18
3. On the Ponderability of Amboceptor.
Having noticed that sensitized RBCs are more quickly sedimented by
centrifugation than unsensitized ones and that this holds good, though in a lesser
degree, even when the RBCs are not visibly agglutinated during the process of
sensitization, it seemed by no means unlikely that it would be possible to show
by means of the balance an increase of weight following sensitization.
Addition of matter to the RBCs must be accompanied by an equivalent loss
from the sensitizing serum. Of several methods attempted the following seemed
to give room for least experimental error and were attended with fairly satisfactory
results:—
(a) Equal quantities of washed sheep RBCs were placed in each of two
tubes : to one was added heated normal goat serum and to the other an equal
quantity of heated goat antisheep serum. The tubes were incubated for an hour
and then centrifugated, and aliquot parts were removed from the supernatant fluid
of each, dropped into small weighed beakers and weighed. Samples of the two
heated serums had been weighed before mixing with the RBCs.
Then, if weight 5 cc. heated normal goat serum
...
5 cc. heated goat antisheep serum..
=
=
a
b
,,
5 cc. normal goat serum after incubation
with sheep RBCs
...
...
= c
, ,
5 cc. goat antisheep serum after incubation
with sheep RBCs
...
...
= d
since sheep RBCs extract no amboceptor from normal goat serum weights
a and c should be equal, but c is always a trifle less than a owing to unavoidable
dilution of the serum by being mixed with RBCs suspended in 0·85 per cent.
NaCl.
The difference between a–c and b–d should on this account be equal, but
the proportion a : c:: b: d is not found to hold good.
Instead, it is found that d has lost more weight than can be accounted for
by the dilution, and this loss expresses the extraction of amboceptor.
(b) By a slight modification not only the loss sustained by the serum but also
the gain by the RBCs can be shown. Two weighed centrifuge tubes are filled,
the one with a suspension of washed sheep RBCs and heated normal goat serum,
the other with like quantities of sheep RBCs and heated goat-antisheep-serum.
After incubation for an hour the RBCs are sedimented in the centrifuge and the
supernatant fluids carefully pipetted off into weighed beakers.
If weight of—
Tube A
= a
Tube B
= b
Tube A + RBCs.
= c
Tube B + RBCs.
= d
Tube A + RBCs + normal goat serum
= e
Tube B + RBCs + antisheep serum
= f
Tube A + RBCs — supernatant fluid after centrifugation = g
Tube B + RBCs — supernatant fluid after centrifugation = h
Beaker
= I
Beaker 2
= i
Beaker 1 + supernatant fluid A
= k
Beaker 2 -I- supernatant fluid B
= 1
Then
c—n
d—b
= Weight RBC suspension.
=
„
,,
,,
e—(c-a) =
„
normal serum.
)—(d-b) = „ antisheep serum.
g—a
=
„
RBCs in normal serum.
h—b
= „
RBCs + amboceptor.
k—i
= ,, unextracted supernatant fluid.
/—j
= „ extracted supernatant fluid, i.e., fluid—amboceptor.
From these data a gain of weight by the RBCs by reason of their
sensitization, and an equivalent loss in the antisheep serum, can be calculated.
Thoukh it is fairly easy to show that a gain of weight occurs to RBCs
during sensitization and that the sensitizing serum undergoes a loss, a number
of very accurate observations would have to be collected before a definite pro-
nouncement could be made as to the percentage weight of an artificial amboceptor
in any particular serum.