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J Immunol Methods. 2016 Aug;435:7-16. doi: 10.1016/j.jim.2016.04.012. Epub 2016 Apr 30.

A simple and robust approach to immobilization of antibody fragments.

Author information

1
Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD 20742, USA.
2
Department of Chemical and Biomolecular Engineering, University of Maryland, 2113 Chemical and Nuclear Engineering Building (#090), College Park, MD 20742, USA. Electronic address: ajkarl@umd.edu.

Abstract

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets β-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to β-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization.

KEYWORDS:

AviTag; BCCP; Biotin; Immobilization; Streptavidin; scFv

PMID:
27142477
DOI:
10.1016/j.jim.2016.04.012
[Indexed for MEDLINE]

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