This year, a bench-top technique was developed at the NASA Glenn Research Center to detect antigenic proteins in fluids. The detection of these proteins is a tremendous concern at NASA because of the importance of ensuring astronaut safety onboard the International Space Station, the space shuttles, and the Crew Exploration Vehicle and in human habitats for the upcoming Moon and Mars missions. The detection of these antigenic proteins is also of great importance in keeping terrestrial waterways, treatment plants, and food-processing centers safe from contamination. The technique involves the use of near infrared (NIR) fluorescent dyes conjugated to antibodies, centrifugation, nanofilters, and spectrometry. The system used to detect the antigens utilizes a miniature spectrometer, fiber-optic cables, a tunable miniature NIR laser, and a laptop computer--making the system portable and ideally suited for desktop analysis (see the photograph).
Bench-top antigen-detection system, including laptop computer, spectrometer, cuvette holder, NIR laser, and fiber-optic cables.
For this technique, antibody/fluorescent dye pairs that absorb in the NIR region and emit an offset emission at a longer wavelength in the NIR region are mixed into a solution containing antigens and are then centrifuged. Each antibody/dye pair is specific to one antigen and absorbs and emits at unique wavelengths, making the pairs distinguishable during analysis.
Because of the size difference between the antibody/fluorescent dye pairs and the large protein antigens used in this experiment, antibody/fluorescent dye pairs can be filtered through the nanofilter, but pairs that have bound with antigens cannot. Consequently, antibody/fluorescent dye pairs that have not bound with any of the antigenic proteins can be filtered away from the fluid sample by centrifugation and nanofilters, leaving only antibody/fluorescent dye pairs that have combined with their specific antigen.
Emission of the anti-IgM + IgM complexes in 1× phosphate buffer saline (1× PBS) solution (after nanofiltration) at 804 nm as excited by the NIR laser.
After filtration, the fluid sample is excited with a tunable NIR laser that causes the fluorescent dyes to emit an offset wavelength. The different wavelengths are detected by spectrometry, thus revealing which antigens are present in the fluid sample. In the proof-of-concept experiment, the IRDye 800 CW (Rockland Immunochemicals) conjugated to the primary antibody, anti-IgM, was combined to the antibody (used as our antigen) IgM. An offset emission of 804 nm was detected when the antibody/dye-antigen complexes were excited by the tunable laser at 778 nm and is shown in the preceding graph. To validate this concept, researchers mixed IRDye 800 CW conjugated to the antibody anti-IgM with the nonspecific antigens β galactosidase and thyroglobulin. For both nonspecific antigens, no signal was observed, as illustrated in the following graphs, thus proving that the anti-IgM did not bind to the nonspecific antigens, which were consequently filtered away.
Anti-IgM + beta galactosidase in 1× PBS solution (after nanofiltration) as excited by the NIR laser.
Anti-IgM + thyroblobulin in 1× PBS solution (after nanofiltration) as excited by the NIR laser.
This technique could potentially be used in fluids to detect multiple antigens from microorganisms onboard spacecraft and in human habitats on the Moon and Mars. Because of its portability, the technique is ideally suited for laboratory and clinical diagnostics as well as field testing. With further development, this novel technique could be used to detect antigenic microorganism cell proteins in more complex fluids, such as blood.
Glenn contact: Maximilian C. Scardelletti, 216-433-9704, Maximilian.C.Scardelletti@nasa.govLast updated: December 14, 2007
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