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Research Article Open Access

Development and Validation of a Dried Serum/Blood Spot Combined Enzyme-Linked Immunosorbent Assay for the Analysis of Anticitrullinated Protein Antibodies: A New Rheumatoid Arthritis Biomarker Platform

Abstract

The objective of the present study was to combine dried serum spot (DSS) and dried blood spot (DBS) techniques with enzyme-linked immunosorbent assay (ELISA) detection for the assessment of anticitrullinated peptide and protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. Two new analytical DSS- ELISA-ACPA and DBS-ELISA-ACPA protocols were developed and validated by comparison to an established serum ELISA-ACPA protocol. Serum samples, from RA patients (n=24) and healthy adults (n=5), were tested. Assay performance in all three protocols was comparable over the ACPA concentration range 20- 1000 U/mL. Intra-assay and inter-assay precision (%CV) of the raw optical density (OD) data ranged from 0.8–4.5% and 13.7-27.3% for the ELISA-ACPA platform (a 10-month period), 5.6–15.3% and 31.0-61.7% for the DSS-ELISA-ACPA platform (a 10-month period), and 7.0-21.2% and 24.3-39.3% for the DBS-ELISA-ACPA platform (a 2-month period), respectively. The raw OD data was fitted using a symmetrical sigmoidal four parameter-logistic (4PL) curve technique. The inter- assay precision (%CV) and accuracy (%Recovery) of the calibration standards ranged from 0.2-12.5% and 85-113% for the ELISA-ACPA platform, 1-32% and 77-106% for the DSS-ELISA-ACPA platform, and 1-19% and 95-105% for the DBS- ELISA-ACPA platform, respectively. Correlation between the ELISA-ACPA and the DSS-ELISA-ACPA platform for 24 RA and 5 healthy control serum samples was high (r=0.9873, p<0.0001). The new DSS/DBS-ELISA-ACPA platforms may be an accurate and economical approach to assess the prevalence and early detection of RA in large-scale population studies.

Gary W. Caldwell, Wensheng Lang, Sarah Lamberth, Mark Rigby, Yong-Qing Lin

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