Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging.
Purpose: Construction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging pro...
| Published in: | Molecular Imaging & Biology Vol. 21; no. 1; pp. 54 - 67 |
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| Main Authors: | , , , , , |
| Format: | research Journal Article |
| Published: |
Springer Nature
Feb2019
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| Online Access: | View this record in EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=134786716&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 134786716 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 15361632 KJU jtl: Molecular Imaging & Biology issn: 15361632 maglogo: N pubinfo: dt: Feb2019 vid: 21 iid: 1 pid: 237 pub: Springer Nature place: New York, New York artinfo: ui: 134786716 134786716 NLM29948640 134786716 10.1007/s11307-018-1222-y NLM29948640 134786716 ppf: 54 ppct: 13 formats: fmt: – @attributes: type: T – @attributes: type: P tig: atl: Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging. aug: au: Alam, Md. Kausar El-Sayed, Ayman Barreto, Kris Bernhard, Wendy Fonge, Humphrey Geyer, C. Ronald affil: Department of Pathology and Laboratory Medicine, College of Medicine, University of Saskatchewan, S7N 5E5, Saskatoon, SK, Canada sug: subj: Acute Lung Injury Diagnosis Luminescent Measurements Methods Diagnostic Imaging Methods Antibodies, Monoclonal Metabolism Animal Studies Human Rats Enzymes Acute Lung Injury Graft Survival Antibodies, Monoclonal Oxidoreductases Enzymes Metabolism Xenografts Cell Transplantation Methods Oxidoreductases Metabolism Fluorescent Antibody Technique Methods Antibodies Pharmacokinetics Cells, Cultured Fluorescent Dyes Pharmacokinetics Genes Proteins Metabolism Acute Lung Injury Therapy Fluorescent Dyes Immunity Proteins Receptors, Cell Surface Acute Lung Injury Metabolism Receptors, Cell Surface Metabolism Antibodies Metabolism Female Validation Studies Comparative Studies Evaluation Research Multicenter Studies Female ab: Purpose: Construction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.Procedures: We used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.Results: Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.Conclusions: These results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities. pubtype: Academic Journal doctype: research Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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