A quantitative assay for concanavalin A-mediated cell agglutination.

A quantitative method was developed for measuring cell agglutination by the plant lectin, concanavalin A. The method, based on an assay for intercellular adhesion, determines the concanavalin A-stimulated rate of attachment of single cells to a cell layer. The rate depended on the number of single cells and the concentration of concanavalin A. Experiments on concanavalin A agglutinability of mouse BALB/c 3T3 cells agreed qualitatively with observations obtained with a visual method presently in use. For example, the concanavalin A-stimulated rate was low when EDTA was used to prepare the single cells, high when trypsin-dispersed cells were employed, and highest when the latter cells were assayed with trypsin-treated cell layers. The latter result was not explicable in terms of concanavalin A binding to the cell layer, since the same quantities of labeled lectin attached to untreated and to trypsin-treated cell layers. Concanavalin A attached to an untreated cell layer in such a manner that it retained high binding activity toward surface receptors on trypsin-treated single cells. Above a threshold density of concanavalin A bound to the cell layer, the stimulation of single cell attachment to the layer was directly proportional to the amount of concanavalin A bound to the layer. The concanavalin A-stimulated rate of attachment was specifically inhibited by methyl a-D-mannopyranoside. However, once single cells were attached to the cell layer in the presence of concanavalin A, they were not released by methyl a-mannoside. Agglutination is considered to be more complex than the simple binding of cells together through lectin bridges.