BIOLOGICAL METHODS 73 



The precipitate formation takes place most readily in neutral 

 solution, and is impeded by the presence of mineral acids or alkalis ; 

 the presence of salts is necessary, those of calcium exerting a speci- 

 ally favourable influence. 



On heating a serum containing a precipitin, its capacity for 

 forming precipitates is lost ; the changed precipitin (precipitoid) 

 retains, in spite of this fact, its capacity for combining with a sub- 

 stance contained in the precipitinogen. 



As to the actual chemical nature of the precipitin, precipitinogen 

 and precipitate but little is known ; the substance of the last-named 

 appears to be derived chiefly from the precipitin containing serum 

 (Welsh and Chapman), although opinions on this point are not 

 unanimous. The precipitates, furthermore, are soluble in excess of 

 the precipitable substances. 



The conditions of chemical equilibrium have been recently 

 investigated by Hamburger and Arrhenius. The precipitin used 

 in their experiments was contained in the serum of a calf which 

 had been immunised against horse's serum. In one set of experi- 

 ments a constant quantity of the calf-serum was added to varying 

 quantities of diluted horse-serum (i in 50) ; the mixture was 

 allowed to remain at 37 C. for one hour, and then centrifuged in a 

 funnel-shaped tube ending in a graduated capillary tube, in which 

 the precipitate formed could be collected and measured. In a 

 second series of experiments the quantity of horse-serum was kept 

 constant and that of the calf-serum varied. If the solubility of the 

 precipitate in physiological saline (the diluent used) were the only 

 factor which caused the diminution of the quantity of the precipitate, 

 then, according to the Guldberg-Waage hypothesis 



Concentration of precipitinogen x concentration of precipitin = 

 K(reaction constant) x concentration of dissolved precipitate. 



The quantity of dissolved precipitate was, however, larger than 

 could be accounted for by the above equation, and Hamburger and 

 Arrhenius came to the conclusion that a soluble substance is formed 

 by the combination of the precipitate with some substance in the 

 precipitinogenous body (i.e., horse-serum), and that the conditions 

 are analogous to those existing in the Ca(OH) 2 : CO 2 reaction. 



A detailed account of the precipitin reaction and of the theories 

 that have been advanced to explain the precipitin formation need 

 not be discussed here ; it remains, however, to consider briefly the 

 practical applications of the reaction and the technique of the 

 methods employed. 



The method for determination of the species origin of a protein 

 is due chiefly to Wassermann and Uhlenhuth. 1 It has been studied 

 in great detail by Nuttall, and has been recently modified by A. 

 Schulz and extended by him to the quantitative estimation of 

 mixtures of proteins. Owing to the fact already mentioned, that 

 the precipitin reaction is not strictly specific for a protein of any 

 given species, great care is required in the application of the re- 

 action. 



1 A recent detailed account of the method for technical purposes has been published 

 by Uhlenhuth and his co-workers (Arbeitcn aus dem KaiserUchcn Gesundheitsamt, 1908, 

 vol. xxviii., pt. 3). 



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