132 Immunity 



tube attached to the cannula into sterile bottles prepared to receive it. A large 

 horse may furnish 7 to 9 liters; small horses, s to 7 liters. 



rv. Preparation of the Serum. — The blood is stood away in a cool place until 

 the clot retracts after coagulation and the clear serum separates. The serum 

 is then withdrawn under strict aseptic precautions. It is variously prepared 

 for the market. Some manufacturers bottle it without any added preservative; 

 some add a crystal of thymol; some Pasteurize it; some add carbolic acid; some 

 add trikresol. 



The plain serum would be ideal, but the danger of subsequent contamination 

 through careless treatment makes it rather better to have an antiseptic added. 

 Trikresol is probably the most satisfactory of these, though it throws down a 

 precipitate that necessitates the filtration of the product, and leaves the serum 

 slightly opalescent. 



V. Determining the Potency of the Serum. — The potency of the serum is 

 expressed as so many "immunizing units." Only one method of testing is in 

 use at the present time, though to understand it, it seems wise to mention the 

 original method from .which it was derived. 



(A) Behring's Method. — Behring's unit was an arbitrary standard chosen in 

 consequence of certain conditions existing at the time it was devised. It is 

 difficult to understand apart from the circumstances governing its creation, but 

 may be defined as " Ten times the least quantity of antitoxin serum that will protect 

 a standard {^oo-gram) guinea-pig against ten times the least certainly fatal dose of 

 toxic bouillon." 



The method of determining it is not difficult to those skilled in laboratory 

 technic, and is as follows: 



1. Determine accurately the least certainly fatal dose of a sterile diphtheria 

 toxic bouillon for a standard guinea-pig. 



2. Determine accurately the least quantity of the serum that will 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 centi- 

 meter and multiply by 10; the result is one unit. 



Example: It is found that o.oi cc. 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 J^^oqo cc, K500 cc, Mooo cc The first 

 two live. The fraction J^soo is now multiplied by 10; J^soo X 10 = Hso = i 

 unit. 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 to.xin, under natural conditions, is changed 

 with varying rapidity into toxoids, of which he demonstrates three groups — 

 prototoxoids, syntoxoids, and epitoxoids. The epitoxoids have a greater anti- 

 toxin-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 important modification of 

 the method, by which the standard of measurement is a special antitoxin of 

 known strength, by which the antitoxin-combining power of the test toxic bouil- 

 lon 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, water, and other injurious 

 agents by which variations in its strength could be initiated. Periodically one 



*"Klinisches Jahrbuch," 1897. 



