Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides

Abstract

IntroductionAutoantibodies 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.MethodsThe 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.ResultsStrong 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.ConclusionsThe 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.

Environmental and genetic factors in the development of anticitrullinated protein antibodies (ACPAs) and ACPA-positive rheumatoid arthritis: an epidemiological investigation in twins

“…The latter is in the higher range of previously reported prevalences in smaller population-based studies13 41 of healthy individuals, but lower than the previously reported prevalence for non-diseased family members of RA patients 12 14 16. In ACPA-positive individuals without RA, specific reactivities against citrullinated antigens were sparse, whereas the frequency of these reactivities in twins with RA was similar to previously reported frequencies in other cohorts of established RA 9 33 43…”

Fine specificity of anti-citrullinated peptide antibodies discloses a heterogeneous antibody population in rheumatoid arthritis

“…In a recent study, almost the same citrullinated epitopes have been used to develop a multiplex immunofluorescence-based assay that proved to be comparable with the ELISA assays, and the recorded serum reactivities were found similar to those observed in our study. However, the citrullinated collagen epitope was slightly different and reactivity reached only 19% [34]. Conversely, both specificity and sensitivity for all six citrullinated peptides were low among SLE and SS patients and among healthy controls.…”

Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants

“…A proof-of-concept study was published in 2012, where we investigated reactivity against 12 different citrullinated peptides in 927 patients with RA and 461 healthy controls from the Epidemiological Investigations in Rheumatoid Arthritis (EIRA) case–control study 22. We described varying sensitivity at the same predefined high specificity (98%) for individual ACPA, with some of them approaching but never surpassing the anti-CCP2 sensitivity.…”