Abstract
Antibody-based sensors have made outstanding contributions to the fields of molecular biology and biotechnology. Our group recently developed a novel powerful fluorescent immunosensor strategy named Quenchbody (Q-body), which has been applied to the detection of a range of antigens in a rapid, simple, and sensitive manner. However, there were some Q-bodies whose fluorescence response was limited, especially for detecting protein antigens. With the aim of improving this issue, here we made twelve types of Q-bodies incorporated with different number and position of TAMRA fluorophore in the single chain Fv of HyHEL-10, an anti-hen egg lysozyme antibody, as a model. By measuring the fluorescence intensity and its antigen dependency, it was revealed that VL-VH type Q-bodies labeled at a non-CDR loop region of the VL shows the highest fluorescence response. This position locates close to the quenching Trp35 in VL, while it is far from Trp residues in the bound antigen. This result clearly suggests the importance of dye position to maximize the fluorescence quenching and antigen-dependent de-quenching. The discovery may open a way to make many other Q-bodies with superior response.
Highlights
Antibody-based biosensors represent practical and precise tools for detecting antigens with their high selectivity, specificity and immediacy
VL-VH type scFv gene incorporated with an additional amber codon in the middle of the interdomain linker was amplified with primers LinkVH(HEL)back, VH(HEL)For, LinkAmbBack, and VL(HEL)linkFor
We reported a novel strategy to increase the response of anti-protein Q-body
Summary
Antibody-based biosensors represent practical and precise tools for detecting antigens with their high selectivity, specificity and immediacy. Those immunosensors have been increasingly demanded in the fields of scientific and analytical applications, and have been widely used in molecular biology and biomedical research, as well as in clinical diagnosis [1,2]. Fluorescent immunosensors, which are made by conjugation of fluorescent probes to antibodies, have merits such as no need of additional reagents and availability of sensitivity without need of long incubation. Fluorescence is an optical signal that allows one to detect molecular interactions with great sensitivity. The transduction is based on a variation of the fluorescence properties of the biosensor when it interacts with its analyte [3]. Intrinsic protein fluorescence can be used to study molecular interactions in purified experimental systems, extrinsic fluorescence is normally preferable to monitor specific interactions in complex media, without interference from other protein components [4]
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