Sequential determination of two proteins by temperature-triggered homogeneous chemiluminescent immunoassay

A novel protocol for performing a sequential dual-protein immunoassay, based on a temperature-triggered separation/mixing process and HRP-catalyzed chemiluminescence (CL) detection, is described. In contrast to current multilabel-based detection techniques, a single HRP label is employed in this proposed method. Herein we introduce poly(N-isopropylacrylamide) (PNIP) and magnetic beads as bimolecular immobilizing carriers to separate different targets by taking advantage of thermal response, as demonstrated by sequential detection of human IgG and IgA. PNIP is known to aggregate and precipitate out of water when the temperature is raised above the lower critical solution temperature (LCST) of 31 degrees C; thus, it can be separated from supernatant by centrifugation. Besides, magnetic beads can be separated from PNIP by magnetic force as the temperature is lower than LCST. A homogeneous noncompetitive ELISA was employed, formed by primary antibodies immobilized onto the surface of magnetic beads and PNIP, antigen as IgG and IgA in the sample, and HRP-labeled second antibodies. Moreover, highly sensitive CL detection of HRP was applied, and the detection limits of IgG and IgA were as low as 2.0 and 1.5 ng/mL, respectively. Within the calibrated amount, the protocol had excellent precision within 11% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual analyte in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for IgG and IgA in 40 human serum samples.