Advances in Next Generation Ligand Binding Assay Technologies

Abstract

In contemporary drug discovery, lead optimization, and biotherapeutic characterisation, real-time ligand–target interaction quantification has become essential. Although they yield accurate affinity values, conventional endpoint assays including ELISA, radioligand binding, and fluorescence polarisation are unable to record kinetic data that are crvery essential for comprehending residence duration and mechanism of action. Under near-physiological settings, next-generation ligand binding assays (LBA) now enable label-free, continuous monitoring of association and dissociation events .Real-time kinetic, affinity, and thermodynamic data are provided by methods like surface plasmon resonance (SPR), bio-layer interferometry (BLI), microscale thermophoresis (MST), mass photometry (MP), electrochemical impedance spectroscopy (EIS), field-effect transistor (FET) biosensors, and nanopore sensing .With a focus on kinetic screening, binding selectivity, and integration with automation and computational modelling, this review examines the relevance of these new technologies in biopharma operations . We provide an overview of the transition from labelled endpoint assays to high-throughput, real-time systems, emphasising its benefits, drawbacks, and potential future developments.

Introduction

Next-generation ligand binding assays (NGLBAs) represent a significant advancement over conventional ligand binding assays such as ELISA and RIA by enabling more sensitive, real-time, and often label-free analysis of biomolecular interactions. These technologies are increasingly important in pharmacokinetics, drug discovery, and biomarker quantification, as they overcome limitations of traditional assays related to sensitivity, speed, and data depth. To promote awareness of modern bioanalytical methods, several emerging NGLBA technologies have been introduced, including SPR-based systems, NanoDLSay, microfluidic lab-on-a-chip devices, and AlphaLISA.

Surface Plasmon Resonance (SPR) enables real-time, label-free monitoring of molecular binding events by detecting changes in refractive index on a metal surface. It is widely used for studying protein–ligand interactions, binding kinetics, equilibrium measurements, and mutant protein analysis. SPR provides detailed kinetic and affinity data but requires careful surface immobilization and expertise in data interpretation.

NanoDLSay adapts dynamic light scattering (DLS) for quantitative bioanalysis by measuring changes in particle size when nanoparticles bind to target analytes. It is applied in cancer biomarker detection, protein interaction studies, and protein conformational analysis. However, interference from background scattering in complex biological samples remains a challenge.

Microfluidic Lab-on-a-Chip (LoC) technologies miniaturize and automate biochemical assays, allowing precise handling of very small liquid volumes. These systems are used in protein crystallization, biotransformation, fermentation, lateral flow diagnostics, and high-throughput screening. Their advantages include reduced reagent use, parallel processing, and improved safety and reproducibility.

AlphaLISA is a bead-based, no-wash immunoassay designed for high-throughput screening. Using oxygen channeling chemistry, it enables sensitive detection of proteins, biomarkers, and drug candidates without multiple washing steps, making it well suited for drug discovery and immunogenicity studies.

Despite these advances, challenges remain, including high instrument costs, surface immobilization artifacts, mass-transport limitations, complex data modeling, and difficulties in analyzing heterogeneous biologics and small molecules. Additionally, the lack of standardized data reporting across platforms limits data integration. Ongoing efforts such as MIAPARIS and MI-TAB aim to address these issues by standardizing kinetic data reporting. Overall, NGLBAs offer powerful tools for modern bioanalysis but require continued technical refinement and standardization for broader adoption.

Conclusion

Technology suppliers and developers alike are faced with a difficult task in light of today\'s constrained capital budgets: how to promote innovative technology buy-up and provide cutting-edge advantages to the groups most in need of them? Finding technological investments that will yield the best possibilities and outcomes throughout time is only one of the many issues that scientists face due to their restricted funding. Our objective in putting together this article on real-time measurement technologies was to highlight information on a number of novel technologies that we think have the potential to help bio analysis and might, with further development, have considerable bio analytical value.

References

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Copyright

Copyright © 2025 Pallavi Badarala, Lalithya Teli, Lohitha Sri Pulapaka, Naga Vaishnavi Ravuri, Bhaskara Raju Vatchavai. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.