Kinetic and Apparent Thermodynamic Analysis of Phaseolus Vulgaris Leucoagglutinin Binding to Cell-Surface Glycans in SR Lymphoma Cells Using a Fixation-Modified Cell-ELISA.

Cell-surface glycosylation plays an important role in lectin-mediated molecular recognition and is frequently altered in malignant cells. In this study, a fixation-modified cell-ELISA was developed to enable stable immobilization of non-adherent cells and to compare the binding behavior of Phaseolus vulgaris leucoagglutinin (PHA-L) toward SR lymphoma cells and normal human peripheral blood mononuclear cells (PBMCs). Saturation binding and time-course assays were performed at 5, 15, 25, and 37 °C, and the resulting whole-cell ELISA data were analyzed using Langmuir, Van't Hoff, Arrhenius, and Eyring models to obtain comparative apparent kinetic and thermodynamic descriptors. Saturation binding analysis showed lower apparent equilibrium dissociation constants (Kdapp) and higher maximum binding responses (ODmax) for SR lymphoma cells than for normal PBMCs under identical experimental conditions, indicating a stronger apparent PHA-L binding response. Time-course analysis demonstrated faster apparent binding kinetics in SR lymphoma cells, characterized by higher apparent observed and association rate constants (kobsapp and konapp), lower apparent dissociation rates (koffapp), and shorter observed association half-times. Model-based thermodynamic analysis suggested that the apparent binding process was endothermic and predominantly entropy-favored in both cell types. Arrhenius and Eyring analyses further provided comparative apparent energetic descriptors, with SR lymphoma cells exhibiting lower apparent Arrhenius-derived energetic parameters and lower apparent activation enthalpy than normal PBMCs under the experimental conditions. Because these parameters were derived from mathematical analysis of whole-cell ELISA measurements rather than direct real-time biophysical techniques, they should be interpreted as apparent comparative descriptors rather than intrinsic molecular kinetic or thermodynamic constants. Overall, the fixation-modified cell-ELISA provided a feasible platform for exploratory comparative evaluation of PHA-L binding to suspension cells and revealed measurable differences in the apparent binding behavior of SR lymphoma cells and normal PBMCs. These findings support the potential utility of this approach for comparative lectin-cell interaction studies and future investigations of cell-surface glycosylation.