Human autoantibodies specific for PAD3 which are cross-reactive with PAD4 and their use in the diagnosis and treatment of rheumatoid arthritis and related diseases
In one or more embodiments, the present invention provides a novel biomarker which provides a link between a distinct clinical phenotype and a biochemical effect of an autoantibody on an enzyme implicated in disease pathogenesis. In particular, the present invention provides an isolated or purified human autoantibody to PAD3 protein. Methods of diagnosis of subjects for rheumatoid arthritis (RA) using these antibodies as well as diagnosis of the severity of RA in the subject, and methods for monitoring treatment of a subject with RA are also provided. The biomarkers provided herein are also useful in the diagnosis of connective tissue-interstitial lung disease (CT-ILD) in patients having or suspected of having RA.
1. A method for the diagnosis of severe rheumatoid arthritis (RA) in a subject comprising:
a) obtaining a sample comprising blood, plasma or serum from a subject suspected of having severe RA;
b) providing a substrate having a first capture probe bound thereto, wherein the capture probe comprises peptidyl arginine deiminase 3 (PAD3) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects suffering from RA;
c) contacting the substrate having the capture probe bound thereto with the sample from the subject;
d) measuring the amount of a complex of the capture probe and the autoantibodies formed by step c);
e) providing a normal reference level sample comprising blood, plasma or serum;
f) comparing the amount of a complex of the capture probe and the autoantibodies formed from the subject suspected of having severe RA to the amount of a complex of the capture probe and the autoantibodies formed from the normal reference level sample; and
g) identifying the subject suspected of having severe RA as having severe RA when the amount of a complex of the capture probe and the autoantibodies formed from the subject suspected of having severe RA is statistically significant increased compared to the amount of a complex of the capture probe and the autoantibodies formed from the normal reference level sample.
2. The method of claim 1 , wherein
step (b) further comprises a second capture probe comprising peptidyl arginine deiminase 4 (PAD4) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects suffering from RA;
step (d) further comprises detecting the formation of a complex of the first and second capture probes and the autoantibodies;
step (f) further comprises comparing the amount of the cross-reactive complex of both capture probes formed from the subject suspected of having severe RA to the amount of a complex of the capture probe and the autoantibodies formed from the normal reference level sample; and
step (g) further comprises identifying the subject suspected of having severe RA as having severe RA when the amount of the cross-reactive complex of both first and second capture probes formed from the subject suspected of having severe RA is statistically significant increased compared to the amount of the cross-reactive complex of both first and second capture probes formed from the normal reference level sample.
3. The method of claim 1 , wherein said method is an enzyme immunoassay method.
4. The method of claim 1 , wherein said method is a radioimmunoassay method.
5. The method of claim 1 , wherein said method is an immunoblotting method.
6. A method for identifying a subject as having connective tissue disease associated with interstitial lung disease (CT-ILD) comprising:
a) obtaining a sample comprising blood, plasma or serum from a subject having or suspected of having severe rheumatoid arthritis (RA);
b) providing a substrate having at least a first capture probe bound thereto, wherein the capture probe comprises peptidyl arginine deiminase 3 (PAD3) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects suffering from RA;
c) contacting the substrate having the at least first capture probe bound thereto with the sample from the subject;
d) measuring the amount of a complex of the capture probe and the autoantibodies formed by step c);
e) providing a normal reference level sample comprising blood, plasma or serum;
f) comparing the amount of a complex of the at least first capture probe and the autoantibodies formed from the subject suspected of having severe RA to the amount of a complex of the at least first capture probe and the autoantibodies formed from the normal reference level sample; and
g) identifying the subject having or suspected of having severe RA as having CT-ILD when the amount of the complex of the at least first capture probe formed from the subject having or suspected of having severe RA is statistically significant increased compared to the amount of the complex of the at least first capture probe formed from the normal reference level sample.
7. The method of claim 6 , wherein
step (b) further comprises a second capture probe comprising peptidyl arginine deiminase 4 (PAD4) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects having or suspected of having RA;
step (d) further comprises detecting the formation of a complex of the first and second capture probes and the autoantibodies;
step (f) further comprises comparing the amount of the cross-reactive complex of both capture probes formed from the subject having or suspected of having RA to the amount of a complex of the capture probe and the autoantibodies formed from the normal reference level sample; and
step (g) further comprises identifying the subject having or suspected of having RA as having CT-ILD when the amount of the cross-reactive complex of both first and second capture probes formed from the subject having or suspected of having RA is statistically significant increased compared to the amount of the cross-reactive complex of both first and second capture probes formed from the normal reference level sample.
8. The method of claim 6 , wherein said method is an enzyme immunoassay method.
9. The method of claim 6 , wherein said method is a radioimmunoassay method.
10. The method of claim 6 , wherein said method is an immunoblotting method.
11. A method for identifying a subject having rheumatoid arthritis (RA) as having an increased risk of connective tissue disease associated with interstitial lung disease (CT-ILD) comprising:
a) obtaining a sample comprising blood, plasma or serum from a subject having or suspected of having severe rheumatoid arthritis (RA);
b) providing a substrate having at least a first capture probe bound thereto, wherein the capture probe comprises peptidyl arginine deiminase 3 (PAD3) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects suffering from RA;
c) contacting the substrate having the at least first capture probe bound thereto with the sample from the subject;
d) measuring the amount of a complex of the capture probe and the autoantibodies formed by step c);
e) providing a normal reference level sample comprising blood, plasma or serum;
f) comparing the amount of a complex of the at least first capture probe and the autoantibodies formed from the subject suspected of having severe RA to the amount of a complex of the at least first capture probe and the autoantibodies formed from the normal reference level sample; and
g) identifying the subject having or suspected of having severe RA as having an increased risk of CT-ILD when the amount of the complex of the at least first capture probe formed from the subject having or suspected of having severe RA is statistically significant increased compared to the amount of the complex of the at least first capture probe formed from the normal reference level sample.
12. The method of claim 11 , wherein
step (b) further comprises a second capture probe comprising peptidyl arginine deiminase 4 (PAD4) protein or a portion or fragment thereof which comprises an antigen recognized by autoantibodies present in the serum of subjects having or suspected of having RA;
step (d) further comprises detecting the formation of a complex of the first and second capture probes and the autoantibodies;
step (f) further comprises comparing the amount of the cross-reactive complex of both capture probes formed from the subject having or suspected of having RA to the amount of a complex of the capture probe and the autoantibodies formed from the normal reference level sample; and
step (g) further comprises identifying the subject having or suspected of having RA as having an increased risk of CT-ILD when the amount of the cross-reactive complex of both first and second capture probes formed from the subject having or suspected of having RA is statistically significant increased compared to the amount of the cross-reactive complex of both first and second capture probes formed from the normal reference level sample.
13. The method of claim 11 , wherein said method is an enzyme immunoassay method.
14. The method of claim 11 , wherein said method is a radioimmunoassay method.
15. The method of claim 11 , wherein said method is an immunoblotting method.
16. The method of claim 1 , wherein said capture probe is bound to said substrate with a binding reagent.
17. The method of claim 3 , further comprising an anti-human antibody.
18. The method of claim 4 , further comprising an anti-human antibody.
19. The method of claim 6 , wherein said capture probe is bound to said substrate with a binding reagent.
20. The method of claim 8 , further comprising an anti-human antibody.
21. The method of claim 9 , further comprising an anti-human antibody.
22. The method of claim 11 , wherein said capture probe is bound to said substrate with a binding reagent.
23. The method of claim 13 , further comprising an anti-human antibody.
24. The method of claim 14 , further comprising an anti-human antibody.