hitachi f 7000 spectrofluorometer  (Hitachi Ltd)

Journal: Biotechnology Reports

Article Title: Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological validation

doi: 10.1016/j.btre.2026.e00955

Figure Lengend Snippet: Fluorescence quenching titration for scFv-MICA affinity determination. (A) Illustration of the analytical technique of tryptophan fluorescence quenching used to measure the binding interactions of biomolecules. Various concentrations of anti-MICA scFvs were incubated with a fixed concentration of MICA (1 μM) in a quartz cell. Titrations were performed by adding aliquots of the titrant to the solution containing MICA, continuing until a molar ratio of [titrant]/[MICA] of ten was achieved. The fluorescence emission spectra were collected using an FS5 Spectrofluorometer with bandwidths of 5.0 nm for both, excitation and emission. Samples were excited at 295 nm. The fluorescence signal from the buffer was subtracted from all analyses. (B) Representative quenching curves resulting from the interaction of scFvs with MICA. The curves represent the reduction in tryptophan fluorescence intensity observed upon binding of WT or Beta mutant scFvs at specific antibody concentrations. Non-linear fit of the fluorescence data of scFvs binding to MICA. The area of fluorescence was plotted as a function of molarity. The affinity constant (K D ) was calculated based on the binding curves, which provided information on scFvs affinity to MICA. Data are presented as mean ± SD from three independent experiments.

Article Snippet: The fluorescence emission spectra were collected using an FS5 Spectrofluorometer (Edinburgh Instruments, Scotland) with 5 nm bandwidths for excitation and emission.

Techniques: Fluorescence, Titration, Binding Assay, Incubation, Concentration Assay, Mutagenesis