in which amphoteric substances reactivate complement is thus clearly
demonstrated.
COMPLEMENT-SPLITTING BY MEANS OF DIALYSIS.
Before Sachs, Liefmann, and others introduced various methods
of splitting the complement, Ferrata discovered that by dialyzing
complement against water through a fish membrane or a celloidin
sac, it could be separated into two inactive components, an albumin
fraction corresponding with the end-piece of the later investigators,
and a globulin fraction (which precipitates during dialysis) corre-
sponding with the mid-piece. This result, although not always
obtained, was nevertheless confirmed by other workers.
On several occasions in the present series of experiments we have
also confirmed this finding. Our explanation for this phenomenon
is, however, entirely different from that given by previous workers.
As will be shown in the following protocols, the supernatant fluid
(albumin fraction) when separated from the precipitate (globulin
fraction) and made isotonic with sodium chlorid, is quite inactive
upon the sensitized corpuscles, but can be reactivated by adding,
not only the globulin fraction of the same serum (isotonized with
sodium chlorid), but also the globulin fraction prepared by the
hydrochloric acid or carbon dioxid methods (table XIV). Indeed,
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620
Biochemical Study of Complement'Splitting.
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Jacob Branfenbrenner cmd Eideyo Nog%u>hi.
621
the reactivation may be even better and more constant with mid-
pieces prepared by these other methods than with its own mid-piece.
Further, it is found that an acid (table XV, B) or certain ampho-
teric substances which bear no relation to the serum (table XIV)
TABLE XV.
Reactivation of Complement Inactivated by Acid {Carbon Dioxid) Compared
to Reactivation of Complement by Alkali (Dialysis or Distilled Water),
Comidement + distilled water (i:zo) at room temperature for i hr.; the whole
made isotonic; 0.5 c.c. used in each tube.
Incabatkm dme.
+ Alanin 7.5 f.
No alanin.
oa C.C.
0.3 c.c.
ihr.
a hrs.
zhr.
a hrs.
zhr.
a hrs.
<-
Z,
u
X
O.I
0.125
o.is
NoH.
NoH.
NoH.
No. H.
Tr.H.
Sl.H.
Tr.H.
S1.H.
Mch.H.
No. H.
S1.H.
S1.H.
Sl.H.
F.C.H.
F.C.H.
Control
F.C.H.
F.C.H.
C.H.
C.H.
NoH.
No H.(?)
o (
Sf 0.1
0.4
1 ("-s
NoH.
NoH.
Tr.H.
NoH.
Tr.H.(?)
Tr.H.
NoH.
NoH.
S1.H.(?)
No. H.
Tr.H.
S1.H.
NoH.
NoH.
NoH.
NoH.
iDcabation tim«.
isotonic ; final dilution xno; 0.3 c.c. in each tube.
+ Alanin 7.5 It
O.S c.c.
0.3 C.C.
xhr.
a his.
zhr.
a hrs.
xhr.
a hrs.
n-
o (
»o
^ 0.1
^ 0.I2S
U 0.15
X [
NoH.
NoH.
NoH.
NoH.
S1.H.
S1.H.
NoH.
Mch.H.
Mch.H.
NoH.
S1.H.
S1.H.
NoH.
Mch.H.
F.C.H.
Control
F.C.H.
C.H.
C.H.
C.H.
NoH.
Tr.H.
o
CI
z
1 il
0.x
0.2
0.3
0.4
0-5
NoH.
NoH.
Tr.H.
NoH.
NoH.
Tr.H.
NoH.
NoH.
NoH.
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622
Biochemical Study of Complement-Splitting.
in each tube.
Incubation time.
4-Alanin7.5j<.
No Alanin
o.a cc.
0.3 cc
ihr.
ahn.
ihr.
ahn.
zhr.
ahn.
o*
Z
u
X
o.i
O.I as
O.IS
NoH.
NoH.
NoH.
NoH.
NoH.
NoH.
NoH.
NoH.
NoH.
Tr.H.
Tr.H.
Tr.H.
Control
SLH.
C.H.
Tr.H.
C.H.
NoH.
S1.H.
w
Z
X'
[ i
O.I
0.2
0.3
0.4
o.s
NoH.
Tr.H.
Mch.H.
Tr.H.
F.C.H.
C.H.
NoH.
Tr.H.
Mch.H.
Tr.H.
C.H.
C.H.
C.H.
C.H.
reactivate this dialyzed end-piece. On the other hand, alkalis do
not reactivate it (table XV, B).
From the experiments given above it becomes highly probable
that dialyzed serum loses its complement activity chiefly by an
alkali inactivation of a reversible character. The cause of this
TABLE XVI.
