H. M. POWELL 829 



PREPARATION OF PRECIPITINOGEN 



For careful precipitin tests, clear precipitinogen is necessary. This may be ob- 

 tained in some cases without any preliminary treatment, or in others by filtration or 

 by extraction with saline. In instances such as the latter it is preferable not to agitate 

 the material while it is being extracted, in order to avoid cloudiness. With bacterial 

 antigens, separation of the filtrate from cultures treated in various ways may be ac- 

 complished by centrifugation or by filtration. 



TEST PROCEDURE 



The precipitin test may be carried out by setting up mixtures containing constant 

 amounts of antigen and diminishing amounts of (preferably) undiluted immune serum, 

 or by holding the precipitin amount constant and diminishing the precipitinogen. In 

 certain quantitative studies of precipitin and precipitinogen Cromwell' notes that in 

 tests with the simpler antigens dilution of antigen gives no direct insight into the 

 potency of precipitin. With simple antigens the antigen titre divided by the antigen 

 dilution at maximum precipitation equals the antiserum titre divided by the anti- 

 serum dilution at maximum precipitation. That is, the natural units of precipitin 

 and precipitinogen at the point of maximum precipitation are approximately equal. 

 However, in practical precipitin tests complex antigens (mixtures) are used and exact 

 quantitative relations are obscured. It is sufficiently exact here to set up diminishing 

 quantities of antigen against constant amounts of immune serum. Very clear-cut 

 readings of tests may be made when a ring test is used; however, an antigen-antibody 

 mixture may be used and the generally distributed precipitate observed on appearing 

 or after settling out. 



SPECIES IDENTIFICATION AND RELATIONSHIP 



The voluminous writings upon the use of the precipitin reaction in identifying 

 blood have been reviewed by Nuttall in his book on Blood Immunity and Blood Rela- 

 tionship. Nuttall's own very extensive tests indicated that the precipitin test is a 

 valuable aid in confirming the biological relationships among species established upon 

 the basis of morphology, comparative anatomy, embryology, etc. Far-reaching con- 

 clusions must, however, be made with caution because immunologically very closely 

 allied species, the guinea pig and rat, are shown to be very different according to the 

 distribution of so-called "heterophile antigen." This is true also of the horse and cow. 

 On the other hand, the guinea pig, the horse, and the chicken are "proved" closely 

 related since they contain the same antigenic material. 



The identification of blood stains, body fluids, and fixed tissues, either native or 

 modified by heat or other agencies, frequently presents difficulties proportional to the 

 extent to which these materials have been denatured. Generally, proteins which have 

 been dried quickly are easily identified by the precipitin test. However, if materials 

 to be identified in this way have undergone spontaneous decomposition or digestion or 

 have been affected markedly by heat or various chemicals, they may give irregular 

 reactions with native antigen-mcited precipitin. It is believed by some investigators 

 that more satisfactory reactions in testing heated proteins may be secured through 



' Cromwell, H.: /. Infect. Dis., 37, 321. 1925. 



