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Anal Biochem. 2009 Feb 15;385(2):346-57. doi: 10.1016/j.ab.2008.11.026. Epub 2008 Nov 27.

Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors.

Author information

1
CSIRO Molecular and Health Technologies, Molecular and Health Technologies, Parkville, VIC, Australia.

Abstract

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V(NAR) single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.

PMID:
19073134
DOI:
10.1016/j.ab.2008.11.026
[Indexed for MEDLINE]

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