Benchtop Antigen Detection Technique Using Nanofiltration and Fluorescent Dyes

John H. Glenn Research Center

The designed benchtop technique is primed to detect bacteria and viruses from antigenic surface marker proteins in solutions, initially water. This inclusive bio-immunoassay uniquely combines nanofiltration and near infrared (NIR) dyes conjugated to antibodies to isolate and distinguish microbial antigens, using laser excitation and spectrometric analysis. The project goals include detecting microorganisms aboard the International Space Station, space shuttle, Crew Exploration Vehicle (CEV), and human habitats on future Moon and Mars missions, ensuring astronaut safety. The technique is intended to improve and advance water contamination testing both commercially and environmentally as well. Lastly, this streamlined technique poses to greatly simplify and expedite testing of pathogens in complex matrices, such as blood, in hospital and laboratory clinics.

The Benchtop Analysis System (a) consists of a medium-power, class IV laser, a four-port cuvette sampler holder, a spectrometer, laptop and software, and fiber-optic cables. (b) The microcentrifuge used in nanofiltration of the complexes.

The approach relies on NIR fluorescent dyes derivatized to specific antibody sets that are selected to bind and differentiate microbial surface proteins, termed antigens. In a solution containing an antigenic slurry, NIR conjugated antibodies are added to the mixture, and will bind to the respective antigens if present. To eliminate any false positives, excess antibodies, i.e., those antibodies not bound to an antigenic protein or those with no respective antigen present, are removed via the nanofiltration process using a portable, table-top centrifuge. The remaining NIR dye/ antibody and antigenic protein pairs left on the nanofilter are transferred to cuvette, excited by an NIR laser, and detected by spectrometer. Using simple computer software, the results are easily interpreted as intensity peaks at the appropriate NIR offset wavelength emission.

Initial data reveal the assay sensitively identified antigens at intensity counts of 100 IC or higher (or roughly 36 pW) with an accuracy of 85 percent for 2-hour incubations and 75 percent for 3-hour incubations. Interestingly, samples incubated for less time (2 hours vs 3 hours) produced an increased percentage of antigen detection. Further testing at incubation times such as 1 hour or lower could potentially increase positive predictability based on the study’s results. Also encouraging were negative control experiments with nonspecific antigens, beta galactosidase and thyroglobulin, which showed results of 100 percent accuracy, with no false positive detection.

This work was done by Maximilian C. Scardelletti and Vanessa Varaljay of Glenn Research Center.

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