METHOD OF EARLY DIAGNOSIS OF IMMUNE-MEDIATED INFLAMMATORY DISEASE

• US 20190317091A1
• Filed: 06/23/2017
• Published: 10/17/2019
• Est. Priority Date: 07/02/2016
• Status: Active Grant

• Current Assignee: Centre Hospitalier Universitaire de Liège, Université de Liège
• Original Assignee: Centre Hospitalier Universitaire de Liège, Université de Liège
• Inventors: MALAISE, Michel, DE SENY, Dominique
• Granted Patent: US 11,047,856 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 2800/105 Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

An in-vitro method for early diagnosing or prediction of immune-mediated inflammatory diseases, comprising: —obtaining a sample from a subject; —quantifying simultaneously by one LC/MS-MS analysis of said sample, a presence of V65 vitronectin fragment or fragment, variant or degradation product thereof; and a presence of a complement C3f or fragment or variant or degradation products thereof;

Claims

1. An in-vitro method for early diagnosing or prediction of immune-mediated inflammatory diseases, comprising:

obtaining a biological sample from a subject;

quantifying simultaneously by one LC/MS-MS analysis of said biological sample, a presence of V65 vitronectin fragment or fragment, variant or degradation product thereof; and

a presence of a complement C3f or fragment, variant or degradation product thereof;

2. The method according to claim 1 wherein the subject is a human;

3. The Method according to claim 1 or 2 wherein the biological sample is blood, serum, plasma, urine, or synovial fluid;

4. The method according to any preceding claims, wherein the V65 vitronectin fragment comprises the following amino sequence:

₃₈₁SQRGHSRGRNQNSRRPS₃₉₇

5. The method according to any preceding claims wherein the complement C3f comprises the following amino sequence:

₁₃₀₄SSKITHRIHWESASLLR₁₃₂₀

6. The method according to any preceding claims wherein the immune-mediated inflammatory diseases is osteoarthritis.

7. The method according to any preceding claims wherein the immune-mediated inflammatory diseases is lupus

8. The method according to any preceding claims wherein the immune-mediated inflammatory diseases is sclerodermia

9. The method according to any preceding claims wherein the LC/MS-MS analysis is a microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry analysis.

10. The method according to any preceding claims comprising the following steps:

mixing the biological sample with an acidic aqueous solution;

loading the resulting mixture on a trapping LC column;

optionally washing the column to remove uncertain component;

eluting the column with an alcoholic mixture and obtaining an eluate;

drying the eluate;

dispersing the dried eluate in an acetonitrile aqueous solution;

loading the resulting dispersion on a reversed phase LC column and separating two mobile phase A and B;

injecting by continuous flux in MS-MS, both phases A and B and quantifying V65 vitronectin fragment or fragment, variant or degradation product thereof simultaneously to quantificating complement C3f or fragment, variant or degradation product thereof.

11. The method according to claim 10 wherein the acidic aqueous solution comprise trifluoro acetic acid (TFA).

12. The method according to claim 11 wherein the acidic aqueous solution comprise 1% v trifluoro acetic acid (TFA).

13. The method according to anyone of claims 10 to 12 wherein the alcoholic mixture is methanol/water/TFA.

14. The method according to claim 13 wherein the mixture methanol/water/TFA is in the ratio from 90:

7;

3 to 90;

9;

1 v/v/v, preferably 90;

9;

1 v/v/v.

15. The method according to anyone of claims 10 to 14 wherein the acetonitrile aqueous solution comprises TFA

16. The method according to claim 15 wherein the acetonitrile aqueous solution comprising TFA in the ratio acetonitrile/water/TFA from 3:

97;

0.1 to 1;

99;

0.1 v/v/v preferably 1;

99;

0.1 v/v/v.

17. The method according to any preceding claims wherein C3f or fragment, variant or degradation product thereof are quantified at m/z 459.3, 530.7 and 646.3 and wherein V65 vitronectin fragment or fragment, variant or degradation product thereof are quantified at m/z 434.9, 445.2, 460.5, 466.2, 469.7, 480.3, 485.3.