9
This experiment was performed by lysing 17 cc washed sheep RBCs in
:85 cc distilled water and adding NaCI to 0 . 85 per cent. Series were then put up,
(A) containing specific sensitizing serum and complement, and (B) containing
.an equal quantity of a non-specific serum with complement. Both were then incu-
bated. Sensitized sheep RBCs were subsequently added and the tubes again
incubated. It was then found that the disrupted RBCs (A) had combined with
the sensitizer and removed complement, so that in series (A) complete lysis was
shown by a tube containing 0 . 25 cc complement, whereas in series (B) 0 .06 cc
complement produced complete lysis.
The occurrence of lysis is, therefore, a purely accidental phenomenon, and the
liberation of hæmoglobin is simply an indication that amboceptor and complement
have united with some part of the substance of the RBC and is not an integral
part of the reaction.
(4) The effect of inspissation and desiccation on a specific amboceptor appears
to be small.
Goat antisheep serum was inspissated at 70°C. On breaking up the jelly
next day and extracting it with distilled water equal in quantity to the original
bulk of serum, the extract was found to contain the sensitizer unimpaired in quality
as far as could be judged by comparing its action with that of the original serum.
Extract prepared from another portion of the inspissated serum which had been
kept in the ice box showed considerable deterioration after 20 days.
Serum desiccated rapidly in vacuo over H2S04 r e t a i n s i t s s e n s i t i z i n g p o w e r
nearly unimpaired for at least six months, when kept at room temperature
(15°-20° C). Even after 20 days such a desiccated serum had not entirely lost
its complement.
*
(5) The sensitizing molecule is attached to the globulin fraction of the serum
and is easily precipitable with the globulin by the saturation of an immune serum
with MgSO 4, or semisaturation with (NH 4)2SO4.
For example, the serum of a pony, which had been immunised with sheep
RBCs was semisaturated with (NH 4 )2SO4 by adding slowly an equal bulk
of saturated solution of (NH 4) 2 SO4. The precipitate was collected on a
filter and redissolved in another portion of saturated (NH 4 )2SO4 and a third time
precipitated. Finally the precipitate was dissolved in a quantity of distilled water
equal to the original serum and dialysed in running water for 72 hours to get rid of
the (NH 4)2SO4. A few drops of chloroform were added to prevent decomposition
during dialysis. The tonicity of the solution was then ascertained by means of
RBCs, and sufficient NaCI was added to bring it up to o . 85 per cent. A series of
tubes, put up with fresh sheep serum as complement, showed that this dissolved
globulin solution contained active sensitizer for sheep RBCs.
The filtrate from which the globulins had been removed was dialysed in run-
ning water and a little chloroform was added to it to prevent decomposition. After
removal of the (NH 4)2SO4 the tonicity of the solution was made to equal
0 . 85 per cent. NaCI, but it had no sensitizing effect whatever on sheep RBCs ; so
it is evident that the whole of a specific amboceptor is precipitated with the globulins
of a serum and that no recognisable quantity is left in the albuminous filtrate.
It was thought to be not unlikely that by fractional precipitation of the
globulins the amboceptor might be obtained in a more concentrated state.
94 cc of pony antisheep serum were taken and a saturated solution
of (NH 4)2SO4 was gradually added till the serum contained the solution
in 20 per cent. concentration. The precipitate (A) was filtered off, and more
'(NH4)2SO4 added to the filtrate till the concentration was 40 per cent.
Precipitate (B) was filtered off and the concentration of the filtrate raised to 50 per
cent. This gave precipitate (C)—
A weighed
o.29 grams.
B
,,
3 . 0 4
, ,
C
,,
0'79
,,