<b>Fundamental Accuracy Constraints in Surface Kinetic Assays and Implications for High-Throughput Screening</b>

<p dir="ltr">The accuracy of equilibrium dissociation constants (Kd) determined from surface-based kinetic assays such as surface plasmon resonance (SPR) and biolayer interferometry (BLI) is often attributed to technical limitations, including mass transport effects, nonspecific binding, and detection sensitivity. However, retrospective analyses have shown that even under optimized conditions, assays can produce markedly different Kd values for the same binding pair, indicating that fundamental, not just technical, sources of inaccuracy are at play. In this study, we conduct an analytical error-propagation analysis to derive closed-form expressions that describe how systematic errors in experimental variables, particularly analyte concentration (T0) and response signal (Rt), affect the accuracy of Kd determination. Our analysis reveals a critical and previously overlooked principle: under fixed levels of systematic error, lowering T0 improves the accuracy of Kd provided that the maximum response (Rmax) can be independently determined. Building on this insight, we introduce the Dual-Sensorgram High-Throughput Assay (DSHTA), which employs two complementary sensorgrams: one at high T0 to determine Rmax and another at low T0 to minimize error amplification in Kd determination. Numerical simulations and experimental SPR measurements confirm that, compared with conventional single-sensorgram analysis, DSHTA substantially reduces systematic error in Kd determination, enabling accurate compound ranking without the need for labor- and resource-intensive steady-state titrations. This strategy provides both scientific and economic advantages for pharmaceutical research by enhancing confidence, accuracy, and efficiency in early-stage drug discovery decisions.</p>