DEMONSTRATION OF PRESENCE OF ANTIBODIES. 197 



A preliminary titration of the serum was made with both hemolytic 

 systems to determine the amount of complement fixed by the serum alone. 

 Guinea-pig serum was used for the complement and such dilutions of this 

 serum were made that 0.1 c.c. of the dilution would equal respectively 0.01, 

 0.02, 0.03 c.c, etc., up to 0.1 c.c. 



Active heloderma serum to the amount of 0.5 c.c. was incubated for 2 

 hours with 0.1 c.c. of each dilution of complement and likewise two other sets, 

 II and III, of 0.5 c.c. of inactive sera with 0.1 c.c. of each dilution of guinea-pig 

 serum. At the expiration of this time, to the first series was added 1 c.c. of 5 

 per cent suspension of washed human cells and 2 units of antihuman ambo- 

 ceptor, to series II the same was added, and to series III, 1 c.c. of 5 per cent 

 washed sheep-cells suspension and 2 units of antisheep amboceptor. After a 

 second incubation of an hour the result was read. In all, three separate titra- 

 tions of the serum were made with the different systems. The complement 

 present in the unheated serum activated the amboceptor and in every tube 

 there was complete hemolysis. The result with the inactive serum and the 

 two hemolytic systems were practically the same. 



(1) 0.5 c.c. 1-3000 dilution of venom and .05 c.c. complement. 



(2) 0.5 c.c. 1-6000 dilution of venom and .05 c.c. complement. 



(3) 0.5 c.c. 1-12000 dilution of venom and .05 c.c. complement. 



(4) 0.5 c.c. 1-24000 dilution of venom and .05 c.c. complement. 



(5) 0.5 c.c. 1-48000 dilution of venom and .05 c.c. complement. 



(6) 0.5 c.c. 1-64000 dilution of venom and .05 c.c. complement. 



(7) 0.5 c.c. 1-80000 dilution of venom and .05 c.c. complement. 



(8) 0.5 c.c. 1-96000 dilution of venom and .05 c.c. complement. 



(9) 0.5 c.c. 1-128000 dilution of venom and .05 c.c. complement. 



units of 



Incubate for 2 hours at 37 C, then add 1 c.c. washed erythrocytes and 2 

 amboceptor to every tube and again incubate for 1 hour at 37 C. 



Result: Result: 



(1) No hemolysis. (6) Partial hemolysis. 



(2) No hemolysis. (7) Partial hemolysis. 



(3) No hemolysis. (8) Complete hemolysis. 



(4) No hemolysis. (9) Complete hemolysis. 



(5) No hemolysis. 



In the three titrations using the sheep system the dose absorbed was 

 between 0.05 + 0.06 c.c. of complement. 



Three different titrations with the two systems were then made, using a 

 fixed dose of 0.05 c.c. of complement and 0.5 c.c. of varying dilutions of the 

 venom. 



Another titration was now made of the venom in dilutions between 40,000 

 and 90,000, fixing the dose causing almost complete hemolysis at 1 to 70,000. 



Having decided on the amount of complement fixed by the serum and 

 venom, four series of fixation tests were made with the two hemolytic systems, 

 using in every case a complement 18 hours old and made up of the blood of 

 three guinea-pigs. 0.5 c.c. of serum was added to 0.5 c.c. of 1 to 70,000 dilu- 

 tion of venom and the sum of the amounts of complement absorbed by each 

 factor alone added; that is, 0.05 c.c. +0.05 c.c. or 0.1 c.c. of complement; con- 