Rate of Dialysis of Sodium Chlorid and NaHCO% from the Mixture of Fifty
Cubic Centimeters of i Per cent. Sodium Chlorid + Fifty Cubic
Centimeters N/50 NaHCO,,
NaCI
NaHCOa
AgNO,
NH4SCN
Combined
Ag
NaCl
N?xoo*
NaOH
N/xoo
Combined
add
NaHCO,
N/50
After"
4.0 cc.
3.0 cc
2.0 CC
0.02 gm.
6.0 CC
1.0 CC
S.O CC
2.5 CC
20 min.
3.0 C.C.
2.0 cc
1.0 CC
o.oi gm.
4.0 CC
1.0 cc.
3.0 CC.
I.S cc
40 min.
2.0 CC.
1.2 cc
0.8 CC.
0.008 gm.
2.SCC
o.s cc
2.0 CC
1.0 cc
I hr.
I.O CC
0.6 cc
0.4 CC
0.004 gm*
2.5 cc.
1.3 cc
1.2 CC
0.6 cc
2 hrs.
o.s cc
0.4 cc
0.x cc
o.ooi gm.
2.0 CC.
1.3 cc.
0.7 CC.
0.3s cc
3 hrs.
o.s cc.
0.5 cc
Trace(?)
i.S cc
0.9 cc
0.6 cc
0.3 cc
4 hrs.
o.s cc.
o.s cc
i.S cc
1.0 cc
0.5 cc
0.2s cc.
Running water
1.0 cc.
1.0 cc
I.S cc
1.0 cc.
0.5 cc
0.25 cc.
^At the time indicated, 5 cc of the contents of the collodion sac were re-
moved and examined for hydrochloric acid and NaHCOt.
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Jacob Bronfenbrenner and Hideyo Noguchi. 623
inactivation may be found in the gradual changes taking place in
the salt content of the serum within the sac during dialysis. As
may be expected, sodium chlorid passes out of the celloidin sac
with a greater velocity than the remaining salts, such as carbonates
or phosphates, and this together with an increase in volimie (by
endosmosis of water) results in removing the restraining power of
the chlorids upon the dissociation of the carbonates. These salts
then inactivate the complement.
A study of the comparative velocity of osmosis of sodium chlorid
and NajCOs through a celloidin membrane is given in table XVI.
At the time indicated, five cubic centimeters of the contents of the
collodion sac were removed and examined for sodium chlorid and
sodium bicarbonate.
SUMMARY.
It is generally accepted that complement may be split into a mid-
piece and an end-piece. The mid-piece is thought to be in the
globulin fraction, and the end-piece in the albumin fraction. The
restoration of complement activity by putting together the albimiin
and globulin fractions does not prove, however, that each fraction
contained a part of the complement, for the albumin fraction can be
reactivated in the absence of the globulin fraction.
Complement-splitting as brought about by hydrochloric acid,
carbon dioxid, and dialysis, is really an inactivation of the whole
complement by certain acids or alkalis, either added in the free
state to the serum, or liberated as a result of the dissociation of
certain electrolytes.
That the whole complement, and not a part only, is present in the
albumin fraction of the serimi can be demonstrated by the removal
of the inhibitory action of the acid or alkali. This can be effected
by the addition, not only of alkali or acid, but also of any amphoteric
substance. When hydrochloric acid, carbon dioxid, or dialysis are
employed to produce the phenomenon known as complement-split-
ting, the complement is merely inactivated, not split.
BIBLIOGRAPHY.
Amako, T., Ztschr. /. Immunitdtsforsch., 1910, viii, 168.
Bernhardt, Wien. klin. Wchnschr., 191 1, xxiv, 606.
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624 Biochemical Study of Complement'Splitting.
Bordet, J., Ann, de I'Inst, Pasteur, 189^ x, 193; iSgS, xii, 688.
Brand, E., BerL klin, Wchnschr,, 1907, xliv, 1075.
Buchner, H., Arch, f, Hyg,, 1893, xvii, 138.
Buchner, H., and Orthcnberger, M., Arch, f, Hyg,, 1890, x, 149.
Ehrlich, P., and Morgenroth, J., Berl, klin. Wchnschr,, 1899^ xxxvi, 481.
Ferrata, A., BerU klin. Wchnschr., 1907, xliv, 366.
Frankel, E., Ztschr. f. Immunitdtsforsch., 191 1, viii, 781; 191 1, x, 388.
Hecker, R^ Arb, a, d, k, Inst, f, exper, Therap,, 1907, No. 3, 39.
