30
Series A had evidently absorbed rather more amboceptor than was compatible with
complete lysis by the minimum active dose of complement as indicated by B & C.
The concentration of amboceptor in the serum was somewhat reduced by the
removal of the amount absorbed by A, considerably reduced by B, yet enough
remained to sensitize C as fully as B.
Some information as to the influence of the available quantity of amboceptor
and of its concentration in the sensitizing fluid was gained in further experiments.
(a) I. 2 cc. washed sheep RBCs were sensitized in 1.8 cc. heated goat B
serum, and another (b) 1. 2 cc. RBCs in 1.8 cc. serum+ 198.2 cc. of 0.85 per cent.
NaCL
The actual quantity of amboceptor in (b) was the same as in (a), but its con-
centration was only 1/1oo.I n t h e t w o s e r i e s p u t u p a f t e r s e n s i t i z a t i o n p r e c i s e l y s i m i l a r
degrees of lysis were given by guinea-pig complement (0.017 = C lysis), so in spite
of the great diminution of concentration the RBCs in (b) had in one hour picked up
sufficient amboceptor for full sensitization.
As long as amboceptor is in excess of the absorptive limits of RBCs it does
not appear to matter whether its concentration in the fluid is greately reduced or
not. It is possible that with a lowered concentration more time would be taken to
reach the limit of absorption, but on this point we have made no observations.
Other facts to be related presently make it seem probable that, if the concen-
tration of amboceptor in an experimental fluid can be raised much above that which
may be considered possible in the serum of a living animal, the absorptive power
of RBCs rises in proportion. There seems to be a certain degree of concentra--
tion of amboceptor below which RBCs cannot fix enough to enable deviation of
complement to be demonstrated, although there may be in actual quantity sufficient
to sensitize many times the number of RBCs to their apparent limit.
Deviation is more easy to demonstrate when the immune serum itself is
present than when RBCs are sensitized and then washed; and it is often clearly
marked during the first 20 minutes of incubation in an experiment in which
1½ hours will render the lysis complete. Deviation could not be demonstrated by
sensitization of sheep RBCs in our pony-antisheep-serum, washing and adding
complement, but was well shown in one time-experiment, performed with another
object, in which the serum itself was present in the hæmolytic system (vide
Chart 4).
When, however, the amboceptor was presented to the sheep RBCs in the
form of a solution of the dried globulins of the antisheep serum, obtained by salting
out, dialysing and drying in vacuo over H 2SO4, complete deviation was readily
shown. It seems as if the amboceptor were more powerful in the absence of the
albuminous and lipochrome elements of the serum than in the whole serum, but
this we have not proved with exactitude. It is easy by this means to obtain a
solution containing amboceptor in 4 times the concentration in which it is found in
the original serum. The oretically a much higher concentration is possible, but in
practice it is found difficult to induce the dried globulins to dissolve in greater
concentration than 4/1 in 0.85 per cent. salt solution. Even then the solution is
turbid and contains much suspended matter. A nearly clear solution can be
obtained by filtration through paper, or centrifugation; and the removal of the
undissolved portion does not seem to reduce the amboceptoral potency of the fluid
appreciably, so it may be presumed that the amboceptor molecules are more
soluble than the globulins with which they are associated.
A similar concentration of amboceptors could be obtained by drying the whole
serum and re-dissolving it in one-fourth of its original volume of distilled water, or
by evaporation, but all the other elements, including the salts, would be concen-
trated likewise, and lysis would be interfered with thereby.