10
The precipitates were dissolved in distilled water, reprecipitated by similar
concentrations of (NH 4) 2 SO4, filtered, re-dissolved in distilled water and
dialysed free from salts. They were then dried in vacuo over sulphuric acid
, a n d s o l u t i o n s w e r e m a d e u p f r o m e a c h w i t h 0 · 8 5 p e r c e n t . N a C l c o r r e s p o n d i n g i n
volume with the original bulk of serum.
It is always difficult to induce globulins which have been thus precipitated
and re-dissolved to give a clear solution in the end, but a preliminary experi-
ment with another portion of dried globulins had shown that clear fluid could
be obtained by filtration through paper which did not differ demonstrably in
amboceptoral content from the turbid liquid. It may be assumed, therefore, that
the amboceptoral molecules are more readily soluble in 0·85 per cent. NaCl.
than the globulins with which they are precipitated.
Series were then put up with fluids A, B and C to test their relative
amboceptoral powers on sheep RBCs with 0 .2 cc guinea-pig complement.
0·75 cc Fluid A was required for complete lysis.
< 0 · 0 5 c c B " "
1·0 cc
C
"
"
If the amboceptor had been equally distributed throughout the three precipi-
tates:—
<0·5 cc A would have given complete lysis, and
<0· 1 9 cc C " "
So precipitate A contained 35 .7 per cent of the total amboceptor.
B
53.5
"
"
C
I 0 . 7 " "
It is clear, therefore, that amboceptor begins to be precipitated before 20 per cent.
saturation with (NH4)2SO4 is reached, that the greater part of it comes down
between 20 per cent. and 40 per cent. saturation and that only 1/10 remains to be
precipitated by 40 per cent.—50 per cent. saturation.
After removal of the globulins complete saturation. with (NH 4)2SO4 precipi-
tates the albumins. These, when dialysed, dried and dissolved, are found to
contain no amboceptor.
As it is not possible in practice to separate the globulins entirely from
the albumins of serum by the process of salting out, i.e., a little albumin is
carried down with the globulins precipitated by 50 per cent. saturation with
(NH 4) 2 SO4 and a little globulin remains unprecipitated in solution with the albumin,
so a fraction of amboceptor seems to cling to the unprecipitated globulins. If,
therefore, a large quantity of the dialysed, dried and dissolved albumin be present,.
in the hæmolytic system, a trace of lysis may be observed.
To find the effect of extraction of pony antisheep globulins by ether and
acetone—
One gram of globulin was extracted with 25 cc ether and one gram with
25 cc acetone for five hours in a shaker, and the vessels were left to stand overnight.
The weight of matter dissolved was very small, amounting to 4/1000 by the ether and
12/1000by the acetone. The ether and acetone were evaporated away in vacuo
and suspensions of the residues were made in 0 ·85 per cent. NaCl with which. hæmolytic
series were put up. The result showed that neither the ether nor the acetone had
extracted any amboceptor from the globulins, and that the globulins which had been
subjected to extraction had not been deprived of any of their sensitizing power.
Amboceptoral molecules are not, therefore, of a fatty nature.
(6) Arrhenius (Immunochemistry) maintains that there is a state of equili
brium between the amboceptor attached to RBCs and the amboceptor in the
sensitizing fluid, and that amboceptor merely diffuses into the RBCs and is not.
in a state of chemical combination.
Muir also holds that amboceptor can diffuse out from sensitized RBCs into
the surrounding fluid and thus tacitly binds himself to a denial of chemical union.