CHAPTER VII 



THERAPEUTIC SERA 



Diphtheria Antitoxin 



When a meat-infusion bouillon culture of the diphtheria bacillus, which has 

 been incubated at 37C. for several weeks, is filtered through a porcelain filter, 

 the filtrate contains the toxin, but no bacteria, and we speak of this filtrate 

 as toxin. It is capable of producing disease and death when injected into 

 susceptible animals in sufficient quantity. 



Different cultures of diphtheria bacillus produce different quantities of toxin, 

 consequently when toxin is obtained by filtering a bouillon culture, its strength 

 must be determined. A unit of diphtheria toxin is the smallest amount that 

 will regularly kill a 25o-Gm. guinea-pig in 4 days; this amount is called the 

 " Minimum Lethal Dose," expressed "M.L.D." 



The first step in the production of diphtheria antitoxin is to produce toxin 

 and determine its M.L.D. The next is to immunize a horse. This is done by 

 injecting a minute quantity of the toxin into a horse. About a week after the 

 first injection the horse is less susceptible to the toxin than before and a second 

 injection, larger than the first in amount, is given. At intervals of about a 

 week subsequent injections of gradually increasing amounts of toxin are given 

 until, in 2 or 3 months, the horse acquires maximum immunity. Then, under 

 aseptic conditions, the horse is bled and the serum obtained from the blood. 

 The antitoxic properties of the serum are confined to the serum pseudoglobulins, 

 the pseudoglobulins are removed from the serum by mixing the serum with an 

 equal amount of saturated ammonium sulphate solution; this is allowed to 

 stand for 12 hours and the precipitate is then collected on hard filter-paper. 



The precipitate is dissolved in sufficient water to bring it up to the original 

 volume of the serum, an equal amount of saturated ammonium sulphate 

 solution is added and, after 12 hours, the precipitate is collected and dried be- 

 tween filter-papers until it has a pasty consistency. It is then dissolved in a 

 saturated solution of sodium chloride. This solution is filtered and 2.5 cc. of 

 80 per cent, acetic acid is added to each liter of the filtrate. The precipitate is 

 collected and dried on filter-papers and dialyzed in running water until dissolved. 

 It is then neutralized and again dialyzed until free of sodium chloride. 



The amount of antitoxin present in the serum of immunized horses varies, 

 hence when serum is obtained its strength must be determined. This is done 

 by mixing various amounts of serum with a M.L.D. of toxin and injecting the 

 mixtures into guinea-pigs, until an amount is found which is just sufficient to 

 neutralize a M.L.D. of toxin. 



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