In complex diseases such as osteoarthritis (OA), the measurement of multiple biomarkers is expected to provide valuable information to improve diagnosis, prognosis and therapy monitoring. Bead-based xMAP technology offers a flexible and open platform to simultaneously quantify proteins. The aim of this study has been to develop sandwich antibody microarrays to validate a panel of 6 potential OA biomarkers in clinical samples.
Antibody pairs and recombinant proteins tested to work in an ELISA were purchased for each of the 6 analytes of interest. First, 1.6 µg of each monoclonal antibody were coupled to 5x105 of an activated fluorescent bead region and the efficiency of the coupling was confirmed by Phycoerythrin-labelled anti-species antibodies. Assay conditions, including the standard curve range and sample dilution, were optimized for each protein individually. Cross-reactivity of antibodies with the non-target analyte was assessed between those proteins requiring the same sample dilution to generate multiplex sandwich immunoassays. Then, analytical characteristics in terms of accuracy, precision and limit of detection (LLOD) and quantification (LLOQ) were evaluated and finally their utility to quantify the potential biomarkers was assessed.
Individual capture immunoassays were successfully converted for all biomarkers to a protein microarray platform. According to the serum sample dilution, the six proteins were finally grouped in three different immunoassays: a duplex, a triplex and a singleplex sandwich immunoassays. No cross-reactivity was observed for the antibody pairs against the non-targeted proteins. Median Fluorescence Intensity of the negative controls in 8 replicates was used to evaluate the LLOD and LLOQ of each microarray. Precision and accuracy were calculated using 3 different known concentrations of the standards. In all cases precision was below 10% and accuracy ranged between 70–130%.
In conclusion, three different sandwich immunoassays have been successfully developed in a suspension beads array format to absolutely quantify six potential OA biomarkers.