ABSTRACT: INTRODUCTION: Autoantibodies directed against citrullinated proteins/peptides (ACPA) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognosis 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. METHODS: The microarray is based on Phadia's ImmunoCAP ISAC(R) system, where reactivity to more than 100 antigens can be analysed simultaneously, using minute serum volumes (< 10 l). 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 3-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, while 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 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 analysed using image analysis software. RESULTS: Strong correlations between ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman's typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anti- cyclic 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. CONCLUSIONS: 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 individualised treatment.