DEMONSTRATION OF SPECIES DIFFERENTIALS 499 



subsequently employed for similar experiments by Uhlenhuth and his col- 

 laborators. They introduced a refinement in the precipitin test by obtaining 

 the precipitins from animals belonging to one of the species whose blood was 

 to be tested or from a related species. For this purpose, cross-immunization 

 was used between two species, the relationship of which was to be studied 

 by the precipitin test, each of the two species being injected with the blood 

 serum of the other species. This method eliminated the coarser reactions 

 which led to the production of less specific precipitins in farther distantly 

 related animals which had been immunized against blood serum. Thus, by 

 immunizing rabbits with hare serum Uhlenhuth could obtain specific precipi- 

 tins for hare blood, although it was not possible to distinguish between in- 

 dividual rabbits. Similarly, through a slight modification of the same method, 

 Black succeeded in differentiating Negro chickens from Italian chickens, 

 individuals belonging to a third chicken species being immunized against 

 Negro and Italian chickens. Subsequently, further refinements were intro- 

 duced by Boyden, Hektoen, Wolfe, and Wilhelmi. The specificity of the 

 precipitin test was enhanced by limiting the amount of protein injected into 

 animals to be immunized, by dilution of the anti-sera, by using reciprocal tests 

 and quantitative methods of determination, by removing the lipids from the 

 antigens and in addition also by employing a nephelometric method of meas- 

 urement. In some instances it was possible to distinguish between the serum 

 proteins of such closely related animal species as ox, sheep and goat, and in 

 some cases even between the proteins of buffalo and ox serum. With these 

 improved methods it was also possible to differentiate between nearly related 

 species of birds and of reptiles, classes of animals which had not been found 

 very suitable in the experiments of Nuttall. By considering also the results of 

 cross-reactions and of absorption or exhaustion tests of immune sera against 

 egg albumins of various species of birds, differentiation between these species 

 was possible (Hooker and Boyd, Landsteiner and van der Scheer). In using 

 these precautions, the ring-test for the detection of precipitates permits not 

 only the determination of relationships, but also the degrees of relationship 

 between various species. 



Still, it was found impossible to distinguish in every case by the ordinary 

 precipitin method between man and anthropoid apes, and between horse and 

 donkey. On the other hand, certain differences were more readily found 

 between man and lower monkeys, and the Old World monkeys were shown 

 to be more nearly related to man than the New World monkeys. It was also 

 possible to distinguish between rat and mouse, and in recent experiments 

 Landsteiner and Levine observed, in one case, that by the use of Uhlenhuth's 

 method precipitins could be produced which differentiate the blood sera of 

 man and chimpanzee. These investigators immunized a chimpanzee with 

 human serum proteins and thus obtained an immune serum which precipitated 

 only human serum but not chimpanzee serum. However, this differentiation 

 could be much better accomplished by applying the Uhlenhuth method to the 

 production of hemagglutinins. Furthermore, Hicks and Little traced, by 

 means of the precipitin test, the relationship and origin of different species 



