Construction of label-free FRET immunoassays using three antibody fragments to insight into the structural basis of sensitivity difference

Intrinsic fluorescence of tryptophan (Trp) in antibody is of particular interest in label-free fluorescence resonance energy transfer (FRET) immunoassay. However, the relationship between the effective Trp proportion of antibody fragment and energy transfer efficiency and the behind structural basis are still unclear. Herein, we constructed three label-free FRET-immunoassays for detecting sulfonamides (SAs) based on intact IgG, fragment of antigen binding (Fab), and single-chain variable fragment (scFv) of antibody 4C7 as donors, respectively. By analyzing the amino acid sequence of three antibody fragments and the interaction between antibody fragments and SAs, the results that the higher effective Trp proportions of antibody fragments lead to higher energy transfer efficiencies can be obtained. Then, the higher detection sensitivity for detecting sulfathiazole (STZ) was achieved, while the limits of detection (LODs) were 1.27, 0.58, and 0.29 ng/mL of IgG, Fab, and scFv-based label-free FRET-immunoassays, respectively. Furthermore, the conclusion derived from 4C7 has been proved by another combination of IgG, Fab, and scFv of 4D11 against sulfamethazine (SMZ) with the LODs of 0.97, 0.32, and 0.15 ng/mL, respectively. In conclusion, apart from the first demonstration of label-free FRET immunoassays for detecting SAs, we have clarified the structural basis of label-free FRET immunoassays, which gives useful clues to other analytes that require rapid screening by label-free FRET platform.