No 6, 2012

Publication of the Month


June 06/12: 

Reference material for anti-CCP assays


Key message:

  • The international standard can improve the comparability of quantitative anti CCP assays and reduce their variability when used as calibrator.
  • The reference material seems to be representative of the general anti-CCP reactivity.


Bizzaro N, Pregnolato F, van Boekel MAM, Villalta D, Tozzoli R, Tonutti E, Antico A, Borghi MO, Wiik A, Meroni PL
Preliminary evaluation of the first international reference preparation for anticitrullinated peptide antibodies
Ann.Rheum.Dis. 2012. doi:10.1136/annrheumdis-2011-200693  



In the diagnosis of rheumatoid arthritis (RA) the lack of a useful reference reagent makes it difficult to compare different anti-CCP assays available on the market, especially regarding the comparison of different studies and the evaluation whether anti-CCP levels are usable for monitoring therapy and for prognostic purposes. Recently a potential reference sample was provided to the Center for Disease Control and Prevention (CDC). This will be made available as the first international anti-CCP reference reagent if it fulfils the criteria for such use.


In the present study the reference reagent and sera from 20 patients with RA were analysed by 12 commercial ELISAs for anti-CCP detection in order to evaluate their reproducibility, linearity and comparability. To establish the cut-off value of each assay, when using the reference serum as calibration curve, we tested samples from 50 normal healthy subjects.
The results of kits calibrated on their own standard curve showed huge variability, up to 85.9% for the negative sera and 82.5% and 76.4% for the low and medium/high positive samples, respectively. By using the reference material for the kit calibration the variability was significantly reduced to 27.9% and 33.5% for the medium/high and negative samples, respectively and to 55.5% for the low positive sera resulting in an overall drop from 81.4% to 39.3% of mean variability values. 


The availability of an international standard and the improved comparability of the quantitative results will increase the diagnostic power of anti-CCPs for RA.
The optimal linearity of the dilution curve of the proposed reference standard obtained in all 12 methods shows that the reference sample can be used as a calibrator in assays using different antigenic substrates or different assay structures.
The reference material is highly reactive with all the autoantigenic preparations in the different kits and seems to be representative of the general anti-CCP reactivity.


Standardization with the help of reference sera is important in autoimmune diagnostics. However, experiences with international standards in autoantibody testing tell us, that this does not help in terms of comparability of test results because of the different conditions in every test system. Different assays are calibrated using different calibration material, and patient samples containing a different composition of antibodies compared to the international standard will give different results. A step forward would be to use the identical calibration material for all assays, as suggested for CCP by the authors. The remaining differences in test results are then due to differences in antigen, reagents and assay performance conditions. However, differences would still be substantial and it would just be unrealistic to use an international standard as calibration material for all assays, not least because of the high volume needed.


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As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.