title: Facile Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy.
abstract: Protein-nanoparticle conjugates are widely used for conventional applications such as immunohistochemistry and biomolecular detection as well as emerging applications such as therapeutics and advanced materials. Nevertheless, it remains challenging to reproducibly prepare stable protein-nanoparticle conjugates with highly similar optical properties. Here we report an improved physisorption method for reproducibly preparing stable antibody-gold conjugates at acidic pH using polyclonal antibodies from a wide range of species (human, goat, rabbit, mouse, and rat). We find that gold particles synthesized using citrate alone or in combination with tannic acid are similar in size but display variable colloidal stability when conjugated to polyclonal antibodies. The variability in conjugate stability is due to differences in the pH and composition of the original gold colloid, which prevents reproducible preparation of stable antibody conjugates without additional purification of the particles prior to conjugation. Sedimentation-based purification of gold particles synthesized using different methods enabled reproducible generation of antibody-gold conjugates with high stability and similar plasmon wavelengths. We also find that antibody conjugates prepared using our improved procedure display excellent performance when applied to a high-throughput immunogold assay (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) for identifying monoclonal antibodies with low self-association, high solubility, and low viscosity. The stable antibody conjugates prepared with various types of gold colloid result in robust and reproducible AC-SINS measurements of antibody self-association using extremely dilute (microgram per mL) and unpurified antibody solutions. We expect that this improved methodology will be useful for reproducibly preparing stable antibody-gold conjugates for diverse applications.
authors: Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Steven B
authors: SB
authors: Geng
authors: Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Jiemin
authors: J
authors: Wu
authors: Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Magfur E
authors: ME
authors: Alam
authors: Eli Lilly Biotechnology Center , San Diego, California 92121, United States.
authors: Jason S
authors: JS
authors: Schultz
authors: Eli Lilly Biotechnology Center , San Diego, California 92121, United States.
authors: Craig D
authors: CD
authors: Dickinson
authors: Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Carly R
authors: CR
authors: Seminer
authors: Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors: Peter M
authors: PM
authors: Tessier
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D006041:Goats; D006046:Gold / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018796:Immunoconjugates / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D051379:Mice; D011817:Rabbits; D051381:Rats; D015203:Reproducibility of Results; D012995:Solubility
chemical_list: D000906:Antibodies; D018796:Immunoconjugates; D006046:Gold