10 BRENT, MEDAWAR AND RUSZKIEWICZ 



applies a fortiori when bivalent cations (e.g. CaClg or MgClg to a 

 final concentration of only o.oim) are added to the crude aqueous 

 "solution". Under these circumstances most of the antigenic 

 activity appears in the sediment, accompanied by DNA-protein, 

 after centrifugation at low speeds (Table I, D). We can take it 

 that the antigenic matter contained in the original crude aqueous 

 preparation consists of particles of very diverse sizes, and that even 

 in the presence of 0.i5M-NaCl some of the material is "soluble" 

 in the sense defined by the above operations. The significance of 

 these facts will be discussed later. The particles could well consist 

 of fragments of cellular membranes, as Dr. Kandutsch has long 

 insisted (Kandutsch and Reiner t-Wenck, 1957; and see also 

 Herzenberg and Herzenberg, 1961). 



The "crude aqueous preparation" referred to in later experi- 

 ments consists of an aqueous homogenate to which NaCl (final 

 concentration o-I5m) has been added, and from which DNA- 

 protein and undispersed matter has thereupon been removed by 

 centrifugation at 5000^ for 10 min. The "antigenic sediment" 

 with which the behaviour of this crude aqueous preparation will 

 be compared is the sediment formed by a further centrifugation 

 at 30,000^ for one hour. This sediment forms a typically "col- 

 loidal" suspension in water or physiological salt solutions, and it 

 flocculates rapidly at pH 5 • 5 or less. The crude composition of the 

 sediment is summarized in Table II. Its activity is such that, if a 

 discriminating test is used, 0*25 nig. dry weight can be shown to 

 sensitize a mouse; something like o- 5 mg. is needed to remove all 

 detectable antibody from i-o ml. of a 1/50 dilution of a homo- 

 logous antiserum of titre ~ 3200. These results do not encourage 

 us to beheve that the material is anything but grossly impure. On 

 compositional grounds no inference can be drawn about the 

 ingredient of the preparation in which activity lies; activity could 

 reside in any one, or in any combination, of carbohydrate, lipid, 

 or protein. 



The solubihzation of this insoluble sediment raises not one 



