The same serum should therefore neutralise 1 milligramme of venom in a dose of 1 c.c., that is to say, that this mixture injected into a mouse ought to be entirely innocuous. Experiments show, however, that in reality it is necessary to mix 1·2 c.c. of serum with 1 milligramme of venom in order that the inoculated mouse may not succumb.
This proves that, in the initial mixture of 0·00005 gramme of venom + 0·05 c.c. of serum, there remained an exceedingly small quantity of non-neutralised venom, and that this quantity of venom in a free state was insufficient to cause the death of the animal, or even any apparent malaise. When multiplied by twenty, however, it becomes capable of producing toxic effects; it is for this reason that, when it is desired to inoculate a mouse with twenty times the lethal dose of 0·00005 gramme neutralised, it is necessary to mix with this twenty times lethal dose a dose of serum a little larger than twenty times that which renders 0·00005 gramme of venom innocuous to the mouse, that is to say, 1·2 c.c.
If, instead of making use of the mouse as test animal, we employ the rabbit, it is found that the same serum, in a dose of 0·75 c.c., neutralises 0·001 gramme of venom sufficiently for the mixture to be innocuous when inoculated. It is clear that, in this mixture, the whole of the venom was not neutralised by the serum, but the small quantity left free is incapable of producing harmful effects.
By this method of employing mixtures of the same dose of venom with variable quantities of antivenomous serum, we are therefore enabled to determine with the greatest exactness the antitoxic power in vitro of each specimen of serum. But it must not be forgotten that the result obtained applies only to the species of animal into which the mixtures were injected.
I have already stated (Chapter VIII.) that a fairly close parallelism exists between the neurotoxic action of venoms and their hæmolytic action, and I have established that, in order that the sensitive red blood-corpuscles may be dissolved under the influence of venom, it is indispensable that the reaction take place in the presence of normal serum, since venoms have no effect upon red corpuscles freed from serum by several successive washings and centrifugings.
Preston Kyes has explained this phenomenon very well by showing that the venom combines with the lecithins in the serum, or with those contained in the stroma of the corpuscle, so as to constitute a hæmolysing lecithide.
The knowledge of this fact enables us to determine, by means of a very neat and simple method, and with a sufficient degree of accuracy for practical purposes, the antitoxic power of an antivenomous serum by measuring its antihæmolytic power.[102]
To this end it is sufficient to cause variable doses of serum to act on a given quantity of defibrinated horse- or rat-blood, to which a constant dose of venom is then added. We employ, for example, a 5 per cent. dilution of defibrinated horse-blood, which is portioned out in doses of 1 c.c. into a series of test-tubes. To each of these tubes in succession is added a progressively increasing quantity of the serum for titration, starting with 0·01 c.c., and continuing with 0·02 c.c., 0·03 c.c., &c., up to 0·1 c.c. A control tube receives no serum. There are then introduced into all the tubes 1 decimilligramme of venom and 0·2 c.c. of normal horse-serum, deprived of alexin by previous heating for half an hour at 58° C. At a temperature of about 16° C. hæmolysis commences to manifest itself in the control tube in from fifteen to twenty minutes. It takes place in the other tubes with a retardation which varies with the dose of serum added. Tubes are to be noticed in which it does not occur even after the lapse of a couple of hours.
Experience shows that we may consider as good for therapeutic use serums which, in a dose of 0·05 c.c., completely prevent hæmolysis by 1 decimilligramme of Colubrine venom, such as that of Cobra, Krait, &c., and those that in a dose of 0·7 c.c., prevent hæmolysis by 1 milligramme of the venom of Lachesis or Vipera berus.
By a method calculated upon the foregoing, it is likewise possible to measure the antihæmorrhagic activity of an antivenomous serum, for the parallelism existing between the antineurotoxic and antihæmolytic actions of serums occurs again, as I have been able to establish in conjunction with Noc, between the antihæmorrhagic and antiproteolytic action of the same serums.