Relative Quantification of Experimental Data from Antigen Particle Arrays

Abstract

Protein arrays typically consist of a capture protein on a matrix, e.g., glass slide, silicon chip, or coded microparticle (1). The latter minimizes steric constraints and enhances reaction kinetics (2). Microarray technologies have been used for detecting allergens (3) and cytokines (4). A primary advantage of microarray technologies over conventional immunoassays is the ability to multiplex assays. Currently, ELISA is the method of choice for autoantibody detection (5). This method, however, is labor-intensive and requires comparatively large sample and reaction volumes. Nonetheless, ELISAs are currently performed to aid differential diagnosis of certain autoimmune diseases. Dermatomyositis (6) is characterized by detection of anti-Jo-1 IgG in patient sera. Both anti-Sm and anti-RNP/Sm IgGs are indicative of systemic lupus erythematosus (7). The presence of anti-Scl70 IgGs aids diagnosis of systemic sclerosis (8). Anti-SSB and -SSA IgGs are present in systemic lupus erythematosus (9) or Sjogren syndrome (10). With microarray technologies, all these autoantibodies can be screened simultaneously. The Luminex particle array platform comprises a hundred microparticles, each possessing a distinct fluorescent signature generated by a blend of two internal fluorescent dyes. Capture protein is conjugated to the bead surface, to assay for the cognate entity within a single reaction vessel. The instrument includes a microfluidics system and two lasers. A 635 nm laser excites the red and infrared classifier fluorophores that form the particular signature of each bead set. The second laser (523 nm) excites phycoerythrin dye used as a molecular tag for detection. Detection of cytokines (11), cancer markers, and allergens (12) has been reported. Use of internal calibration curves for cytokine quantification has been documented (13), but with antigen arrays, quantification is more difficult because of the time and expense required for antibody synthesis. Where quantification of antibodies has been cited, competitive immunoassays are described that use labeled forms of the …