Characterization of purified CD24. (A) Affinity-purified CD24 antigen isolated from human neutrophils was analyzed by Western blotting. Antigen was detected by MoAbs to CD24 (mixture of ML-5, SWA11, 32D12) followed by peroxidase-conjugated secondary antibody and ECL detection. Antigen was also detected on the membrane after treatment with digoxigenin-3-O-succinyl-ε-amino-caproic acidhydrazide followed by Digoxigenin-specific antibodies. Antigen was also coated to microtiter plates and analyzed by ELISA using MoAb ML-5 hybridoma supernatant or tissue culture medium for control. (B) Detection of CD24 after coating to polystyrene beads. Beads were stained using MoAb ML-5 specific for CD24 or control MoAbs W 6/32 to MHC class I followed by PE-conjugated rabbit-antimouse IgG. Background control was the PE-conjugated antibody alone. Stained beads were analyzed by FACScan.