Evaluation of Near Infrared Dyes as Labels for Immunoassays Utilizing Laser Diode Detection: Development of Near Infrared Dye Immunoassay (NIRDIA)

Abstract

Abstract Many recent studies of near infrared (NIR) dyes have shown their usefulness as probes for many analytical applications. Since these dyes have absorption maxima between 600 and 1000 nm, a region of low interference by most bioorganic molecules, their use as a label for biomolecules is practical. We report here the first study to evaluate the use of near infrared dyes as a quantitative label for immunoassays. A near infrared dye was derivatized with an isothiocyanate functional group and conjugated to goat anti-human immunoglobulins (GAHG). After purification by G-25 size exclusion chromatography, the conjugate was used in an immunoassay to detect and quantitate human immunoglobulins (Ig), the antigen for GAHG. Polystyrene microtiter plate wells were coated with varying amounts of human Ig. The Ig-coated wells were then exposed to an excess of NIRD-GAHG, and were subsequently read and quantitated by laser diode spectroscopy. An identical immunoassay was performed which utilized an enzyme(peroxidase...