Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights

Gray ER, et al., 3(7):479-491, ACS Infect Dis., 2017

The p24 capsid protein is one of the earliest biomarkers of HIV-1 infection and widely used in HIV-1 diagnostic tests. Described herein is the development of high affinity nanobodies to detect HIV-1 p24. An initial semiquantitative screen was performed to identify lead nanobody candidates using ELISA. Bio-Layer Interferometry (BLI) was used to better understand the biophysical characteristics, thermodynamics and kinetics of the identified lead nanobody candidates. The binding interactions of nanobodies and mAbs with p24 subtype B was compared by BLI using a Pall ForteBio Octet RED96 system equipped with Amine Reactive 2G (AR2G) Biosensor probes. AR2G Biosensor tips were activated by incubating in a freshly mixed sulfo-NHS-EDC solution. Subsequently, p24 subtype B was immobilized onto sensor tips. Ethanolamine-HCl (pH 8.5) was used to cap any residual active sites. Biosensor tips immobilized with p24 subtype B were then dipped in various concentrations of analyte (nanobodies and mAbs) samples. BLI data obtained were fitted to a 1:1 binding model. Kinetic rate constants (kon and koff) and the Equilibrium Dissociation Constant Values (KD values) were determined. Overall results of this study indicate that the high affinity HIV-1 anti-p24 nanobodies identified have broad implications for use as capture tools in HIV-1 diagnostics.

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