Analytical and diagnostic characteristics of 11 2nd- and 3rd-generation immunoenzymatic methods for the detection of antibodies to citrullinated proteins.

BACKGROUND: Measurement of antibodies to citrullinated peptides or proteins (CP) is a new test for the diagnosis of rheumatoid arthritis (RA). We analyzed the analytical characteristics and diagnostic accuracy of commercially available methods.
METHODS: We studied 11 commercially available 2nd- and 3rd-generation methods that used various citrullinated antigen substrates: synthetic cyclic peptides, recombinant rat filaggrin, mutated human vimentin, and Epstein-Barr virus- or IgG-derived peptides. We assessed imprecision by measuring samples with low, intermediate, and high concentrations 5 times on each of 5 days. We measured CPs by each of the assays in 100 serum samples from patients with RA and in 202 samples from healthy persons or patients with other autoimmune, viral, or neoplastic diseases.
RESULTS: The between-run imprecision (CV) of the methods was between 0.4% and 22%, and the repeatability (within-run imprecision) was 0.5%-19%. The areas under the ROC curves varied between 0.79 (95% CI, 0.72-0.85) and 0.92 (0.88-0.95). At a fixed specificity of 98.5%, the sensitivities ranged from 41% (95% CI, 31%-51%) to 74% (64%-82%). Sensitivities and specificities varied markedly at the manufacturer's suggested cutoffs. Most false-positive results were recorded in patients with viral infections. The methods that use the original synthetic cyclic CP gave the best and very similar performances, although these methods use different components in their reagent sets (conjugate, type of substrate, dilution, and washing buffers). This finding shows that the antigenic source is the most important variable in determining the diagnostic accuracy of the methods.
CONCLUSIONS: The analytical imprecision and diagnostic accuracies of commercially available methods for the detection of anti-CP antibodies differ. Careful selection of methods is needed.