No abstract

SmartBead Technologies has developed a multiplexed immunofluorescence assay, the UltraPlex ANA Profile, which determines nine antinuclear antibodies simultaneously. The UltraPlex assay platform uses bar-coded microparticles to track analytes through assays. These bar-coded microparticles were used to tag and track key ANA markers: Jo-1, Scl-70, Sm, SmRNP, SSA, SSB, U1RNP, Centromere B, dsDNA, and a blank control microparticle. The immunofluorescence assays are fully automated and are performed on a Perkin-Elmer multiprobe II liquid handling system that performs all sera dilutions, additions of reagents, washes, and incubation steps. Results were determined by the automated UltraPlex plate reader. This fully automated multiplex antinuclear antibody (ANA) immunoassay was used to screen commercially available ANA-positive sera and negative control samples. The UltraPlex ANA Profile enables the panels of samples to be screened simultaneously for nine ANA antoantibodies, requiring significantly less labor and fewer reagents, with performance equivalent to existing gold-standard methods.

To enable the detection of IgG class, anti-IgA antibodies (IgG-aIgA) and to investigate the possible occurrence of IgE class, anti-IgA antibodies (IgE-aIgA), we developed a solid phase immunoradiometric assay (IRA), which uses purified IgA coupled covalently to microcrystalline cellulose as an immunosorbent. Radiolabeled, Fc specific anti-IgG and anti-IgE antibodies were used to detect specific aIgA after incubation of test sera or controls with the immunosorbent. IgG-aIgA were detected by the IRA in 100 and 67 per cent of control sera with class specific and limited specificity aIgA. The IRA was sensitive to approximately two ng of class specific IgG-aIgA. IgG-aIgA also were detected by IRA in 7.9 per cent of sera from patients with urticarial transfusion reactions and 73 per cent of sera from patients with ataxia telangiectasia and IgA deficiency. Sera from 50 normal blood donors did not have detectable IgG-aIgA. Tests for IgE-aIgA were negative in all cases, including control sera with class specific IgG-aIgA. We conclude that the IRA is a sensitive and reproducible method for detection of class specific and limited specificity IgG-aIgA, and that IgE-aIgA do not mediate urticarial transfusion reactions.