title: Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use.
abstract: Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.
authors.affiliation: 1] Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Central Clinical School, Monash University, Melbourne, Victoria, Australia.
authors.forename: Christoph E
authors.initials: CE
authors.lastname: Hagemeyer
authors.affiliation: 1] Vascular Biotechnology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
authors.forename: Karen
authors.initials: K
authors.lastname: Alt
authors.affiliation: Department of Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.
authors.forename: Angus P R
authors.identifier: 0000000156114515
authors.initials: AP
authors.lastname: Johnston
authors.affiliation: Department of Chemistry, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Georgina K
authors.initials: GK
authors.lastname: Such
authors.affiliation: 1] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia. [2] Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Hang T
authors.initials: HT
authors.lastname: Ta
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Melissa K M
authors.initials: MK
authors.lastname: Leung
authors.affiliation: 1] Central Clinical School, Monash University, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Prabhu
authors.affiliation: 1] Central Clinical School, Monash University, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Frank
authors.initials: F
authors.lastname: Caruso
authors.affiliation: 1] Central Clinical School, Monash University, Melbourne, Victoria, Australia. [2] Atherothrombosis and Vascular Biology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia.
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Peter
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D006860:Hydrogen Bonding; D057054:Molecular Imaging / Q000379:methods*; D015394:Molecular Structure; D053758:Nanoparticles / Q000378:metabolism*; D011092:Polyethylene Glycols; D011109:Polymethacrylic Acids; D011145:Polyvinyls; D011759:Pyrrolidines; D057127:Single-Chain Antibodies / Q000378:metabolism / Q000627:therapeutic use*
chemical_list: D001426:Bacterial Proteins; D011109:Polymethacrylic Acids; D011145:Polyvinyls; D011759:Pyrrolidines; D057127:Single-Chain Antibodies; C505150:poly(N-vinylpyrrolidine); D011092:Polyethylene Glycols; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases