Citrullinated peptides for diagnosing and prognosing rheumatoid arthritis

Abstract

The present invention provides novel citrullinated peptides, their use in methods for aiding, assisting, improving, or facilitating the diagnosis or prognosis of rheumatic diseases such as rheumatoid arthritis (RA), and methods for identifying novel citrullinated peptides that are immunoreactive with anti-citrullinated protein antibodies (ACPAs). The present invention also provides methods for detecting rheumatoid factor (RF) using novel RF detection reagents as a means to aid, assist, improve, or facilitate the diagnosis or prognosis of rheumatic diseases such as RA. Kits comprising at least one of the novel citrullinated peptides and/or RF detection reagents of the present invention are also provided.

What is claimed is: 
     
         1 . A method for identifying a peptide that is immunologically reactive with an anti-citrullinated protein antibody, said method comprising:
 (a) identifying at least one antigenic peptide epitope in at least one synovial fluid polypeptide, wherein said antigenic peptide epitope is predicted to be immunologically reactive with an anti-citrullinated protein antibody, wherein said antigenic peptide epitope contains at least one arginine residue, and wherein at least one of said arginine residues is citrullinated;   (b) synthesizing a peptide that comprises at least one of said antigenic peptide epitopes;   (c) contacting a biological sample from an individual having rheumatoid arthritis (RA) with said peptide under conditions suitable to transform said peptide into a complex comprising said peptide and said anti-citrullinated protein antibody; and   (d) identifying said peptide as being immunologically reactive with said anti-citrullinated protein antibody based on the presence or level of said complex.   
     
     
         2 . The method of  claim 1 , wherein said at least one synovial fluid polypeptide is selected from the group consisting of vimentin, fibrinogen alpha-chain, fibrinogen beta-chain, fibrinogen gamma-chain, alpha-enolase, β-actin, aggrecan, gelsolin, lumican, fibronectin, tropomyosin, cartilage oligomeric matrix protein, glucose-6-phosphate isomerase, lamin B1, lamin B2, lamin A/C, myeloblastin (proteinase 3), phospholipid (PL) scramblase 1, apolipoprotein (a), BiP (heat shock 70 kDa protein 5), histone H2A, histone H 2 B, histone H3, histone H4, COL2A1, COL9A1, COL10A1, COL11A1, COL11A2, syndecan 1, syndecan 3, CD44, intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), glypican 1, glypican 2, glypican 4, glypican 5, glypican 6, vitronectin, nidogen, and combinations thereof. 
     
     
         3 . The method of  claim 1 , wherein said antigenic peptide epitope comprises at least 9 contiguous amino acids of said synovial fluid polypeptide sequence. 
     
     
         4 . The method of  claim 1 , wherein said antigenic peptide epitope is predicted to be immunologically reactive with an anti-citrullinated protein antibody when a score calculated for said antigenic peptide epitope is greater than or equal to a predetermined score. 
     
     
         5 . The method of  claim 4 , wherein said score for said antigenic peptide epitope is calculated by adding up a value given to each amino acid in said antigenic peptide epitope based on the position and side-chain of said amino acid. 
     
     
         6 . The method of  claim 5 , wherein said value given to each amino acid at a particular position (P 1-P9) in said antigenic peptide epitope is shown in  FIG. 10 . 
     
     
         7 . The method of  claim 4 , wherein said predetermined score is +2.0. 
     
     
         8 . The method of  claim 1 , wherein said sample is human serum. 
     
     
         9 . The method of  claim 1 , wherein step (b) comprises synthesizing a peptide that comprises at least two of said antigenic peptide epitopes, wherein said antigenic peptide epitopes are linked together by a peptide bond. 
     
     
         10 . The method of  claim 1 , wherein step (b) comprises synthesizing a peptide that comprises three of said antigenic peptide epitopes, wherein said antigenic peptide epitopes are linked together by a peptide bond. 
     
     
         11 . The method of  claim 1 , wherein said complex in step (c) is detected with a detectable moiety conjugated to said peptide. 
     
     
         12 . The method of  claim 11 , wherein said detectable moiety is a fluorescent moiety. 
     
     
         13 . The method of  claim 1 , wherein step (c) comprises the sub-steps of:
 (i) contacting said complex with a detection reagent comprising a reporter group to transform said complex into a labeled complex; and   (ii) detecting the presence or level of said labeled complex.   
     
     
         14 . The method of  claim 13 , wherein said detection reagent is selected from the group consisting of an anti-IgA antibody, an anti-IgG antibody, an anti-IgM antibody, Protein L, Protein A, Protein G, and mixtures thereof. 
     
     
         15 . The method of  claim 13 , wherein said reporter group is selected from the group consisting of radioactive groups, fluorescent groups, luminescent groups, enzymes, biotin, and dyes. 
     
     
         16 . The method of  claim 13 , wherein detecting the presence or level of said labeled complex comprises detecting the presence or level of a signal generated from said reporter group. 
     
     
         17 . The method of  claim 16 , wherein detecting the presence or level of a signal generated from said reporter group comprises the use of a detection device. 
     
     
         18 . The method of  claim 17 , wherein said detection device comprises a spectrophotometer. 
     
     
         19 . The method of  claim 1 , wherein said anti-citrullinated protein antibody comprises an IgA anti-citrullinated protein antibody, IgG anti-citrullinated protein antibody, IgM anti-citrullinated protein antibody, or mixtures thereof. 
     
     
         20 . The method of  claim 1 , wherein step (c) comprises an enzyme-linked immunosorbent assay (ELISA), a fluorescence polarization assay, or a fluorescence anisotropy assay. 
     
     
         21 . A synthetic peptide identified by the method of  claim 1 . 
     
     
         22 . The synthetic peptide of  claim 21 , wherein said peptide is about 15 to about 40 amino acids in length. 
     
     
         23 . The synthetic peptide of  claim 21 , wherein said peptide further comprises a tag. 
     
     
         24 . The synthetic peptide of  claim 23 , wherein said tag is biotin. 
     
     
         25 . A kit comprising:
 (a) at least one synthetic peptide identified by the method of  claim 1 ; and   (b) a detection reagent comprising a reporter group.   
     
     
         26 . The kit of  claim 25 , wherein said at least one synthetic peptide is immobilized on a solid support. 
     
     
         27 . The kit of  claim 25 , wherein said detection reagent is selected from the group consisting of an anti-IgA antibody, an anti-IgG antibody, an anti-IgM antibody, Protein L, Protein A, Protein G, and mixtures thereof. 
     
     
         28 . The kit of  claim 25 , wherein said reporter group is selected from the group consisting of radioactive groups, fluorescent groups, luminescent groups, enzymes, biotin, and dyes.