PRODUCTION OF H^MOLYTIC AMBOCEPTORS. 245 



to activate it by the addition of normal rabbit serum, the comple- 

 ments of the latter will be made inert by the auto-anticomplement 

 present. Since these auto-anticomplements, however, have no in- 

 fluence on the binding of the amboceptor, the rational mode of pro- 

 cedure is at once indicated. The blood-cells are mixed with the serum 

 of the immunized rabbits and the mixture allowed to stand until 

 the amboceptors present have been bound by the blood-cells. r ihe 

 latter are then separated by centrifuge, the supernatant fluid which 

 contains the cause of the trouble, the auto-anticomplement, being 

 removed. If the blood-cells are now mixed with fresh normal rabbit 

 serum, the haemolysis which ensues in the incubator will show the 

 presence of the anchored amboceptor. Should this method, which 

 guards against all errors, prove successful, one can also get round 

 the difficulty in an easier manner by using guinea-pig serum as com- 

 plement. Against this serum, according to our experience, the auto- 

 anticomplement is ineffective. This method, however, does not 

 suffice if we wish to obtain results which permit of only one inter- 

 pretation. In order surely to avoid another source of error it is 

 well to modify the test still further. 



It has been found that normal rabbit serum possesses a con- 

 siderable though variable haemolytic action for goat blood (see 

 Table I). The question whether we are dealing with an amboceptor 

 artificially produced or with one which was originally present requires 

 detailed preliminary examination and control tests, and even then is 

 very uncertain because the amboceptor normally present finds a 

 supply of complement in guinea-pig serum more plentiful even than 

 that in rabbit serum itself, as can be seen on reference to the table. 

 This difficulty is avoided without further trouble if the amboceptors 

 produced by immunization and which it is desired to find are taken 

 out of the fluid by means of ox blood-cells instead of goat blood-cells. 

 Because of the partial community of receptor of these two blood- 

 cells this is perfectly allowable. As a rule, too, normal rabbit serum 

 dissolves ox blood only very little, even though considerable comple- 

 ment is present. (See Table I.) 



The experiments from which the conclusions are drawn in this 

 study were therefore always made with ox blood. One cc. of a 5^ 

 suspension of ox blood-cells is mixed with varying amounts of serum 

 from a rabbit immunized with goat serum, the mixture kept at 38 C. 

 on a water-bath for one hour, then centrifuged, and either fresh rabbit 

 serum added after the supernatant fluid had been decanted, or acti- 



