Development of a solid-phase immunoassay for the detection of human immunoglobulin G: A novel bio-analytical application of semiconductor laser induced near-infrared fluorescence

The limitations of laser induced fluorescence in the development of immunoassays have been widely reported. Recent advances in semiconductor laser technology have made the use of lasers as an excitation source more practical. These semiconductor lasers typically excite in the near infrared region (650-1200 nm). When coupled with near infrared laser dyes that are spectrally compatible, in theory, several bioanalytical applications become more feasible. An immunoassay that uses antibody labeled with these near infrared laser dyes should have a detection sensitivity comparable to what is currently available with the enzyme linked immunosorbent assay (ELISA) technique. Based on simple models, a solid phase the immunoassay to detect trace amounts of human immunoglobulin G (HuIgG) was developed in three major steps.;In the first step, noncovalent and covalent methods of labeling protein with near infrared (near-IR) polymethine cyanine dyes were evaluated for use in analyzing human serum albumin (HSA) by high performance liquid chromatography (HPLC) with near infrared absorbance detection. While noncovalent labeling was faster than covalent labeling, and took place in the physiological pH range, covalent labeling was more stable under conditions encountered in many of the widely used types of HPLC and demonstrated the stability and selectivity required for most bioanalytical applications of near-IR fluorescence.;In the next step, the construction of a near-infrared fluorescence detector for measuring picomolar levels of near-IR laser dyes was completed. The detector was designed for use in an immunoassay technique that employs antibodies labeled with near-IR polymethine cyanine dyes, and possessed the capability to measure fluorescence in both solution and solid phase environments. The instrumentation was characterized. This included its hardware and data acquisition software components.;Finally, solid phase near-infrared fluorescence was used to develop an immunoassay that detected trace amounts of HuIgG, bound to a solid phase surface, with goat anti-human antibody (GAHG) labeled with a heptamethine cyanine dye that absorbs and fluoresces above 780nm. Detection was done with a near-IR fluorescence instrument previously developed in the laboratory. Labeling of GAHG was optimized under aqueous conditions to detect HuIgG on a nitrocellulose matrix. Solutions of HuIgG with concentrations as low as 10...