8
method of decomplementing a serum. We have found that equally good results
can be obtained by using RBCs heated for a shorter time to a higher temperature.
All that is necessary is to heat the RBCs in a tube in a waterbath until they
are thoroughly browned and aggregate in clumps, leaving the fluid clear or slightly
brownish.
Provided with such heated RBCs it can easily be shown that, unless they
have absorbed amboceptor, they will not absorb complement;e . g . ,
h o r s e s e r u m ,
which contains little or no amboceptor for rabbit RBCs, cannot be decomple-
mented by rabbit RBCs, whereas sheep serum which normally lyses rabbit RBCs
strongly can be completely decomplemented by treatment with heated rabbit RBCs.
In heating various kinds of RBCs for these experiments we noticed that
there were considerable differences in the temperatures required for the browning
and in the time the heat had to be maintained. Slight lysis is the first effect of
the heat on all RBCs, but it is more marked in some species than in others. We
thought it might be possible to arrange the RBCs of different species in order,
with regard to the degree of temperature required to brown and clump them and
the duration of its application, but after some trials it appeared that the experi--
ments would require more time than could be spared for them. The variations
are, however, so well marked that such an arrangement would probably be quite
feasible, in spite of the non-existence of a definite coagulation temperature.
The coagulation of hae moglobin is said to take place logarithmically, the coagula-
tion rate at any moment being proportional to the concentration of uncoagulated
protein. The temperature coefficient for Hb = 1·3 per degree C., i.e., increase
in amount coagulated. There is therefore no definite coagulation temperature,
as coagulation proceeds at different rates at different temperatures.
(3) The union of RBC receptor and specific amboceptor is generally held to
be a chemical one, and the only method of dissociation hitherto published
is to place unsensitized RBCs in contact with fully sensitized ones, which results
in the transference of a portion of the sensitizer from the latter to the former.
It seemed of interest to determine whether the destruction of sensitized RBCs by
hypotonicity leads to a disruption of this union, or whether the resulting fluid still.
retains the power of absorbing complement.
Muir (Studies on Immunity) has shown that the stromata of lysed RBCs
retain the power of absorbing both amboceptor and complement when separated by
centrifugation from a lysed suspension, but his experiment does not cover the
possible dissociation of the amboceptor molecule by distilled water from its attach-
ment to a RBC.
We took 10 cc washed sheep RBCs, sensitized in goat antisheep serum,
and lysed them in 48 cc distilled water (Fluid A). 10 cc of unsensitized sheep
RBCs were similarly lysed (Fluid B). NaCl was added to both fluids to
0·85 per cent.
Series were put up of the two fluids, with varying quantities of fresh sheep
serum as complement, and were incubated for half an hour at 37°C. A sus-
pension of sensitized sheep RBCs was then added and the series again incubated.
No complement was abstracted from the series containing fluid B, and the
several tubes showed precisely the same degree of lysis as did the control series,
(0·1 cc complement = C lysis), whereas, in the series containing fluid A, so much
complement had been fixed by the fluid that 1·0 cc did not give complete lysis
the added RBCs.
It is evident that hypotonic lysis of sensitized RBCs neither dissolves the union
between sensitizer and RBC receptor, nor does it prevent the subsequent absorption
of complement.
The converse of this experiment, as was to be expected, led to the conclu-
sion that lysed RBCs unite with amboceptor and complement just as well as un-
injured RBCs and that their disruption is a purely mechanical phenomenon which
does not alter their combining affinities in the least.