176 . PRACTICAL METHODS IN IMMUNITY 



About one-half of these antigens are lacking in power to absorb 

 complement in the presence of syphilitic sera. More rarely they may 

 absorb complement with a nonsyphilitic serum (anticomplementary) 

 or they may have a haemolytic action. Consequently a new stock 

 antigen should be tested as to its reliability 



1. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, and o.i 

 c.c. of a 10% suspension of washed red cells when incubated at 37C. for two hours 

 should not show any haemolysis. 



2. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, o.i c.c. 

 of a 40% solution of fresh guinea-pig serum, and 2 units of amboceptor and incubated 

 at 37C. for one hour should show haemolysis when we now add o.i c.c. of a 10% 

 washed red-cell emulsion and the whole then again incubated for two hours at 37C. 

 (The antigen did not absorb complement in the absence of syphilitic antibodies.) 



3. A mixture of 0.2 c.c. of a i to 10 dilution of working antigen emulsion, 0.8 

 c.c. of salt solution, i drop of syphilitic serum, o.i c.c. of a 40% dilution of fresh 

 guinea-pig serum, and 2 units of amboceptor should be incubated at 37C. for one 

 hour. When we then add o.i c.c. of a 10% suspension of washed red cells and 

 again incubate for two hours we should fail to obtain haemolysis. (The antigen can 

 absorb complement through the intermediation of syphilitic antibodies.) 



Preparation of Amboceptor Paper. In order to secure blood from the vein of a 

 man or the heart of the immunized rabbit, the most convenient method is with the 

 use of an Erlenmeyer flask with a rubber stopper having two perforations in the 

 stopper. To one of the projecting pieces of glass tubing a stout hypodermic needle 

 is attached through the medium of about 8 inches of rubber tubing, and the second 

 piece of glass tubing is bent at an angle as it leaves the stopper to provide a suction 

 tube. With a man, constrict the upper arm sufficiently to stop venous return with 

 an Esmarck rubber bandage or a towel. Paint tincture of iodine over a prominent 

 vein at the bend of the elbow. Gentle suction will cause the blood to flow into the 

 needle tube and thence into the flask when the vein is entered. 



The blood as it is taken from the arm should be received in about 50 c.c. of normal 

 salt solution containing i% of sodium citrate. About 28 to 30 c.c. are usually suffi- 

 cient. Now throw down this red-cell suspension in three or four centrifuge tubes. 

 The resulting sediment should be washed and rewashed with salt solution. Two 

 to three washings with salt solution suffice. 



Now take a large healthy rabbit, shave the lower abdomen and paint the surface 

 with tincture of iodine. The easiest way to inject the rabbit is to hold the animal 

 head down and plunge the needle of a large glass hypodermic syringe containing the 

 washed red-cell sediment into the abdominal cavity in the median line. The intes- 

 tines gravitate downward and by entering the needle below the limits of the bladder 

 we avoid injuring any vital part. 



Make the injections at intervals of five days and give increasing amounts at each 

 successive injection. Thus, first injection, 5 c.c.; second injection, 8 c.c.; third 

 injection, 10 c.c.; fourth injection 12 c.c.; and at the fifth injection give about 15 to 

 20 c.c. of washed red-cell sediment. It is well to dilute the cell sediment with an 

 equal amount of salt solution. About ten days after the last injection, we take some 

 blood in a Wright's tube from a vein of the ear and dilute the serum to make a i to 



