7
(6) Experiments were made to determine whether the inhibition of specific lysis
by different metallic salts is related to their respective molecular weights. Calcium
-chloride and magnesium sulphate were heated to 200°C to render them anhydrous,
and equimolecular solutions of these and of dried sodium chloride were prepared.
I per cent. NaCl = 1 . 89 per cent. CaC12.= 2.06 per cent. MgSO4.
Proportionally increasing strengths of these solutions were used as the diluting
agents in a haemolytic system consisting of sheep RBCs sensitized in goat-
antisheep-serum with fresh sheep serum complement.
The inhibitory action as shown by the diagrams (chart No. 2) was greatest
in the case of CaC12 next of MgSO4 and least of NaCl. It is evident that the
prevention of lysis is not a function of the molecular weight of a salt, but depends
on some other property such as the degree of ionisation.
As regards lysis by hypotonicity the following strengths of the above salts
are approximately equivalent :—
NaC1.
. . .
...
...
...
per cent.
CaC1 2
...
...
...
...
...
...
... I.4 per cent.
MgSO4. 7 H 2 0 ...
. . .
. .
.
. . •
...
. . .
. . .
5.8 per cent.
2. Some Facts relating to Amboceptors.
(I) Washed sheep RBCs were placed in a thin collodion sac and the latter
was suspended in heated goat antisheep serum. The tube was kept at 37°C for
two hours and in the ice box overnight. The RBCs were then removed from the
sac and complemented with fresh sheep serum. No lysis occurred, showing that
no diffusion of amboceptor had taken place through the membrane.
The sensitizing molecules of an immune serum are evidently of a colloidal
nature and will not pass through a semipermeable membrane, even when RBCs,
for which they possess a high degree of affinity, are on the other side.
(2) Washed rabbit RBCs were extracted with acetone for 24 hours, then
centrifugated and washed free from acetone. The brown residue was mixed with
10 cc fresh sheep serum, kept at 37°C for two hours and in the ice box overnight
to enable it to extract both amboceptor and complement from the serum. After
centrifugation the treated serum was put up with fresh rabbit RBCs, using another
portion of the same sheep serum, which had also been kept in the incubator and
ice box as a control. 0•1 cc of the untreated serum dissolved 0 .05 cc rabbit
RBCs nearly completely, whereas a similar quantity of the treated serum gave only
slight lysis.
It is evident that the lytic receptors of the RBCs had not been dissolved by
the acetone and that their combination with the amboceptor and complement of
the sheep serum still remained possible. It is to be inferred that the lytic receptors
belong to the albuminous portion of the .RBC and not to the acetone soluble portion.
Treatment with acetone had so altered the constitution of the h ae moglobin
that it could no longer diffuse out from the RBC, so that the exhaustion of the
serum was not accompanied by lysis which would have rendered the latter part of
the experiment more difficult to perform.
RBCs, unlike other body cells, do not remove complement from a serum
unless they have been sensitized. In this experiment there was nothing to show
that complement had actually been removed from the serum, the reduction of its
lysin content for rabbit RBCs only was proved. Amboceptor must, therefore, have
been absorbed by the acetone extracted RBCs but it is probable that complement
also was attached.
Muir has shown (Studies on Immunity) that RBCs washed and suspended
in 0 . 85 per cent. NaC1 and heated to 55°C for 24 hours, still retain their power of
combining with amboceptor and complement, and this constitutes a convenient