Automated support-resolution strategy for a one-way chemiluminescent multiplex immunoassay

An automated support-resolution strategy was designed to couple with a flow-through immunosensing system for performing a one-way chemiluminescent (CL) multiplex immunoassay. Different from multilabel and multichannel-based detection techniques, this immunoassay method employed a single horseradish peroxidase (HRP) label in one way. With the use of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes, the capture antibodies for CEA and AFP were immobilized on the inner wall of a glass tube and the surface of paramagnetic microspheres (PMs), respectively. The on-line incubation could be performed in the glass tube after introducing the mixture of CEA, HRP-labeled anti-CEA antibody, AFP, anti-AFP immobilized PMs, and HRP-labeled anti-AFP antibody. With the use of a wash step, the formed sandwich immunocomplexes were separated automatically and the immunocomplex immobilized PMs were captured in another unmodified glass tube with a magnet. The CL signals from the two glass tubes were near-simultaneously collected with the aid of an optical shutter to perform quantitative detection. CEA and AFP could be rapidly assayed in the ranges of 1.0-60 and 1.0-80 ng/mL within 27 min. The assay results of clinical serum samples with the proposed method were in an acceptable agreement with the reference values. This system provides a promising multiplex immunoassay approach for clinical applications.