Abstract
A Q-body capable of detecting target molecules in solutions could serve as a simple molecular detection tool. The position of the fluorescent dye in a Q-body affects sensitivity and therefore must be optimized. This report describes the development of Nef Q-bodies that recognize Nef protein, one of the human immunodeficiency virus (HIV)’s gene products, in which fluorescent dye molecules were placed at various positions using an in vivo unnatural amino acid incorporation system. A maximum change in fluorescence intensity of 2-fold was observed after optimization of the dye position. During the process, some tryptophan residues of the antibody were found to quench the fluorescence. Moreover, analysis of the epitope indicated that some amino acid residues of the antigen located near the epitope affected the fluorescence intensity.
Highlights
A Q-body is a recombinant antibody incorporating a fluorescent dye that can be used as a biosensor to detect proteins or small molecules based on an antigen-dependent change in quenching [1,2]
Some mutagenesis positions were within four residues of the complementarity-determining regions (CDRs)
Ofthe theantibody antibody were changed to the DBCO-Cy3 modification the were changed to the unnatural amino acid (UAA) 3-azido-L-tyrosine with DBCO-Cy3 modification of theofazido azido group
Summary
A Q-body is a recombinant antibody incorporating a fluorescent dye that can be used as a biosensor to detect proteins or small molecules based on an antigen-dependent change in quenching [1,2]. The most common methods employing antibodies to detect antigens are the enzymelinked immunosorbent assay (ELISA) and western blotting (WB). Both methods require time-consuming incubation, multistep treatments and antigen immobilization procedures. Assays using Q-bodies require only a fluorescence measurement after the antigen and Q-body are mixed in solution as a single step process. This would offer great advantages available for Kd measurements, some in-line readout in a process, etc. The Q-body is considered a new type of immune-biosensor [1]
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