AN ISOANTIGENIC LIPOPROTEIN 73 



(PVP) test (Stimpfling, 1961). The use of PVP as a developing 

 agent is necessitated by the incomplete character of haemagglu- 

 tinins in mouse isoantisera. Varying amounts of each preparation 

 were added to a constant quantity of antisera and incubated for 

 two hours at room temperature followed by an additional 18 

 hours' incubation at 4°c. The absorbed antisera were then titrated 

 against erythrocytes collected from appropriate strains. All 

 titrations were done in triplicate and the mean log of the reciprocal 

 titres was determined. 



As preliminary steps towards the isolation of the antigens our 

 initial efforts were directed towards determining the distribution 

 of the antigens and their stability to a variety of factors which 

 might be applied in an isolation procedure or provide a clue to 

 their identity. The results of these experiments led us to conceive 

 of the antigens as molecules formed in some part of an essential, 

 labile protein, possibly with carbohydrate as a second component, 

 ' localized in the membranes of cells, and most concentrated in 

 tumour (sarcoma I), spleen, and parotid glands (Kandutsch and 

 Reinert-Wenck, 1957; Kandutsch, i960). Further investigations 

 carried out in the light of this information have led to the isolation 

 of an isoantigenic lipoprotein which, in studies conducted so far, 

 appears to be electrophoretically homogeneous. 



Isolation procedure 



The isolation procedure shown in Fig. i can be divided into 

 three phases. 



(A) Preparation from sarcoma I {ascites jorm) oj a particulate 

 fraction which may consist principally oj cellular membranes {Kan- 

 dutsch, i960). Initially the procedure for obtaining a membrane 

 fraction was essentially that used by Smith and co-workers 

 (1957) to obtain a membrane fraction from liver cells. The pro- 

 cedure has been rather extensively modified to omit repeated 

 extractions with highly concentrated salt solutions. 