Guggenheimer, H., Ztschr, f, Immunitdtsforsch,, 191 1, viii, 29s
Kiss, J., Ztschr, f, Immunitdtsforsch,, 1909-10^ iv, 703; 190ft iii, 558.
Landstciner, Centralhl, f, Bakteriol,, Ref., 1910, xlvii, SuppU 88.
von Liebermann, L., Arch, f, Hyg,, 1907, Ixii, 306 ; Biochem, Ztschr,, 1907, iv, 2S
Liefmann, H., Centralbl, f, Bakteriol,, Ref,, 1910, xlvii, Suppl., 87; Munchen.
med. Wchnschr., 1909, Ivi, 2097.
Liefmann, H., and G>hn, M., Ztschr. f. Immunitdtsforsch., 1910, vi, 562; 1910,
vii, 669; 191a viii, 58.
Markl, Ztschr. f. Hyg,, 1902, xxxix, 86.
Marks, H. K., Ztschr, f, Immunitdtsforsch,, 1910-11, viii, 508; 1911, xi, 18;
Jour, Exper, Med,, 191 1, xiii, 590.
Meyer, K., Arch. f. Hyg., 1908, Ixvii, 114.
Michaelis, L., and Skwirsky, P., Ztschr, f, Immunitdtsforsch., 190^10, iv, 357,
629; Berl, klin. Wchnschr,, igio, xlvii, 139.
Mutermilch, S., Compt, rend. Soc. de hiol., 191 1, Ixx, 577.
Nogachi, H., Proc, Soc. Exper, Biol, and Med,, 1907, iv, 45, 157; Biochem,
Ztschr., 1907, vi, 327.
Noguchi, H., and Bronfenbrenner, J., Jour. Exper. Med,, 191 1, xiii, 43.
Rusznyik, S., Ztschr. f, Immunitdtsforsch,, 1910-11, viii, 421.
Sachs, H., Centralhl, f, Bakteriol,, Ref,, 1908, xiii, Suppl., 174.
Sachs, H., and Altmann, K., Kraus and Levaditi's Handbuch der Technikund
Methodik der Immtmitatsforschung, Jena, 1909, ii, 965; Berl, klin,
Wchnschr,, 1908, xlv, 699.
Sachs, H., and Bolkowska, G., Ztschr, f, Immunitdtsforsch,, 1910, vii, 778.
Sachs, H., and Teruuchi, Y., Berl. klin, Wchnschr,, 1907, xliv, 467, S20, 602.
Skwirsky, P., Ztschr, f. Immunitdtsforsch., 1910, v, 538.
Tsurusaki, H., Biochem. Ztschr., 1908* x, 345-
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preprinted from the JoimsAL of Expbrimbntal Mbdionb, Vol. XV. No. 6, 191 a.]
A BIOCHEMICAL STUDY OF THE PHENOMENA
KNOWN AS COMPLEMENT-SPLITTING.
Second Paper: Sputting of the Complement without a
Visible Alteration of the Proteid Constituents.*
By JACOB BRONFENBRENNER and HIDEYO NOGUCHI.
(From the Laboratories of The Rockefeller Institute for Medical Research,
and the Laboratory of Biological Chemistry of Columbia University,
New York,)
In the preceding communication we have dealt with various phe-
nomena of complement-splitting in which the globulin fraction of the
serum was precipitated and subsequently separated from the albumin
fraction. In all these processes, according to current conceptions,
the globulin fractions contained the mid-piece of complement, and
the albumin fractions, the end-piece. Yet, as has already been
pointed out, the so-called end-piece of the albumin portion depends
in no way upon the introduction of the mid-piece in order to regain
its original complement property, but can be reactivated by any
reagent carrying the ions opposite in sign to those keeping the active
atom-complex of complement temporarily inactive. The precipita-
tion of the globulin fraction, therefore, was not essential in splitting
the complement, but was merely an associated phenomenon. The
correctness of the above deduction will be established by the experi-
ments recorded in the following pages.
The so-called splitting of complement may be effected by certain
procedures without causing the separation of the globulin fraction
of complement. In one of these procedures, distilled water in cer-
tain proportions is used, and in another serum is treated with phos-
phates. The inactivation of complement by distilled water is doubt-
less due to the action upon the complement of dissociated ions of
electrolytes contained in the serum, and the inactivation by a mix-
♦ Received for publication, April i, 1912.
625
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626 Biochemical Study of Complement'Splitting.
ture of acid and alkaline phosphates (the acid being in excess)
seems similar to the action of any other acid, but the presence of an
alkaline phosphate modifies the reaction of the medium in such a
manner that the globulin is kept in solution.
