CD99 supports aggregation of CD99-transfected CHO cells. Mock-transfected CHO cells (CHO) or CD99-transfected CHO cells (CHO-CD99) were allowed to aggregate (A) in the presence of 30 μg/mL of the following antibodies: control preimmune IgG (co-IgG), affinity-purified antibodies against mouse CD99 (anti-CD99), F(ab′)2, or Fab fragments of affinity-purified anti-CD99 antibodies (as indicated); P < .0001, CHO-CD99 + co-IgG versus CHO + co-IgG; P < .0001, CHO-CD99 + anti-CD99 versus CHO-CD99 + co-IgG; P < .0005, CHO-CD99 + F(ab′)2 versus CHO-CD99 + co-IgG; P = .25, CHO-CD99 + Fab versus CHO-CD99 + co-IgG. (B) Mock-transfected CHO cells (CHO) or CD99-transfected CHO cells (CHO-CD99) were allowed to aggregate in the absence of antibodies (w/o Ab), or in the presence of affinity-purified anti-CD99 antibodies or control antibodies against β1-integrin (as indicated); P < .01, CHO-CD99 + anti-CD99 versus CHO-CD99 w/o Ab; P = .48, CHO-CD99 w/o Ab versus CHO-CD99 + anti–β1-integrin. (C) After cells were harvested in PBS with 5 mM EDTA, they were washed in Mg2+/Ca2+-free HBSS and then allowed to aggregate in HBSS either without (–) or with (+) 1 mM Mg2+/Ca2+; P < .001, CHO-CD99 without Mg2+/Ca2+ versus CHO-CD99 with Mg2+/Ca2+. (D) Reactivity of affinity-purified anti-CD99 antibodies (anti-CD99) and the corresponding F(ab′)2 and Fab fragments with immobilized CD99-Fc in an ELISA assay, which was developed using peroxidase-labeled polyclonal antibodies to rabbit IgG F(ab′)2. Optical density at OD 492 is plotted against antibody concentration. The results are presented as the mean ± SEM, and are representative of at least 3 separate experiments.
