Evaluation on the use of reactive dye-modified polylysine as the biomarker in immunochromatographic test application

Abstract

An improved dye immunochromatographic test (DICT) using polylysine (PL) as conjugate spacer loading dye molecules to enhance chromophor color intensity with the potential of a simultaneous multicolored assay has been developed. To construct this new effective chromophor, a dyeing process coupling a reactive dye, Procion Blue MX-7RX (PB7RX), with PL of different molecular weights was performed. The optimal conjugate condition between PB7RX and PL was studied. It showed that under the optimized dyeing conditions, a PL molecular weight of 189.4kDa and a molar ratio (mol dye/mol amine group in PL) of 1.5 were obtained. The resulting dyed PL chromophor, used as both a spacer and a color intensifier, was further labeled to a model antibody, anti-human serum albumin (anti-HSA), to build a PB7RX–PL–anti-HSA (PPA) conjugate. The PPA obtained in this way generated the highest color intensity of 19,455 assayed by immunochromatographic test strip under densitometer scanning. A competitive DICT for determination of HSA was carried out. A linear range between 0 and 18.77μg/ml with a detection limit of 0.49μg/ml was observed. A test for using dyed PL chromophors as biomarkers was also performed to demonstrate the feasibility of a multianalyte immunoassay.