Although the phenomena of splitting complement as brought
about by these chemical or physical interferences appear to be simi-
lar in mechanism to those effected by complement fixation, the
complex biological reactions of the latter are difficult to analyze,
and will be left for future study.
Under separate headings, we shall give the results of the other
ways of splitting complement.
COMPLEMENT-SPLITTING WITH WATER.
Complement, as long as it is handled with an isotonic solution of
sodium chlorid, retains its activity on dilution. On the other hand,
complement rapidly loses its activity if it is diluted to a certain
extent with an isotonic sugar (saccharose) solution, as was first
pointed out by Sachs and Teruuchi. Thus these authors found that
complement becomes completely inactive within one hour when
diluted ten times with an isotonic sugar solution. The inactivation
was considered by them to be due to a ferment which in a sodium
chlorid- free mediimi manifests its activity upon complement.^
The above phenomenon appears, however, to be caused by some
other factors than a ferment. In the case of inactivation by means
of distilled water we must consider first what occurs if a solution
containing two different salts of varying dissociabilities are diluted
ten or more times with water. In the original concentration, the
stronger electrolyte hinders the dissociation of the weaker, but when
sufficiently diluted the dominating salt permits the weaker elec-
trolyte to dissociate more fully. Sodiimi chlorid is a much stronger
electrolyte than NaHCOg or NagCOg and it doubtless interferes
with the free dissociation of the latter salts in the concentration
present in serum; but when diluted ten times, its concentration no
longer hinders the free dissociation of the carbonates. Thus, it
becomes evident that the complement which may remain unaffected
* Bronfenbrenner, J., and Noguchi, H,, Jour. Exper, Med,, 1912, xv, 598
(tables XIV and XV, A).
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Jacob Bronfenbrenner cmd Hideyo Noguchi.
«27
by the carbonates in the presence of sodium chlorid in the original
concentration will be exposed to the action of the latter as soon as
the sodium chlorid concentration is much less, and this is exactly
what happens when serum is diluted with water. This, of course,
will not occur as long as a sufficient amount of the chlorid is con-
tained in the diluent (table I). That the change in the tonicity of
the mixture (serum and diluent) has nothing to do with the loss of
complement activity is shown by the fact that the use of an isotonic
solution of a non-electrolyte, such as cane sugar, does not prevent
the inactivation of complement.
We shall next see if the inactivation of complement by dilution
with water or cane sugar solution is really an alkali inactivation.
That this is the case is shown by the fact that an acid and also a
few amphoteric substances may reactivate the inactive complement*
TABLE !•
The InacHvation of Complement in a Salt-Free Isotonic Medium,
Complement diluted to 40 ^ in 0.9 ^
Complement diluted to 40 < with y.a i
sugar and incubated at 37** C. for x hr.
NaCl and incubated at 37° C. for x hr.
o.a C.C. or this dilution is used with vary-
0.3 C.C. of this dilution is used with vary-
ing amounu of 7.2 f sugar.
ing amounts of 0.9 f NaCl.
Totol volume made equal to 1 c.c. with
0.9 f NaCl.
7 a i^ sugar.
s
0.1
C.H.
NoH.
I'J
o.is
C.H.
S1.H.
0.2
C.H.
C.H.
§ll.
0.3
C.H.
C.H.
arying
of SU]
NaCl
0.4
0.$
C.H.
C.H.
C.H.
C.H.
0.7
C.H.
C.H.
>
[icc.
C.H.
C.H.
COMPLEMENT-SPLITTING BY MEANS OF PHOSPHATES.
Michaelis and Skwirsky described a method by which comple-
ment can be split into end- and mid-pieces. In this method one
cubic centimeter of complement (i:io) is mixed with two cubic
centimeters of mixture A-B, which consists of sixteen parts of M/7
NaH2P04 (solution A) and one part of M/7 Na2HP04 (solution
B), and to this are added one cubic centimeter of amboceptor (fifty
* Bronfenbrenner, J., and Noguchi, H., loc, cit., tables XIV and XV.
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628
BioehenUcdl Study of Complement-SpUtting,
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Jacob Bronfenbrenner and Hideyo Noguchi.
629
units) and one cubic centimeter of 5 per cent sheep corpuscles.
After incubation for one hour at 37° C. the mixture is centrifu-
galized and the supernatant fluid is separated from the deposited
corpuscles. To the supernatant fluid, two cubic centimeters of solu-
tion B are added and the whole is called the end-piece. The cor-
puscles are suspended in salt solution and are used as persensitized
corpuscles (sensitized and carrying the mid-piece of complement).
