2011 AACR, MOLECULAR DIAGNOSTICS IN CANCER THERAPEUTIC DEVELOPMENT, DENVER, CO
David Wilson1, David Duffy1, David Rissin1, Cheuk Kan1, Todd Campbell1, Stuart Howes1, David Fournier1, Tomasz Piech1, Gail Provuncher1, Purvish Patel1, Evan Ferrell1, Andrew Rivnak1, Jeff Randall1, Linan Song1, David Walt2.1Quanterix Corporation, Cambridge, MA, 2Tufts University, Boston, MA.
Develop a method for measuring prostate specific antigen (PSA) in the serum of patients who have undergone radical prostatectomy (RP). The method is based on Single Molecule Array (SiMoA) technology in which individual molecules of analyte are trapped in femtoliter microwells and counted. Measurement of PSA in post RP patients 4–6 weeks after surgery (nadir PSA levels) has potential prognostic value.
The assay starts as a sandwich ELISA except that 2.7 µm paramagnetic beads serve as the solid phase rather than the ELISA plate. The assay is initiated by mixing sample with beads coated with anti–PSA antibody, and the mixture is incubated for two hours. During the incubation, PSA molecules are captured with an excess of beads, such that the ratio of bound PSA per bead is much less than one. Following a wash in which beads are separated using a magnet, biotinylated detector antibody is incubated with the beads for 45 minutes. After a second wash, a conjugate of streptavidin–ß–galactosidase is incubated with the beads for 30 minutes to form the final enzyme immunocomplex. Upon completion, the beads are loaded onto the ends of bundles of optical fibers etched with an array of 50,000 microwells, each with a volume of 50 femtoliters (4.5 µm wide x 3.25 µm deep). Single beads are introduced into each well via a 10–minute centrifugation at 1,300 g. Excess beads are removed, and the bundles are sealed against a solution of resorufin ß–D–galactopyranoside (RDG). Wells containing an enzyme immunocomplex convert RDG to a fluorescent product, which becomes concentrated in the small volume of the wells. Concentration of the fluorescent product permits ready imaging by a CCD camera and discrimination from wells that contain no enzyme immunocomplex. Poisson statistics predict that each well will contain either one PSA molecule or no PSA molecules when the ratio of bound PSA per bead is much less than one. This results in digitized quantification. Raw signal is recorded as “% active wells”, which can be converted to “average enzymes/bead” to correct for non–Poisson behavior at higher PSA concentrations. The output is related to a standard curve and converted to a PSA concentration of the sample.
In preliminary validation studies across multiple runs and reagent batches, the AccuPSA assay exhibited a limit of detection (LOD) of 0.01 pg/mL, and a limit of quantification of less than 0.05 pg/mL. Linearity was demonstrated across a calibration range from 0 to 100 pg/mL, giving a five–log assay range relative to the LOD. The assay exhibited good agreement with a commercially available PSA test. PSA levels were measured in 60 post–RP specimens selected as being below the detection limit of a commercially available chemiluminescent immunoassay. All specimens gave measurable PSA results in the SiMoA PSA assay, with results ranging down to 0.014 pg/mL (0.4 fM).
The results suggest that PSA can be reliably measured in post RP patients with SiMoA technology. Recent results by others suggest measurement of nadir PSA following surgery may have prognostic value for recurrence–free survival. Measurement of PSA in post RP patients with AccuPSA could affirm a good prognosis, reduce unnecessary adjuvant radiation, and enable earlier detection of recurrence for earlier, more effective treatment.
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