Magnetic beads-based chemiluminescence substrate-resolved duplex immunoassay for sequential detection of two ischemic stroke markers with two orders of concentration difference.

Herein we have demonstrated a novel protocol to integrating two immunoassay procedures for performing a sequential dual-protein determination, based on a chemiluminescence (CL) substrate-resolved technology. We evaluated our method for the sequential determination of S100β and neuron-specific enolase (NSE) by using alkaline phosphatase (ALP) and horseradish peroxidase (HRP) as two different labels. Especially sharply different and suitable linear ranges and detection limits were successfully obtained for these two markers. Briefly, a "sandwich-type" detection strategy is employed in our design, where capture antibodies against S100β and NSE were coupled to magnetic beads. The quantification of NSE was obtained by further reacting with ALP modified antibodies and measurements by catalyzed chemiluminescence while the determination of S100β was accomplished with HRP-labeled anti-rabbit IgG. A simple CL setup was employed to perform our novel multiplexed protein assays in a single experiment. No obvious cross-reaction was observed. S100β and NSE were found to be suitably assayed in the ranges of 0.02 - 1 and 1 - 20 ng/mL, and the limits of detection were 0.005 and 0.2 ng/mL for S100β and NSE, respectively. It is straightforward to adapt this strategy to detect a spectrum of other biomarkers, which can provide important information about the early-stage diagnosis of diseases.