Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA. The enhancement of precision and sensitivity using fluorogenic substrates was demonstrated in a direct-binding ELISA in a low-analyte concentration range compared with commonly used chromogenic substrates. The enhancement in precision was demonstrated quantitatively with lower coefficients of variation in measurements of signal intensities, approximately a five- to six-fold enhancement in signal-to-noise ratio at a given analyte concentration with fluorogenic substrates. Similarly, the amplitude of the enhancement in sensitivity, as reflected by relative limits of detection or quantitation, is approximately two- to five-fold when compared with commonly used chromogenic substrates. Additional advantages of a fluorescence-based ELISA format include the continuous monitoring of initial rates of enzymatic reactions, the measurement of fluorescence changes in the presence of particulate materials, the absence of a quench step, and a larger quantifiable analyte range.