title: On-bead antibody-small molecule conjugation using high-capacity magnetic beads.
abstract: Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.
authors: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States. Electronic address: nidhi.nath@promega.com.
authors: Nidhi
authors: N
authors: Nath
authors: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States.
authors: Becky
authors: B
authors: Godat
authors: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States.
authors: Hélène
authors: H
authors: Benink
authors: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States.
authors: Marjeta
authors: M
authors: Urh
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D018796:Immunoconjugates / Q000737:chemistry* / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000737:chemistry; D018189:Immunomagnetic Separation; D058185:Magnetite Nanoparticles; D051379:Mice; D008863:Microspheres; D051381:Rats; D013194:Staining and Labeling; D013205:Staphylococcal Protein A / Q000737:chemistry*; D000068878:Trastuzumab / Q000737:chemistry
chemical_list: C569686:Alexa Fluor 647; D000906:Antibodies; D001426:Bacterial Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; C034284:IgG Fc-binding protein, Streptococcus; D018796:Immunoconjugates; D007074:Immunoglobulin G; D058185:Magnetite Nanoparticles; D013205:Staphylococcal Protein A; D000068878:Trastuzumab
keywords: Antibody drug conjugates; Antibody internalization; Antibody labeling; Antibody purification; Cell based assay