David C. Duffy

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations

The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as ∼10–20 enzyme-labeled complexes in 100 μl of sample (∼10−19 M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10−15 M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).

Measurement of Single Protein Molecules Using Digital ELISA

Digital ELISA is one of the most exciting recent innovations in immunoassay development, leading to demonstrated improvements in sensitivity and low-concentration precision by several orders of magnitude across a broad range of analytes. The principles, theory, methodology and instrumentation are clearly described. The importance and derivation of the average number of enzyme-labeled protein molecules per bead (AEB) are explained. The theory relating to analytical sensitivity and dynamic range is described, with experimental analysis of the performance of the digital ELISA methodological elements. The fundamental limitations of the technique are reviewed, along with possible approaches to overcome them. There is then a step-by-step theoretical analysis of each stage of the assay (analyte capture onto the antibody-coated beads, labeling of the captured analyte with biotinylated detection antibodies, labeling of the biotinylated detection antibodies with enzyme conjugate, and detection of the enzymes). Background and specificity are also reviewed. Assay development is then described for reagents, assay optimization, calibration, and background and interference minimization. Actual assay performance from Digital ELISA assays for several analytes is presented, demonstrating dose-response linearity, sensitivity, reproducibility and accuracy. A number of potential fields that will benefit from the exceptional performance of this technique are reviewed.