336 BLOOMBERGH. 



one-half hour. The invariable dose was one cubic centimeter of the diluted serum. 

 The amboceptor has remained stable for over four months, preserved after in- 

 activation in sealed ampoulles. 



The blood serum of the patients as a rule was removed from the veins at the 

 bend of the elbow on the day preceding the test and the serum from the clot 

 was inactivated on the same or following day. It was found later that 0.6 or 

 0.8 cubic centimeter of serum could be obtained from the blood removed by deep 

 puncture from the finger and collected in small tubes. The serum resulting from 

 the clot was centrifugalized and later placed in similar small tubes, floated on 

 corks in water and inactivated. All inactivation was done at 55° to 56° C. for 

 one-half hour. The serum at first was tried in the two doses of 0.1 and 0.2 

 cubic centimeter diluted with the necessary quantity of salt solution. Later the 

 dose of 0.2 cubic centimeter was used exclusively. 



The antigen was made by grinding guinea-pig heart together with sand in a 

 mortar and adding to this 50 cubic centimeters of 95 per cent alcohol. This 

 was heated to 60° C. for two hours, shaken from time to time, and then filtered. 

 The dose was found to be 0.1 cubic centimeter, twice this amount inhibiting 

 haemolysis. The antigen has apparently remained a constant factor. 



The actual technique of the reaction as carried out by us does not 

 differ from that usually employed. On all occasions on which the reac- 

 tion was made, a positive syphilitic and a normal serum were used as 

 the main controls of the effective specific action of our reagents. The 

 sera were likewise tested to determine whether they, independently of 

 the antigen, bound the complement or whether they were actively hsemo- 

 lytic with cells alone, or in the presence of complement. While it was 

 found in actual practice that one hour was a suiScient length of time 

 for the reaction, the final reading was always made at the end of two 

 hours after the addition of the hsemolytic amboceptor and red blood 

 cells. 



At the time of our preliminary titration to establish the strength of 

 our amboceptor, we made controls of cells plus complement, amboceptor 

 and antigen, to show that the antigen did not inhibit haemolysis; and 

 of cells plus complement and of cells plus amboceptor to .show that 

 hsemolysis did not occur under these conditions. 



It is to be noted first of all that the results reached by us depend on 

 the action of an antigen derived from normal heart and the question 

 of the specificity of action of our antigen comes immediately into 

 question. For our tests we have been unable to obtain syphilitic foetal 

 organs in the city of Manila, and we may add that the work of others 

 being conducted along lines similar to our work at the present time in 

 Manila, is likewise undertaken with a similar antigen from normal 

 organs. In July, 1910, Wasserman(2) still advocated the use of syphi- 

 litic organs in the preparation of the antigen and the good resiilts 

 reached by Dean(3) on the reaction in idiots, referred to by Wasserman 

 in his address, might indicate that in certain lines of research such an 

 antigen is still a necessity. Bayly (4) used an alcoholic extract of 

 rabbit's heart and obtained as good results as with an alcoholic extract 



