The separation and purification of V and W antigens is described. The methods that gave the best results were:
The precipitation of both antigens from the supernatant fluid of a 36°C-grown culture of strain M23 by use of ammonium sulfate.
Chromatography on DEAE-cellulose.
V antigen was eluted with 0.1 M NaCl and W antigen with 0.5 M NaCl. Recycling on DEAE-cellulose resulted in a sample containing approximately 20 units of V antigen/mg of protein (100-fold purification) and no W antigen, and a sample containing 600 units of W antigen/mg of protein (1000-fold purification) and no V antigen.
V antigen is a protein with a molecular weight of 90,000 and W antigen is a lipoprotein with a molecular weight of 145,000.
Both antigens were stable at 60°C, but not at 80°C, for 30 min. W antigen, but not V, was lost upon extensive dialysis against distilled water, or pervaporation. Both antigens were reduced in titer by prolonged storage at 5°C or by lyophilization, but not by storage at -20°C.
Based on the use of rabbit antisera containing only V antibody or only W antibody, the conclusion was drawn that V antibody, but not W antibody, can protect mice against plague.