Session Information
Date: Monday, November 9, 2015
Title: B cell Biology and Targets in Rheumatolid Arthritis and other Autoimmune Disease Poster
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose:
Antibodies targeting carbamylated proteins (anti-CarP) were recently identified in serum samples from patients with rheumatoid arthritis (RA). The presence of anti-CarP antibodies was associated with a more severe disease course in RA patients and correlated with the development of RA in arthralgia patients and healthy blood donors. It may therefore serve as a new biomarker in the management of early RA. Carbamylated-fetal calf serum (Ca-FCS), a complex mixture of proteins, is currently used in ELISA to identify anti-CarP antibodies. It is not yet clear which of the proteins in Ca-FCS are responsible for the reactivity of the anti-CarP antibodies. Therefore, we set out to investigate which protein(s) within the Ca-FCS mixture may function as an antigen for anti-CarP antibodies. This might serve as a method to optimize the ELISA, using a known and consistent antigen and may provide novel insights in the pathophysiological mechanisms of anti-CarP antibodies.
Methods:
Ca-FCS was fractionated using anion-exchange chromatography. Fractions containing sufficiently high protein concentrations were coated onto ELISA plates and used for the detection of anti-CarP antibodies. Fractions containing a high anti-CarP antibody binding capacity and low protein content were selected and analysed by SDS-PAGE gel-electrophoresis. Coomassie stained bands were excised and analysed by mass-spectroscopy. Human A1AT was obtained from commercial sources, carbamylated and used in ELISA. Receiver operating characteristic (ROC) analysis was used to analyse the discriminatory power of different assays.
Results:
We observed high anti-CarP binding capacity in several fractions obtained after fractionation of Ca-FCS using anion-exchange chromatography. The fraction with the best signal (ELISA) to protein concentration ratio was further separated by SDS-PAGE gel-electrophoresis, proteins bands were excised and analysed by mass-spectrometry. The most promising band was identified as bovine alpha 1 anti-trypsin (A1AT). Purified human A1AT from commercial sources was in-vitro carbamylated and used as antigen to coat ELISA plates. In sera of RA patients, we found a strong reactivity towards Ca-A1AT and only limited reactivity against non-modified A1AT. We detected anti-CarP antibodies directed against Ca-A1AT in around 45% of the ACPA positive RA patients. Importantly, as for the anti-CarP FCS assay, we observed around 14% anti-Ca-A1AT positive patients in the ACPA negative stratum. Using ROC analyses comparing RA patients to healthy controls we observed for anti-Ca-A1AT an AUC of 0.72 (0.65-0.78) as compared to an AUC of 0.69 (0.63-0.76) for anti-Ca FCS. We observed a good quantitative correlation between anti-Ca-FCS and anti-Ca-A1AT reactivity of anti-CarP antibodies (r=0.76 (0.67-0.86)), making A1AT a suitable new antigen for the detection of anti-CarP antibodies.
Conclusion:
Carbamylated-A1AT is a promising antigenic target of anti-CarP antibodies as an aid in the diagnosis of RA.
To cite this abstract in AMA style:
Verheul MK, Yea A, Seaman A, Cordfunke RA, Drijfhout JW, Janssen GM, de Ru A, van Veelen PA, Toes REM, Mahler M, Trouw LA. Identification of Carbamylated Alpha-1-Anti-Trypsin (A1AT) As an Antigenic Target of Anti-Carp Antibodies in Patients with Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/identification-of-carbamylated-alpha-1-anti-trypsin-a1at-as-an-antigenic-target-of-anti-carp-antibodies-in-patients-with-rheumatoid-arthritis/. Accessed .« Back to 2015 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-carbamylated-alpha-1-anti-trypsin-a1at-as-an-antigenic-target-of-anti-carp-antibodies-in-patients-with-rheumatoid-arthritis/