26 



presented in the figure legends. The general format for 

 determining the extent of conjugation over a period of time 

 was to first establish the specific reaction conditions 



desired. The different parameters investigated included 



3 

 buffer concentration and pH, Con A, H-DM-ADGG and EDC con- 

 centrations. All reactions were initiated by the addition 

 of carbodiimide . At the desired sampling point, duplicate 

 50 or lOOyl samples were removed to small tubes containing 

 200pg each of RNA and BSA in 0.02M Na P in 0.1ml and 

 placed on ice. Cold 10% TCA, 2.0ml, was immediately added 

 and after 15 minutes on ice the precipitates were collected 

 on GF/C glass fiber discs and processed for scintillation 



counting as described by Preston et al. (1975). 



3 

 Production of H-DM-ADGG-Con A for cell binding was 



accomplished by reaction of 0.0032nmoles of Con A with 

 0.22ymoles of 3 H-DM-ADGG (specific activity = 7.4 x 10 dpm/ 

 ymole) with 75ymoles of EDC in 1.5ml of 0.01M phosphate buf- 

 fer pH 5. After 12 hours of reaction, the preparation was 

 applied to a column of Sephadex G-75 (0.2ml bed volume) pre- 

 equilibrated with 0.15M PBS , pH 7.4 and eluted with the 

 same buffer. Following elution of 80ml, 0.1M D-glucose was 

 added to the eluting buffer. Individual 1.0ml fractions were 

 sampled for determination of total radioactivity in Bray's 



solution and for TCA precipitable activity as described above. 



3 

 The H-DM-ADGG-Con A peak was pooled after analysis and 



dialyzed versus three two liter volumes of PBS at 5°C. 



