Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use

Christoph Eugen Hagemeyer, Karen Maria Alt, Angus Johnston, Georgina Kate Such, Hang T Ta, Melissa K M Leung, Sandeep Prabhu, Xiaowei Wang, Frank Caruso, Karlheinz Peter

Research output: Contribution to journalArticleResearchpeer-review

32 Citations (Scopus)

Abstract

Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano-or microparticlesor 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.
Original languageEnglish
Pages (from-to)90 - 105
Number of pages16
JournalNature Protocols
Volume10
Issue number1
DOIs
Publication statusPublished - 2015

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