Fifth-generation digital immunoassay for prostate-specific antigen by single molecule array technology

Abstract

Background: Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays.

Methods: We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients.

Results: The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years.

Conclusions: The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

Fig. 1. Dose-response and linearity of SiMoA PSA assay

Y-axis refers to the average number of label enzymes per individual microbead captured in the array. Fitting for optimal read-back utilized four-parameter logistical regression. Fig. 1A highlights the low background obtained with digital quantification. 20 calibration curves gave a mean signal:background ratio at 0.1 pg/mL of 4.33. Fig. 1B depicts linearity obtained from admixtures of high and low female serum samples.

Fig. 2. Limit of Quantification (LoQ) of SiMoA PSA assay

LoQ was defined as the concentration of PSA at which measurement variation over time reached 20%. LoQ was estimated by non-linear power fit of sample replicate CVs across six weeks of testing. The equation of the fit gave a LoQ of 0.0352 pg/mL (standard error 0.0340 – 0.0387 pg/mL). Female serum samples are highlighted in pink.

Fig. 3. Reproducibility of SiMoA PSA assay

Total imprecision was estimated by repeated measurement of a panel of prepared PSA samples over a 10-day period with two runs/day. Variation sources included fiber strips and processing, inter-calibration, and day-to-day reproducibility. The lowest sample was prepared to approximate the LoQ, and the total imprecision obtained was consistent with the LoQ estimate (20% CV at 0.035 pg/mL).

Fig. 4. Post RP PSA results

Longitudinal samples were tested from 13 recurring (red) and 20 non-recurring (black) RP patients. All samples were well above the LoQ of the assay and were measured with good precision. Horizontal lines depict LoQs. The initial PSA value (nadir PSA) was a significant predictor of 5-year biochemical recurrence-free survival (p<0.01), while PSA slope was not a significant predictor in this study (p>0.05).

Fig. 5. Select longitudinal PSA trends

Fig. 5A depicts PSA results from non-recurring patients from one of the clinical sites. Most patients exhibited extremely low, stable PSA levels over the first year following surgery. The early stages of BCR for patient 9908 (red) are also depicted. The LoQ of ultrasenstive PSA methods is off the scale (arrow). Fig. 5B compares the same non-recurring patients with three examples of recurring patients (red) on a broader scale. Exponential projections for the appearance of 200 pg/mL PSA for patients 9908 and 0138 (curved fits, R² 0.999) were consistent with actual BCR. Inset depicts PSA results from a patient in remission who later recurred.

Fig. 5. Select longitudinal PSA trends

Fig. 5A depicts PSA results from non-recurring patients from one of the clinical sites. Most patients exhibited extremely low, stable PSA levels over the first year following surgery. The early stages of BCR for patient 9908 (red) are also depicted. The LoQ of ultrasenstive PSA methods is off the scale (arrow). Fig. 5B compares the same non-recurring patients with three examples of recurring patients (red) on a broader scale. Exponential projections for the appearance of 200 pg/mL PSA for patients 9908 and 0138 (curved fits, R² 0.999) were consistent with actual BCR. Inset depicts PSA results from a patient in remission who later recurred.