Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides
1 Rheumatology Unit, Department of Medicine, Building D2:01, Karolinska Institutet, Stockholm, SE-17176, Sweden
2 Department of Immunology, Genetics and Pathology, Rudbeck Laboratory C5, Uppsala University, SE-75185, Sweden
3 Phadia AB, P.O. Box 6460, Uppsala, SE-75137, Sweden
4 Phadia Multiplexing Diagnostics GmbH, Tech Gate Vienna, Donau-City-Strasse 1 Vienna, AT-1220, Austria
5 Laboratory of Epidermis Differentiation and Rheumatoid Autoimmunity, UMR 5165, Centre National de la Recherche Scientifique, U 1056 Inserm, Toulouse III University, Hôpital Purpan, Place du Dr Baylac - TSA 40031, 31059 Toulouse cedex 9, France
6 Medical Inflammation Research, Medical Biophysics and Biochemistry, Scheeles väg 2, Building B2:04, Karolinska Institutet, Stockholm, SE-17176, Sweden
Arthritis Research & Therapy 2012, 14:R201 doi:10.1186/ar4039Published: 1 October 2012
Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.
The microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.
Strong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.
The multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.