ANTITRYPSIN DETERMINATION 



I0 3 



If to this trypsin an amount of serum is previously added, which fully 

 neutralizes the digestive action, no clear zone appears upon Loffler's plate. 



The details of this procedure are as follows: The ferment solution consists of o.i gm. 

 trypsin, well shaken with 5 c.cm. of undiluted glycerin and 5 c.cm. of distilled water, 

 then left in an incubator for a half hour at 55 C., then again shaken and filtered. 



The serum is mixed in small test-tubes or upon a glass slide with varying amounts 

 of the trypsin; thus i loopful of serum is mixed with 1/2, i, 2, 3, 4, etc., up to 20 loopfuls 

 of the trypsin solution and of each of these mixtures one loopful is placed upon Loffler's 

 plate. (Ox serum plate, should be three days old). The plates are then placed into 

 the incubator for twenty-one hours at 55 C. The presence or absence of the clear 

 zones determines the quantities of ferment which respectively have not or have been 

 neutralized by the one drop of serum (e.g., i : 6 means that in the mixture of 6 loopfuls 

 of trypsin and i loopful of the serum for examination the digestive power of the trypsin 

 was still interfered with). 



The inequality in the strength of the Loffler plates, their variability in the degree of 

 alkalinity, the measurement by loopfuls, all, might prove to be sources of error which 

 may greatly influence the results. Thus the latter can only be taken as approximate, 

 relative values. 



The second, more exact and satisfactory method was introduced pri- 

 marily by Gross and Fuld for presenting the action of trypsin, and was 

 modified by v. Bergmann together with Bamberg and Meyer for the deter- 

 mination of an ti trypsin. Numerous workers have found it thoroughly 

 reliable. Its principle is based on the digestion of a clear casein solution. 

 If the entire amount of casein is digested, no more is left to be precipitated 

 by the addition of acid and therefore the solution remains clear. If, how- 

 ever, casein has been left undigested, the addition of acid will produce a 

 turbid solution or even a white precipitate. 



The necessary reagents are: 



1. Casein Solution. One gm. of casein is dissolved under slight heating in 100 c.cm. 

 of N/io NaOH; this solution is next neutralized by N/io HC1, litmus being used as 

 indicator, and diluted with physiological salt solution up to 500 c.cm. (If sterilized, 

 it can be kept for a long while.) 



2. Trypsin Solution. 0.5 gm. of trypsin (purissimum Grubler) is dissolved in 50 

 c.cm. of normal NaCL+o.o5 c.cm. of normal sodium hydrate solution and then diluted 

 with physiological saline up to 500 c.cm. 



3., Acid Solution. Five c.cm. of acetic acid +45 c.cm. of alcohol +50 c.cm. of 

 water. 



First the titration of the trypsin solution is undertaken in order to find out how 

 much trypsin is required to fully digest a constant quantity of casein. Gradually 

 increasing amounts of trypsin (from o.i to 0.6 c.cm.) are placed in six test-tubes and 

 to each 2.0 c.cm. of casein are added. These tubes are placed in an incubator at 

 37 C. for one-half hour, and then several drops of the acid solution are placed into 

 each tube. The first tube, and all those above it that remain absolutely clear, 

 contain enough trypsin to fully digest the 2.0 c.cm. of casein. 



Now comes the second part of the test. 



In each of eight to ten test-tubes are placed 2 c.cm. of the casein solution and 0.5 



