Screening autoantibody profiles in systemic rheumatic disease with a diagnostic protein microarray that uses 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 10(3) 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 beta-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.


CONCLUSIONS
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.
McBride
Jeffrey D
JD
Department of Immunology and Molecular Pathology, University College London, London, UK.
Gabriel
Francis Guy
FG
Fordham
John
J
Kolind
Torsten
T
Barcenas-Morales
Gabriela
G
Isenberg
David A
DA
Swana
Marlene
M
Delves
Peter J
PJ
Lund
Torben
T
Cree
Ian A
IA
Roitt
Ivan M
IM
D001323:Autoantibodies / Q000097:blood*; D006801:Humans; D007118:Immunoassay; D008163:Luminescent Measurements; D049329:Nanostructures; D040081:Protein Array Analysis; D012216:Rheumatic Diseases / Q000276:immunology*; D012680:Sensitivity and Specificity
D001323:Autoantibodies