A new two-color Fab labeling method for autoantigen protein microarrays

Abstract

Antigen microarrays hold great promise for profiling the humoral immune response in the settings of autoimmunity, allergy and cancer. This approach involves immobilizing antigens on a slide surface and then exposing the array to biological fluids containing immunoglobulins. Although these arrays have proven extremely useful as research tools, they suffer from several sources of variability. To address these issues, we have developed a new two-color Fab labeling method that allows two samples to be applied simultaneously to the same array. This straightforward labeling approach improves reproducibility and reliably detects changes in autoantibody concentrations. Using this technique we profiled serum from a mouse model of systemic lupus erythematosus (SLE) and detected both expected and previously unrecognized reactivities. The improved labeling and detection method described here overcomes several problems that have hindered antigen microarrays and should facilitate translation to the clinical setting.

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

“…To address these potential complications, we are working to incorporate several technological advances developed by our group and our collaborators into the serum factor array platform. We have developed methods that allow for the detection of reactivity on multiple fluorescent channels and may ultimately enable non-fluorescence-based detection (17,49). We have also recently reported the development of array platforms that enhance the level of fluorescence-based detection more than 100-fold (50).…”

IRF9 and STAT1 are required for IgG autoantibody production and B cell expression of TLR7 in mice

“…In the pristane model of SLE, BALB/c mice given a single i.p. injection of the mineral oil pristane develop a lupus-like disease characterized by the production of anti-U1snRNP/Sm, anti-RiboP, and anti-DNA autoantibodies and the development of inflammatory kidney disease (14)(15)(16). Pristane treatment induces apoptosis (17), formation of peritoneal lipogranulomas that express high levels of IFN-I-inducible genes (18), and hypergammaglobulinemia characterized by high levels of the pathogenic isotype IgG2a (19).…”

Expanding Assay Dynamics: A Combined Competitive and Direct Assay System for the Quantification of Proteins in Multiplexed Immunoassays

“…Although this situation certainly applies for highly parallel assays, the amount of fluorophore introduced into systems of low to medium complexity (up to 20 analytes) is well below the amount of label found in systems that feature complete labeling of the sample (8,15 ). The limitations we have observed may be comparable to those of multiplexed assays, and comparable assay performance is expected.…”

“…These systems benefit from increased throughput and from the potential for testing a wide variety of autoantigens in a single assay, which lowers assay times and costs. Nevertheless, assay systems that give diagnostically relevant information are very demanding, and although several interesting approaches have recently been developed (13)(14)(15), standardization and validation are still required before such assays can be used in clinical diagnostics.…”