32 



[ P tl 1 



A plot of . . versus y-y generates a straight line with a 



slope equal to the reciprocal of the association constant, 



K a- 



Hemagglutination . The hemagglutinating ability of 



various Con A conjugates was examined with human type A red 



blood cells. Standard microtiter plates (Cooke Engineering) 



and dilutions were used throughout. Agglutinations were 



determined for serial two-fold dilutions of protein in 0.15M 



+ 8 



PBS , pH 6.8 and red blood cells at 1 x 10 cells per milli- 

 liter. After complete mixing and suspension of cells, 

 hemagglutination plates were allowed to stand at room tem- 

 perature until a control well containing only red blood 

 cells in PBS had formed a well defined pellet. Agglutina- 

 tion titers were read by visual inspection and are expressed 

 as the reciprocal of the largest dilution yielding positive 

 agglutination. 



Interaction of Conjugates with Cultured Cells 



Maintenance of Cell Cultures 



Cultured mammalian cells used for these studies were 

 maintained at 37°C in a 5% CO_:95% air humidified atmosphere. 

 Tissue culture media, sera and antibiotics were obtained 

 from Flow Laboratories, Rockvillc, MD or International 

 Scientific Industries, Avon Park, IL. All media were 

 supplemented with 10% fetal calf serum (FCS), pennicillin 



(100 units/ml) and streptomycin (lOOpg/ml) . Stock cultures 



2 2 

 were maintained in 25cm or 75cm disposable plastic culture 



