoid arthritis, and is dependent on the number of tender joints (0-28 joints), the number of swollen joints (0-28), the erythrocyte sedimentation rate (mm/h) and the evaluations of the disease status by the patient. The higher the DAS28, the higher the disease activity. FIG. 1a relates to the spontaneous IL-1 secretion, FIG. 1b relates to the spontaneous IL-1 secretion, and FIG. 1c relates to the spontaneous secretion of the dissolved cytokine receptor TNFR1.

(3) An increased spontaneous monocytic in vitro cytokine production along with a spontaneous monocytic secretion of dissolved cytokine receptors is a diagnostic parameter associated with RA.

(4) FIG. 2 shows that the increase in spontaneous monocytic IL-8 secretion in vitro, induced by soluble TNFR2:Ig, correlates with the decrease in the disease during treatment with the TNF inhibitor, measured by the fall in the DAS28. The higher the factor by which IL-8 secretion increases, the greater the fall in the DAS28 after 12 weeks of treatment, and so the better the response to the treatment.

(5) FIGS. 3a and 3b show that the level of the monocytic cytokine production in vitro before treatment, induced by incubation with soluble TNFR2:Ig, is positively correlated with the response to a TNF inhibitor treatment after 4 weeks, measured by the fall in the DAS28. FIG. 3a relates to the TNFR2:Ig-induced TNF- secretion and FIG. 3b relates to the TNFR2:Ig-induced IL-8 secretion before the start of treatment.

(6) FIG. 4 shows that the TNF- production triggered by tmTNF cross-linking and reverse signalling has a significant correlation with the response to therapeutic TNF inhibition, measured by the fall in the DAS28 after 4 weeks of treatment. The results show that a strong TNF- response to tmTNF reverse signalling, induced by bonded TNFR2:Ig, is a predictor of a very good response to a treatment using TNF- inhibitors.

(7) FIG. 5 shows that the level of the monocytic IL-12 secretion in vitro before treatment, induced by incubation with bonded TNFR2:Ig, is negatively correlated with the response to a TNF inhibitor treatment after 8 weeks.

(8) FIGS. 6a and 6b show that the level of monocytic secretion of soluble cytokine receptors in vitro before treatment, induced by incubation with bonded TNFR2:Ig, is positively correlated with the response to a TNF inhibitor treatment after 4 weeks. FIG. 6a relates to the TNFR2:Ig-induced TNFR1 secretion and FIG. 6b relates to the TNFR2:Ig-induced IL-1R1 secretion before the start of treatment.

(9) FIG. 7 shows that the stimulation of GPRC6A by way of increased extracellular calcium concentration is suitable as a prognostic parameter for the prognosis of the expected response to the treatment using a TNF inhibitor. The Ca.sup.2+-induced IL-1 production is correlated with the fall in the DAS28 after 12 weeks of treatment, in other words with the response to the treatment.

EXAMPLE 1

Analysis of the TNF- Concentration After Incubation with Bonded TNFR2:Ig

(10) 5 ml parent solution containing 100 g/ml TNFR2:Ig as a stimulating agent are provided and incubated at 37 C. for 3 h. The residue is subsequently disposed of. It is expected that 125 g TNFR2:Ig will bond to the surface. Subsequently, a soluble form of TNFR2:Ig is additionally added, for a final concentration in the culture medium of 40 g/ml.

(11) 12 ml full blood are mixed with heparin and 0.1 ng/ml LPS immediately after being taken.

(12) 4 ml full blood in each case are subsequently added to the test tube containing bonded TNFR2:Ig, to an empty test tube as a negative control, and to a test tube containing lipopolysaccharide as a positive control.

(13) This is followed by incubation at 37 C. for 16 h. Subsequently, the samples are centrifuged at 2000 rpm for 30 minutes and the concentration of TNF- is determined from the cell-free residue by ELISA. An increased concentration in the residue of the sample from the test tube comprising TNFR2:Ig by comparison with the negative control indicates an increased risk of the presence of RA. A correlation with the response to therapeutic TNF inhibitors, measured by the fall in the DAS28 after 4 weeks of treatment, is shown in FIG. 4.

(14) Table 2 shows TNF- concentrations of five healthy people and five people having rheumatoid arthritis, in the test tube for the negative control and in the test tube comprising the stimulating agent in each case.

(15) TABLE-US-00002 TABLE 1 TNF- concentration pg/ml Healthy control 1 Negative control 1 w/ TNFR2:Ig 701 Healthy control 2 Negative control 0 w/ TNFR2:Ig 363 Healthy control 3 Negative control 14 w/ TNFR2:Ig 157 Healthy control 4 Negative control 4 w/ TNFR2:Ig 248 Healthy control 5 Negative control 29 w/ TNFR2:Ig 404 RA patient 1 Negative control 31 w/ TNFR2:Ig 422 RA patient 2 Negative control 49 w/ TNFR2:Ig 309 RA patient 3 Negative control 19 w/ TNFR2:Ig 717 RA patient 4 Negative control 19 w/ TNFR2:Ig 901 RA patient 5 Negative control 3 w/ TNFR2:Ig 1298

EXAMPLE 2

Triggering the GPRC6A Signal Path

(16) 12 ml full blood are mixed with heparin and 0.1 ng/ml LPS immediately after being taken.

(17) In a test tube, 100 ng/ml LPS are mixed with 2.5 mM calcium chloride.

(18) 4 ml full blood are subsequently added to the test tube containing 2.5 mM calcium chloride, resulting in a final concentration of 1.7 mM calcium chloride. A further 4 ml in each case are added to an empty test tube as a negative control and to a test tube containing lipopolysaccharide as a positive control.

(19) This is followed by incubation at 37 C. for 16 h. Subsequently, the samples are centrifuged and the concentration of IL-1 is determined from the cell-free residue by ELISA. FIG. 7 shows that the Ca.sup.2+-induced IL-1 production is correlated with the fall in the DAS28 after 12 weeks of treatment.

CITED NON-PATENT LITERATURE

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