IMMUNOAFFINITY ANALYSIS OF SUBSTANCE P IN COMPLEX BIOLOGICAL FLUIDS: ANALYSIS OF SUB-MICROLITER SAMPLES

Abstract

ABSTRACT There is a growing need for systems capable of measuring specific analytes in biological samples, especially complex biological fluids. Additionally, this need extends to applying such analyses to precious clinical or research samples, for many of which only micro quantities are still in existence. Immunoaffinity separations provide a useful approach to isolating specific analytes from complex matrices, as well as being capable of miniaturization. In the present communication, we have designed an immunoaffinity system capable of measuring a single analyte in 50 nL samples. We have chosen the clinically important neuropeptide, substance P (SP), as our model analyte and applied it to studying the ability of a micro-chromatography system to measure SP in a number of different biological matrices. The system is capable of isolating SP from biological fluids, including cell cytosols, with reasonable efficiency and reproducibility. The incorporation of laser-induced fluorescence detection enabled the system to achieve a lower limit of detection around 500 femtograms/mL and could isolate the specific analyte in under 5 min, thus, making it potentially useful for high throughput clinical studies.