8 BRENT, MEDAWAR AND RUSZKIEWICZ 



Solubility and solubilization of sensitizing antigens 



The solubilization of transplantation antigens is important not 

 only as a prerequisite of some forms of physical and chemical 

 analysis, but also because the sensitizing activity of antigens may 

 be expected to vary with the forms in which they are presented 

 to the responding subject. 



It will be recollected that the preparation of Billingham and 

 co-workers (1958) begins with the total disruption of A-strain 

 lymphoid cells in distilled water, a process assisted by the exposure 

 of the mechanically prepared homogenate to graded doses of 

 ultrasound. Some old experiments of ours, of which only those 

 with full internal controls are cited in Table I, show that these 

 crude watery extracts contain some antigenic matter in a very 

 fmely subdivided form, and that its state of aggregation is pro- 

 foundly influenced by the presence of electrolytes. If the crude 

 aqueous extract is spun at 27,700 g (max.) for 30 to 45 min., 

 sensitizing activity is about equally divided between sediment and 

 supernatant fluid (Table I, A). If this supernatant fluid is now 

 again spun for three or four hours at 134,000 to 173,000^, it is 

 still possible, mainly by the histological analysis of grafts, to 

 discern a trace of sensitizing activity in the second supernatant 

 (Table I, B). However, sensitizing matter behaves quite diflerently 

 in the presence of electrolyte. If NaCl to a fmal concentration of 

 0.15M is added to the supernatant fluid after centrifugation at 

 27,700^ (procedure I, A), the first effect is the formation of a heavy 

 precipitate of antigenically inert DNA-protein, and this may be 

 removed by centrifugation at low speeds. After its removal the 

 preparation is again spun at 27,700^ for 30 min.; most of the 

 activity now appears in the sediment (Table I, C), and experience 

 has shown that the sediment sensitizes more powerfully than the 

 mother liquor from which it was derived. Evidently antigenic 

 fragments tend to aggregate in the presence of electrolyte, and, as 

 colloid theory would lead one to predict, the same consideration 



