Homogeneous temperature- and substrate-resolved technology for a chemiluminescence multianalyte immunoassay

A novel dual-resolution chemiluminescence (CL) immunoassay platform for the homogeneous determination of four proteins is proposed. The immunoassay is based on temperature- and substrate-resolved technologies. As a proof-of-concept, we evaluated our method for the simultaneous determination of four model proteins (i.e. immunoglobulin A (IgA), IgG, IgM and PEGylated recombinant growth hormone (GH, protein drug)) by using two homogeneous carriers (thermo-sensitive poly-N-isopropylacrylamide (PNIP) and magnetic beads (MB)), and two different CL substrates for alkaline phosphatase (ALP) and horseradish peroxidase (HRP). Briefly, one pair of capture antibodies was immobilized on the PNIP and another pair of capture antibodies was bound to the MB. The four carrier–antibody conjugates were mixed and reacted with the four proteins in a single vessel, and the mixture of two ALP- and two HRP-labeled tracer antibodies was subsequently added to form the four sandwich immunocomplexes. After washing, the two MB conjugates could be easily separated from the two PNIP conjugates by magnetic force while ensuring that the temperature was lower than the lower critical solution temperature (LCST). The CL substrates for ALP and HRP were delivered sequentially into the resultant MB and PNIP solutions. A simple CL setup was employed to perform our novel multiplexed protein assays in the measurement order of ALP and HRP. No obvious cross-reaction was observed. IgA, IgG, IgM and GH were found to be suitably assayed in the ranges of 1.0–50, 0.50–50, 0.50–50 and 0.50–50 ng/mL with limits of detection of 0.50, 0.25, 0.25 and 0.25 ng/mL, respectively. Overall, this simple and homogeneous technique will find applications in areas such as genomics, combinatorial chemistry, drug screening and clinical diagnosis.