Screening autoantibody profiles in systemic rheumatic diseases with a diagnostic protein microarray using a filtration-assisted Nanodot Array Luminometric ImmunoAssay (NALIA)

Background: We developed a cost-efficient modular system for multiplex analysis of the multiple autoantibodies that characterize systemic rheumatoid diseases.
Methods: The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls. The test fluid, control sera, and subsequent reagents are drawn through the membrane. The captured analytes are quantified by monitoring chemiluminescence with a charge-coupled device (CCD) and analyzed with commercial array software.
Results: The assay can detect <20 x 103 IU/L of anti-dsDNA. The interwell CV was 10%–14%. There was an 83% concordance ({kappa} = 0.56) between the NALIA results obtained for anti-dsDNA assayed by β-testing in a routine immunology diagnostic laboratory and the results obtained with a conventional ELISA reagent set. The concordance values for Ro, La, Sm, and RNP were 98% ({kappa}, 0.92), 93% ({kappa}, 0.41), 97% ({kappa}, 0.62), and 97% ({kappa}, 0.73), respectively.
Conclusion: The NALIA approach promises to provide a highly economical platform for a wide range of applications that require assays of multiple analytes. The degree of concordance of our results with a conventional reagent set was no less than that occurring between different commercial products. A sample of serum from a finger stick provides a volume sufficient to perform the array assay.