Amplification of microsphere-based microarrays using catalyzed reporter deposition

Assay sensitivities using three fluorescent signal generation schemes were evaluated on the Luminex flow cytometer. Following microsphere capture of antigen by immobilized antibodies, bound targets were quantified by use of (1) Cy3-labeled “tracer” antibodies (30 min total time), (2) biotinylated tracers followed by streptavidin-R-phycoerythrin (60 min total time), or (3) biotinylated tracers followed by avidin-peroxidase conjugates and tyramide signal amplification (TSA; 90 min total time). Use of TSA for signal generation in three individual toxin assays improved performance up to 100-fold over Cy3-antibody-based detection, and while streptavidin-R-phycoerythrin provided equivalent sensitivities, TSA produced dramatic increases at low concentrations simplifying positive sample identification. Detection limits for TSA-interrogated assays for ricin, cholera toxin, and staphylococcal enterotoxin B were 64 pg/ml, 4 pg/ml, and 0.1 ng/ml, respectively, using optimized conjugates; analogous detection limits for Cy3-antibody-interrogated assays were 8 ng/ml, 1 ng/ml, and 1 ng/ml, respectively. No improvement was observed in botulinum toxoid A assays when TSA amplification was used. As unique preferences for specific avidin-peroxidase conjugates were observed in the individual assays, improvements in multiplexed assays utilizing a single conjugate were significantly lower (3–10-fold improvements). Furthermore, increases in variability resulted in poorer performance of TSA-interrogated assays for botulinum toxoid, indicating that assay-specific optimization should be performed, especially prior to multiplexing.