Diphtheria Antitoxin 439 



protect the guinea-pig against ten times the above determined least 

 fatal dose of toxin. 



3. Express the required dose of antitoxic serum as a fraction of a 

 cubic centimeter and multiply by 10 ; the result is one unit. 



Example: It is found that 0.01 c.c. of a toxic bouillon kills at least 

 9 out of 10 guinea-pigs, and is therefore the least certainly fatal dose. 

 Guinea-pigs receive ten times this dose of the toxic bouillon plus 

 varying quantities of the serum to be tested, measured by dilution 

 say 3oW c.c., ^W c.c., goW c.c. The first two live. The fraction ^~ 

 is now multiplied by 10; ^Vo" X 10 = ^o = * unlt - So we find 

 that each cubic centimeter of the serum contains 250 units. 



This method would be satisfactory were it not for certain variations 

 in the toxic bouillon by which the strength is worked out. Ehrlich,* 

 in an elaborate investigation of these changes, has clearly proved that 

 an ever-changing toxin cannot be a satisfactory standard, because 

 it does not possess uniform combining affinity for the antitoxin. He 

 shows by a labored scheme that the toxicity of the bouillon is no 

 index to its antitoxin-combining power, which, of course, must be 

 the foundation of the test. The toxin, under natural conditions, is 

 changed with varying rapidity into toxoids, of which he demonstrates 

 three groups prototoxoids, syntoxoids, and epitoxoids. The epi- 

 toxoids have a greater antitoxin-combining power than the toxin 

 itself, yet have no toxic action upon the guinea-pigs, hence cause 

 confusion in the results. 



To secure a satisfactory measure of the antitoxic strength of a 

 serum, it is therefore more important to first determine the antitoxin- 

 combining power of the toxin or toxic bouillon to be used, than to 

 determine its guinea-pig fatality, and this is what Ehrlich endeavors 

 to do. 



(B) Ehrlich's Method. In this method the unit is the same as in 

 Behring's method, but its determination is arrived at by a very im- 

 portant modification of the method, by which the standard of measure- 

 ment is a special antitoxin of known strength, by which the antitoxin- 

 combining power of the test toxic bouillon is first determined. Ehrlich 

 began by determining the antitoxic value of a serum as accurately as 

 possible by the old method and then used that serum as the standard 

 for all further determinations. The serum was dried in a vacuum, and 

 two grams of the dry powder were placed in each of a large number 

 of small vacuum tubes, connecting with a small bulb of phosphoric 

 anhydride. In this way the standard powder was protected from 

 oxygen, v/ater, and other injurious agents by which variations in its 

 strength could be initiated. Periodically one of these tubes was 

 opened and the contained powder dissolved in 200 c.c. of a mixture 

 of 10 per cent, aqueous solution of sodium chloride and glycerin. 

 The subsequent calculations are all based upon the strength of the 

 antitoxin powder. In Ehrlich's first test serum 1 gram of the dry 

 powder represented 1700 units. Of the solution mentioned, 1 c.c. 

 represented 17 units; y 1 ^ c.c., one unit. 



Having by dilution 1 c.c. of the first dilution in 17 of water 

 secured the standard unit of antitoxin in a convenient bulk for the 

 subsequent manipulations, it is mixed with varying quantities of the 

 toxic bouillon to be used for testing the new serums, until the least 

 quantity is determined that will cause the death of a 250-gram guinea- 

 pig in exactly four days, when carefully injected beneath the skin 

 of the animal's abdomen. This quantity of toxin is the test dose. 

 If the toxic bouillon was "normal" in constitution, it should represent 

 100 of the least certainly fatal doses that formed the basis of the old 



* "Klinisches Jahrbuch," 1897. 



