OPTIMIZATION OF CONDITIONS FOR SPECIFIC BINDING OF ANTIBODY-COATED BEADS TO CELLS

It has previously been demonstrated that cells can bind antibody-coate d beads; this effect can be used to enhance the fractionation of cells using magnetic fields or by centrifugation on isopycnic, isotonic den sity gradients. As a general rule, the higher the expression of surfac e antigens the more beads bind to cells. However, we have also noted t hat other factors also affect the number of beads found bound to cells . Experiments have been carried out to determine what factors affect b inding of antibody-coated beads to cells. The optimum conditions for b inding of antibody-coated beads to MOLT-4 T cells were found to be, na mely, a 20:1 bead to cell ratio in a 1 ml incubation volume, with cont inuous end-over-end mixing for 1 h at 25 degrees C. Furthermore, the o ptimum centrifugation conditions at which the samples were separated o n isopycnic, isotonic density gradients were determined as 220 x g(max ) for 90 min, at 20 degrees C. The results indicate the preferred cond itions that are necessary to achieve optimum bead binding by cells and their subsequent fractionation. Different antibody-coated beads were examined including Dynabeads M-450, used as a known standard. In addit ion we describe, as a possible alternative to Dynabeads, dense polysty rene beads, for the separation of cells on the basis of the immunologi cal identity of the surface of cells using density perturbation method s.