Bovine β-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry

Abstract

Flow cytometry was used to demonstrate the presence of β-lactoglobulin (βLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-βLG conjugate (FITC-βLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-βLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled βLG. The on-rate constant was 3.2×102 M−1 min−1 and the off-rate constant was 0.002 min−1. Scatchard plot analysis gave a dissociation constant (Kd) of 44±21×10−7 and 39±24×10−5 M (n=3). Flow cytometry indicated that at least 15% of the FITC-βLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan™ system). Author Keywords: β-Lactoglobulin; Receptor; Hybridoma; FITC; Flow cytometry