Protein A- and Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant

Abstract

Detection of disease-related biomarkers in plasma provides a possibility for early clinical diagnosis. However, highly abundant proteins in plasma, such as human immunoglobulin (hIgG) are a main impediment to biomarker discovery and analysis. Therefore, rapid and easy depletion of hIgG in the plasma is beneficial for biomarker discovery. In this work, citrate-capped gold nanoparticles (GNPs) were synthesized and conjugated with cysteine-tagged recombinant Protein A (rProtA) and Protein G (ProtG), respectively. The resultant protein-GNP bioconjugates were thoroughly characterized by surface plasmon resonance spectroscopy, hydrodynamic light scattering (DLS), electrophoretic light scattering (ELS) and rotary metal shadowing transmission electron microscopy (TEM) measurements. In order to quantitatively control the amount of the rProt A and ProtG on the GNP surface, binding studies and isotherm measurements have been performed. rProtA-GNP conjugate exhibited better binding capacities towards hIgG. Its surface coverage with rProtA molecules was determined by protein quantification after hydrolysis of the rProtA-GNP conjugate, GNP removal and subsequent amino acid assay by HPLC with fluorescence detection. Binding isotherms acquired with hIgG revealed their maximal capacity for depletion experiments. Depletion efficiency of around 90% could be achieved in a standard solution. With optimized amount of rProtA-GNP and ProtG-GNP, respectively, hIgG could be efficiently extracted from real samples (human plasma and hIgG-spiked cell culture supernatant). A benchmarking study with ProteinA-modified magnetic particles (Dynabeads) was performed as well. The results document that these rProtA-GNP and ProtG-GNP affinity nanoparticles could be a promising alternative to magnetic bead based immunoaffinity trapping and constitutes a flexible platform for both depletion of hIgG from human plasma and antibody affinity capture from cell culture supernatants in process control of biopharmaceuticals by simple solution handling (via pipetting) and centrifugation steps.

Keywords: Antibody; Bioconjugate; Gold nanoparticle; Nano-immunoaffinity trapping; Protein A and Protein G; Rotary metal shadowing transmission electron microscopy.