ArticleBio-protocol2019
Conjugation of Fab' Fragments with Fluorescent Dyes for Single-Molecule Tracking On Live Cells.
Article in Bio-protocol, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 4 citations in OpenAlex.
- Development of label-free light-controlled gene expression technologies using mid-IR and terahertz light.Frontiers in bioengineering and biotechnology · 2024Review
- MAxSIM: multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells.Communications biology · 2023Article
- Enabling the formation of native mAb, Fab' and Fc-conjugates using a bis-disulfide bridging reagent to achieve tunable payload-to-antibody ratios (PARs).Chemical science · 2023Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have become valuable tools to discern how endogenous proteins control cell biology. Yet, some technical challenges remain; in SMT, these revolve around the characteristics of the labeling reagent. A good reagent should have neutrality (in terms of not affecting the target protein's functions), tagging specificity, and a bright fluorescence signal. In addition, a long shelf-life is desirable due to the time and monetary costs associated with reagent preparation. Semiconductor-based quantum dots (Qdots) or Janelia Fluor (JF) dyes are bright and photostable, and are thus excellent candidates for SMT tagging. Neutral, high-affinity antibodies can selectively bind to target proteins. However, the bivalency of antibodies can cause simultaneous binding to two proteins, and this bridging effect can alter protein functions and behaviors. Bivalency can be avoided using monovalent Fab fragments generated by enzymatic digestion of neutral antibodies. However, conjugation of a Fab with a dye using the chemical cross-linking agent SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) requires reduction of the interchain disulfide bond within the Fab fragment, which can decrease the structural stability of the Fab and weaken its antigen-binding capability. To overcome this problem, we perform limited reduction of F(ab')2 to generate Fab' fragments using a weak reducer, cysteamine, which yields free sulfhydryl groups in the hinge region, while the interchain disulfide bond in Fab' is intact. Here, we describe a method that generates Fab' with high yield from two isoforms of IgG and conjugates the Fab' fragments with Qdots. This conjugation scheme can be applied easily to other types of dyes with similar chemical characteristics.
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