We performed several experiments with the view of demonstrating the
passage of amboceptor from sensitized RBCs into the medium of suspension.
Sheep RBCs were sensitized to their limit in goat antisheep serum, washed and
suspended in 0·85 per cent. NaCl. Whether incubated at 38° C for 2 hours and
tested immediately, or kept in the ice-box for 24 hours after incubation for 4½
hours, or kept in the ice-box for 24 hours without previous incubation, no
amboceptor could ever be extracted by fresh sheep RBCs from the fluid after the
sensitized RBCs had been removed by centrifugation, showing that there had
been no appreciable diffusion and that the amboceptor originally attached to the
RBCs remained attached. The sensitized RBCs, moreover, required precisely
the same quantity of fresh sheep serum as complement to lyse them, after being
given every opportunity of losing amboceptor by diffusion, as was required by
another portion of sensitized RBCs which had been tested at once.
For this reason alone it must be considered that the union of RBC and
amboceptor is a close one, and that a state of equilibrium between corpuscles and
sensitizing fluid as regards amboceptor does not exist.
In the case of goat RBCs obtained from goat-antisheep-blood, which were
washed and incubated in contact with an equal quantity of sheep RBCs, no lysis
was obtained with fresh sheep serum complement, showing that no transference of
sensitizer had occurred from goat RBCs to sheep RBCs in appreciable quantity.
It may therefore be taken as a fact the RBCs of an immune serum contain none
of the immune body.
A further experiment shows that amboceptor, far from being in equilibrium
between RBCs and the surrounding fluid, has such a powerful affinity for RBCs
that it can be extracted from the fluid almost completely, even when the latter is
highly diluted. For example 1·5 cc sheep RBCs were suspended in 2 cc of goat-
antisheep-serum and a similar quantity in other 2 cc of the same serum diluted in
vari ous degrees up t o 1 i n 40. The suspensi ons, aft er one hour i n t he i ncubat or
with one or two shakings, were centrifugated and the sensitized RBCs put up with
fresh sheep serum complement. All the series showed precisely the same degree
of lysis, showing that the affinity between RBCs and sensitizer in the case of immune
serum is so great that for experimental purposes it does not matter whether the
sensitizing- fluid is pure or is diluted with 0.85 per cent. NaCl 40 times (in another
experiment 98 times) the sensitizer is picked out by the RBCs from the dilute
fluid just as surely as from the pure serum.
Given high dilutions the factor of time would doubtless have to be considered ;
but the result would be in the end in all probability the same.
(7) It is generally held that complement cannot be removed from a serum by
RBCs unless they have been previously sensitized. Sheep serum does not lyse dog
RBCs and it follows that treatment of sheep serum with washed dog RBCs will not
result in abstraction of complement. 5 cc of washed dog RBCs were suspended
in 15 cc fresh sheep serum and the tube was incubated for 1½ hours at 37° C.
No lysis occurred. After centrifugation the treated sheep serum was put up with
guinea-pig RBCs, using as a control another portion of the same serum which had
been incubated for the same time. Precisely similar degrees of lysis followed in
both the series showing that no complement had been removed by the dog RBCs.
To test whether any sensitization had occurred in the dog RBCs they were
put up with fresh guinea-pig serum as complement. There was no lysis, so it may
be concluded that there had been no sensitization.
Even when. RBCs have been sensitized they do not always extract complement.
If the available complement fits the sensitizer it is abstracted and lysis occurs; if it
does not fit the sensitizer it is not abstracted and there is no lysis. Abstraction of
complement by sensitized RBCs can be most clearly demonstrated by using heated
RBCs (vide section 2, p. 7) for the subsequent tests are not then interfered with by
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