CHAPTER XIV.

_NEUTRALISATION OF VENOM BY ANTITOXIN._

It is difficult, in the present state of our knowledge on the subject of toxins and antitoxins, to determine the precise nature of the reactions that are produced in the living organism as the result of serum injected for the purpose of preventing the toxic action of venom.

I maintained, some years ago,[100] that the phenomenon in this case was a purely physiological one, which I considered to be proved by the fact that, if we mix _in vitro_, in determinate proportions, venom and antivenomous serum, and if we heat this mixture at 68° C. for half an hour, the injection of the heated mixture kills animals as if they were inoculated with venom alone, although with a considerable retardation. I concluded from this that, in all probability, antitoxic serum does not modify the toxin with which it is mixed, but that it confines itself to displaying a parallel and opposite action by preventing the noxious effects. I therefore supposed that no chemical combination is produced between these two substances, or, at least, that the combination effected is very unstable.

My experiments were subsequently repeated by Martin and Cherry,[101] who showed that the results as stated above were perfectly correct when the mixture of venom and antitoxin was heated less than ten minutes after it had been made, but that, if the heating did not take place until twenty or thirty minutes later, the toxicity of the venom no longer reappeared.

On the other hand, the admirable researches of Kyes and Sachs, and subsequently those of Morgenroth, pursued under the direction of Ehrlich at the Laboratory of Experimental Therapy at Frankfort, have proved the readiness of venom to enter into chemical combination with certain elements of normal serums, in particular with _lecithin_, a combination which results in the formation of _hæmolysing_ and non-toxic _lecithides_, the _neurotoxin_ being left free.

It therefore seemed impossible to deny the existence of a chemical reaction between the venom and the serum, which was until quite recently considered as proved. We shall see presently that this is not the case. But let us first endeavour to determine the laws that govern the neutralisation of variable quantities of venom by antivenomous serum.

If, in a series of test-tubes, we bring the same quantity of cobra-venom (_e.g._, 0·00005 gramme, a dose which is invariably lethal to the mouse in two hours) into contact with progressively increasing quantities of an antivenomous serum (_e.g._, 0·01 c.c., 0·02 c.c., &c., up to 0·1 c.c.), and, after thirty minutes of contact, inject these different mixtures subcutaneously into a series of mice, we find that all those that have received the mixtures containing less than 0·05 c.c. of serum succumb after variable intervals, while all the rest survive. It is evident that, under these conditions, the serum experimented upon has shown itself capable of neutralising _in vitro_, in a dose of 0·05 c.c., 5 centimilligrammes of venom.

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.

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.

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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.

It is therefore possible, by causing hydrochloric acid (in a solution not stronger than 3 per cent.) to act on a neutral mixture of cobra-hæmolysin (toxin) and antitoxin, to set the former at liberty in the form of _lecithide_, to withdraw the latter from the action of the antitoxin, and to demonstrate its presence, owing to its hæmolytic properties.

It has been found by Kyes and Sachs that, under the influence of hydrochloric acid, cobra-hæmolysin becomes resistant to heat to such an extent that it is not destroyed even by prolonged heating at 100° C.

If to a neutral mixture of toxin + antitoxin we add a small quantity of hydrochloric acid, and then heat the mixture at 100° C., the antitoxin being in this case destroyed, we shall recover the whole of the toxin originally employed.

Therefore, as was shown by me so long ago as 1894, if the mixture of toxin + antitoxin produces a chemical combination between the two substances, this combination is unstable, and can be effectively broken up into these two constituent elements by various influences.