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Near-Infrared Heptamethine Cyanine Dyes: A New Tracer for Solid-Phase Immunoassays

Volume 51, Number 6 (June 1997) Page 836-843

Williams, Richard J.; Peralta, Jose Mauro; Tsang, Victor C.W.; Narayanan, Narasimhachari; Casay, Guillermo A.; Lipowska, Malgorzata; Strekowski, Lucjan; Patonay, Gabor

Near-infrared (near-IR) fluorescence has been used to develop a solid-phase immunoassay that detects trace amounts of human immunoglobulin (HuIgG). Various concentrations of HuIgG bound to a nitrocellulose surface were determined from the fluorescence generated by near-IR labeled goat anti-human antibody (GAHG) bound to the HuIgG. The GAHG was labeled with a heptamethine cyanine fluorophore that has spectral properties in the near-IR region (above 780 nm). These fluorophores are versatile because they can be modified for several bioanalytical applications. Fluorescence was detected with a near-IR fluorescence instrument previously developed in the laboratory. Two cyanine fluorophore labels were evaluated for the ability to selectively bind to GAHG on a nitrocellulose matrix with a minimal amount of background interference. After the most appropriate near-IR fluorophore was selected, the labeling of GAHG was optimized under aqueous conditions. The most effective GAHG-dye conjugates were used to develop an immunoassay to detect various concentrations of HuIgG. The results are presented, here. Solutions of HuIgG with concentrations as low as 10-10 molar have been detected with a minimum of interference.