Characterization of the Interaction Between the sdAb-mrh-IgG Nanobody and Immunoglobulin G

Nanobodies (VHHs) are the single variable domains of heavy-chain-only antibodies that contain the antigen-binding site. Due to their small size (12–16 kDa), high stability, specificity, and solubility, nanobodies have emerged as promising tools for biotechnology, diagnostics, and therapeutics. Among them, the sdAb-mrh-IgG nanobody, developed by Pleiner et al. through alpaca immunization and phage display selection, recognizes the Fc region of mouse, rat, and human IgGs.

Despite the availability of numerous nanobody structures in the Protein Data Bank, structural information on VHH–IgG complexes remains unavailable. Therefore, this study aimed to characterize the interaction between sdAb-mrh-IgG and human IgG using complementary biophysical approaches.

The sdAb-mrh-IgG sequence was cloned into the pET-25(b)+ expression vector and expressed in Escherichia coli BL21 (DE3). For NMR studies, isotopically labeled protein was produced in M9 medium supplemented with ¹³C-glucose and ¹⁵NH⁴Cl. The nanobody was purified by nickel-affinity and size-exclusion chromatography, yielding a final concentration of 0.49 mM.

Analytical gel filtration demonstrated that sdAb-mrh-IgG is predominantly monomeric in solution. Circular dichroism (CD) spectroscopy revealed a secondary structure rich in β-strands and a melting temperature (Tm) of approximately 70.5 °C, indicating high thermal stability.

The interaction with human IgG was investigated by Localized Surface Plasmon Resonance (LSPR) and Nuclear Magnetic Resonance (NMR) spectroscopy. LSPR measurements revealed an affinity (KD) of 0.24 µM. Two-dimensional [¹H,¹⁵N] HSQC spectra were acquired at temperatures ranging from 298 to 333 K in the absence and presence of 14.8 µM human IgG. Temperature coefficients were determined from changes in resonance intensities, allowing the evaluation of conformational stability and ligand-induced structural effects. Additional NMR experiments, including heteronuclear NOE and relaxation measurements (T1 and T2), were performed to characterize protein dynamics.

Overall, sdAb-mrh-IgG displayed a highly stable three-dimensional structure and was successfully produced with high purity, enabling detailed biophysical characterization of its interaction with human IgG and its conformational behavior in both free and bound states.

This work was supported by Conselho Nac. Des. Cient. Tecnológico (CNPq), Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ) and by the Coordination for the Improvement of Higher Education Personnel (CAPES) .