Immunotargeting of small (100 nm) and large (1000 nm) anti-PECAM/SA–β-gal conjugates to REN/PECAM cells.

Small and large anti-PECAM/SA (121 ± 12 nm versus 1183 ± 199 nm) and IgG/SA–β-gal (109 ± 12 nm) were incubated with REN/PECAM cells. The β-gal enzyme activity in the conjugate preparations and cell lysates was determined by means of a β-gal enzyme assay kit. Cells were incubated in reporter lysis buffer, then harvested by scraping, and centrifuged. Enzymatic β-gal activity was measured in the supernatants at various dilutions. In the other parallel wells, for X-gal staining, cells were fixed with paraformaldehyde, washed with magnesium chloride, and stained in the dark with a solution containing 1 mg/mL X-gal. Both large and small conjugates delivered equivalent total β-gal enzymatic activity to REN/PECAM cells (A), while control cells incubated with nonimmune counterpart showed no detectable activity (B). Small anti-PECAM/SA–β-gal provided a homogenous intracellular pattern of β-gal activity (C), while large conjugates were associated with a particulate matter apparently localized on the cell surface (D). Bar, 25 μm.