Now, the antiproteolytic action is easily determined by means of a series of test-tubes containing the same quantity of 20 per cent. gelatinised bouillon, rendered imputrescible by the addition of a small quantity of thymol. The gelatine being kept liquid in the incubating stove, a progressively increasing quantity of serum is poured into each tube. The same dose of venom, say 1 milligramme, is then added in each case. The tubes are placed in the stove for six hours at 36° C. They are then withdrawn and immersed in a bath of cold water. Those in which the gelatine solidifies are noted, and thus we establish the dose of antivenomous serum that inhibits the proteolysis of this substance.
These different methods of control enable us to verify the activity of antivenomous serums with great exactness, without the necessity of having recourse to experiments upon animals.
In a very important memoir on the reconstitution of the toxins from a mixture of toxin + antitoxin, J. Morgenroth[103] has shown that the venom, after being naturalised by the antivenomous serum, can be dissociated from its combination by means of a method which consists in adding to the latter a small quantity of hydrochloric acid.
Previous experiments by Kyes had established:—
(1) That antivenomous serum, the antitoxic action of which is so manifest when it is mixed in vitro with cobra-venom, remains entirely inert when brought into contact with the combination lecithin + venom, that is to say, with cobra-lecithide.
(2) That the addition of lecithin to a neutral combination of venom + antivenomous serum does not set the venom free again, and that under these conditions no lecithide is formed.
If, in a neutral mixture of cobra-hæmolysin and antitoxin we could succeed in dissociating the two constituent elements, and in then making the cobra-hæmolysin combine with the lecithin, we should have a toxin and antitoxin side by side; for the reasons indicated above, this toxin (lecithide) and antitoxin (antivenomous serum) would be no longer capable of combining; but the toxin (lecithide), thanks to its hæmolytic properties, could easily be demonstrated.
It is precisely this desideratum that J. Morgenroth has succeeded in realising, by means of hydrochloric acid, which renders it possible to dissociate the neutral mixture, toxin + antitoxin, into its constituent elements, and then to obtain a lecithide.
Experiments show that the quantity of lecithide thus restored absolutely corresponds to that of the cobra-hæmolysin originally added to the antitoxin, and that the antitoxin set free is not injured by the hydrochloric acid, even after twenty-four hours of contact. It is sufficient to add the quantity of soda or of ammonia necessary for the neutralisation of the acid, in order to see the antitoxin reappear in its original strength.