148 PRACTICAL METHODS IN IMMUNITY 



lytic amboceptor when added to the tubes, and hemolysis shows itself 

 almost immediately in tubes when the complement has not been 

 absorbed by the antigen through syphilitic antibodies. 



Noguchi has called attention to the fact that protein constituents of certain 

 aqueous or alcoholic extracts may have the power to fix complement through certain 

 intermediaries existing in fresh serum which, however, does not obtain for inacti- 

 vated sera (sera heated to 56 C. for 15 minutes). 



Pure lipoidal substances as contained in Noguchi's acetone insoluble antigen, 

 however, do not act in this way. 



Consequently by using such an antigen we eliminate the objection to employing 

 fresh human serum in the test for syphilitic antibodies. 



As giving more uniform hemolytic results and as being more stable and easier 

 of employment, I have made use of Noguchi's directions for taking up the serum of 

 the rabbits, immunized to human red cells and his method of standardizing this 

 '' amboceptor " paper. In practice, I measure off the length of paper correspond- 

 ing to 8 to 10 Noguchi units and dissolve the dried serum in such paper in i c.c. of 

 salt solution. This makes a satisfactory and uniform substitute for the sterile 

 immune serum used by Emery. It has been noted that the dried rabbit serum on 

 the paper may contain a certain amount of complement even when several months 

 old, consequently, to avoid confusion, I invariably inactivate this serum paper solu- 

 tion by heating to 56 C. for 5 minutes. 



Method : i. Take blood from the finger or ear in a large Wright U tube (1/4 inch 

 in diameter). Place in 37 C. incubator for 15 minutes (to increase yield of serum) 

 and then centrifuge. 



2. Graduate a capillary pipette for i volume and 4 volumes. 



3. Into each of a series of small test-tubes put 4 volumes of normal salt solution. 

 (These tubes are most conveniently made by breaking off 2 1/2 to 3 inch lengths of 

 i/4-inch soft glass tubing and then fusing one end in the flame to make a small test- 

 tube.) 



Make a distinguishing mark, e. g., X, on end of tube with blue- wax pencil and use 

 this tube to hold control. Mark the other tubes I, II, III, and so on. When differ- 

 ent sera are to be tested they may be distinguished by lines either above or below, 

 or with circles, also marks with red-wax pencil may be used. 



4. Make a i to 10 dilution of stock antigen solution in salt solution. 



To Tube I add 4 volumes of i to 10 antigen, thus making 8 volumes of i to 20 

 antigen in Tube I. Mix thoroughly by manipulating bulb of pipette. Then trans- 

 fer 4 volumes of the i to 20 from Tube I to Tube II, and so on through the series. 

 When the dilution in the last tube has been made throw 4 volumes away. 



The 4 volumes of dilution of the antigen in the respective tubes will then be: 

 In Tube I, i to 20; in Tube II, i to 40; in III, i to 80; in IV, i to 160, and so on. 



5. Add i volume of serum to be tested to control tube X, and to each of the tubes 

 I, II, III, etc., in succession. (If the serum be added to the antigen tubes before the 

 control tube, -antigen might be carried over to the control.) 



6. If the serum has been inactivated restore complement by adding i volume 

 of a 40% fresh guinea-pig serum. Also use 2 volumes of this inactivated human 

 serum instead of i. 



