BIOLOGICAL METHODS 75 



Method. 



Schulds Method of Protein Estimation by Means of the Precipitin 

 Reaction. The experimental basis of the method depends upon the 

 fact that if an antiserum be added to varying dilutions of an extract 

 of its precipitinogen in physiological saline solution the turbidity due 

 to the precipitate formation will appear earliest in the most concen- 

 trated solutions ; the more dilute a solution the longer will be the 

 time interval before turbidity appears. The strength (value) of an 

 antiserum can be determined, therefore, by ascertaining the greatest 

 dilution of the precipitinogen, which is just sufficient to give a tur- 

 bidity within a given time interval, which for the purposes of experi- 

 mental work has been chosen as sixty minutes. If the precipitinogenic 

 protein be mixed with other proteins a more concentrated extract 

 will have to be employed to yield a turbidity within sixty minutes, 

 than would be the case if the pure precipitinogenic protein, unmixed 

 with others, had been employed. By determining the ratio of this 

 dilution to the dilution of the extract of the pure precipitinogenic body 

 necessary to produce a turbidity within one hour, the amount of this 

 substance in a given mixture can be ascertained. The principle of 

 the method can be best illustrated by the example given by Schulz. 

 A given amount of a mixture of horse flesh and other muscular 

 tissue, weighing 50*57 grams, containing x grams of the former, 

 was extracted with 100 c.c. of normal saline (fe). This extract gave 

 a turbidity within one hour with a given quantity of a serum pro- 

 duced by immunising a rabbit against horse flesh, when it was diluted 

 1 60 times. An extract of pure horse flesh gave a turbidity under the 

 same conditions and with the same quantity of the same serum, when 

 the extract was of such dilution that 820 parts corresponded to I part 

 of the meat. The value of the serum used, W, was, according to 



Schulz's method of expression, = . From these data the amount of 



horse flesh present in the mixture can be calculated from the equation 



= - , whence;tr = 19*5. That is to say, 50*57 grams of tissue 



lOOA? o2O 



contained 19*5 grams of horse flesh. The amount actually added 

 to the mixture was 19*0 grams. 



For the purposes of experimental work a serum not too rich in 

 precipitin should be employed. It should be prepared by intra- 

 peritoneal injection of the protein, the quantity of which is to be 

 estimated in a given mixture. The antiserum should be steril- 

 ised by filtration through a clay filter and kept in hermetically 

 sealed glass tubes of O'6 c.c. capacity. Its value should be freshly 

 tested against a pure precipitinogen whenever a quantitative estima- 

 tion is carried out. The extracts of the precipitinogen and sample 

 under investigation should be carefully filtered and perfectly clear. 

 Physiological saline is used for the extraction of the material, which 

 should be finely disintegrated, and kieselguhr serves as a good filter- 

 ing medium. As there is a loss of protein during filtration, the 

 precipitinogen and the sample under investigation should be treated 

 in as nearly as possible the same way and the extracts filtered through 

 exactly similar filters the same number of times. Various dilutions 

 of the extracts can be readily prepared in series. To 0*9 c.c. of each 



