The dissociation constant (KD) of the Thy1-scFv were measured using yeast as described (28 ). In brief, yeast cells (1 × 105) transformed with pCT plasmid containing Thy1-scFv gene were incubated with 0.01 to 100 nM recombinant human and murine biotinylated Fc-conjugated Thy1 (B-Thy1, Abcam), respectively, overnight at room temperature. Cells were stained with the chicken anti-c-myc antibody (eBioscience). After FACS analysis, mean fluorescence values for streptavidin-allophycocyanin (APC; Biolegend) and Alexa 555-conjugated goat anti-chicken IgY (eBioscience) in double-positive populations of each yeast sample were acquired. KD values were analyzed by determining the ratio of the mean fluorescence intensity of [APC] and fluorescence intensity of [A555] using FACS. The ratio was plotted against the used concentration of Thy1 using Prism 5 software (Graphpad, CA). The KD was determined using a nonlinear least squares curve fit (16 (link),29 (link),30 (link)).
Protocol detail
Measuring Thy1-scFv Binding Kinetics
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Allophycocyanin
Anti c antibody
Cells
Chicken
Fluorescence
Gene
Goat
Human
Murine
Plasmid
Populations
Prism
Streptavidin
Yeast
Corresponding Organization : Stanford Medicine
Other organizations : Stanford University
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Variable analysis
independent variables
- Concentration of recombinant human and murine biotinylated Fc-conjugated Thy1 (B-Thy1)
- Ratio of the mean fluorescence intensity of [APC] and fluorescence intensity of [A555]
- Yeast cells (1 × 10^5) transformed with pCT plasmid containing Thy1-scFv gene
- Overnight incubation at room temperature
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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