The discovery (1) that there are many substances with the sensitizing properties hitherto believed peculiar to cholesterol and its derivatives, and (2) that sensitizer can be added to antigen in very large quantities, many times those currently used, and yet continue to increase its complement-fixing efficiency with no danger of giving falsely positive tests has made possible the preparation of an antigen much more sensitive than any now available for use in the Wassermann reaction.
100 gm. of dry powdered beef heart muscle are extracted with 500 cc. ether for 15 minutes at 37°C. with shaking. After filtration with suction, the ether filtrate is discarded. The powder is then dried and extracted for 3 to 5 days with 500 cc. of 95 per cent alcohol, with intermittent shaking. The mixture is filtered, and the moist powder washed on the filter paper with two portions of alcohol, each 100 cc. The alcoholic filtrate and washings are combined and evaporated on the steam bath down to 250 to 300 cc. Cholesterol (0.8 per cent), and sitosterol (0.6 per cent) are then added, and dissolved at 65°–75°C. The excess sensitizer which crystallizes out upon cooling is dissolved just before using by immersing the antigen in a 56°C. bath for a few minutes. It is diluted by pouring the saline rapidly into the antigen. A 1:40 dilution is recommended for use in water bath fixation (½ hour at 37°C.), as well as for the short ice box fixation (8°, 4 hours) and a 1:120 dilution for use in the overnight ice box method (16 to 24 hours at 8°C.), as being well beyond its anticomplementary range; the anticomplementary quantities being 1:5 and 1:25 respectively.
There is reason to believe that this antigen possesses almost the maximum sensitivity obtainable. The method of preparation insures its being almost saturated with antigen-lipoids; and more sensitizer could not be added without increasing the turbidity of its dilution in saline to a point where it would interfere with the reading of hemolysis. Any further improvement must await the discovery of better sensitizers.
Preliminary experiments indicate that this new sensitizer, sitosterol, will find an immediate application, not only in the Wassermann reaction, but also in a more sensitive flocculation test to be described in a following paper.