When the persensitized corpuscles are mixed with the end-piece
they undergo hemolysis, while the sensitized corpuscles are not acted
upon by the end-piece.
The above phenomenon is probably explained by the fact that
complement is highly sensitive to the action of various acids, alkalis,
and salts, and undergoes a reversible inactivation. It is not at all
improbable, therefore, that the mixture of solutions A and B, which
is strongly acid to phenolphthalein, renders the complement inactive,
while the persensitization goes on without hinderance in the same
medium. The acidity of the supernatant fluid is, in this instance,
to a certain degree reduced by the later addition of solution B so
that the inactivated complement, which is near the border of rever-
TABLE IIL
Reaction and Anticomplementary Activity of Different Mixtures of Phosphates.
Reaction of z cc.
Anticomplementary
activity upon 0.04 cc of
complement + a units of
amboceptor.
IndigCMdizarin.
Pbenolphthaleiii.
Complete
hemolytic
dose.
Non-hemo-
lytic dose.
Solution A.
27.4 gm. NaHtP04 per z.ooo
cc
Add
+0.4 CC
NaOH N/ioo
Acid
24 cc
NaOH N/ioo
0.07 CC
0.2 CC
Solution B.
51.4 gm. NasHPOi per z.ooo
cc
Alkali
15 cc
HCl N/ioo
Alkali
0.7 cc
HCl N/IOO
0.5 CC
I CC
Mixture A -B.
x6 cc A+i cc B.
Alkali
0.4s cc
HCl N/ioo
Add
22 cc
NaOH N/IOO
O.I CC
0.25 CC
NeutXBlized Mixture.
X ccA-B+i cc B.
1
0.5 CC I cc
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680
Biochemical Study of Complement-Splitting.
sion, regains its activity whenever an adequate quantity of certain
alkali (table IV) or amphoteric compounds is introduced (table V).
TABLE IV.
Reactivation of Complement Inactivated by Phosphates.
NaOHN/iincc
' ■ ^
a I 0.07 0.05 ao3 0.02 0.01 aoo5 0.001 0.0007 0.0005
Complement* 0.04 cc. '^ Alkali hemolysis.
-f27.4/i,oooNaH,PO^ I
0.2 C.C. in each tube+ [
3 units of amboceptor. J C.H.
Zone neutral to
phenolphthalein.
C.H. C.H. CH. NoH. No H. No H.
HClN/iincc
Zone neutral to alizarin-
indigo-carmin.
0.2 0.1 0.07 0.05 0.03 0.0275 0.025 0.02 o.oi 0.007 0.005
Complement* 0.04 cc
+51.4/ 1,000 Na,HPO^
I cc in each tube -f
3 units ofambocq)tor.
Add he-
molysis.
NoH. S1.H. CH. SI. H. NoH. NoH. NoH. NoH. NoH.
Zone neutral to all-
sariU'indigo-carmin.
Zone neutral
to phenol-
phthalein.
From the foregoing experiments it will be seen that the inactivated
complement is to be found in the supernatant fluid in a reversible
state. The reversion is accomplished by relieving the complement
of the acid reaction by any suitable substance. The reason that the
corpuscles digested in the Michaelis-Skwirsky mixture are capable
of reversing the inactivated complement may be due to the absorp-
tion of a certain amount of serum constituents (probably the globu-
lin fraction), also the absorption of the alkaline phosphate by
certain constituents of the cells. It appears from the following
experiments that the presence of amboceptor has a certain influence
in enabling the corpuscles to absorb them, because without the am-
boceptor the persensitization is seen to be less complete (table VI).
That the end-piece of complement as obtained by the method of
Michaelis and Skwirsky is in a general way similar to those obtained
by other processes is well shown in the following experiments. It
is most striking that alanin plays beautifully the role of the mid-
piece with all end-pieces here studied (table VII, C).
• The complement was incubated with the phosphates at 37* C. for 30 minutes,
then alkali and acid, respectively, were added and the whole was reincubated
for 30 minutes.
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^acoh Bronfenbreimer and Hideyo Noguchi.
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682
Btochemical Study of Complement-Splitting.
s
<
o
oj
S
o
U
at
>o
Complement
(i:io) 0.2 c.c
Mixture A-B o
Amboceptor o
Corpuscles
(S%) 0.2 cc
NaCl (0.9%) 0.6 c.c
u
*n
Complement
(1:10) 0.2 cc.
Mixture A-B 0.4 cc
Amboceptor
(20 units) 0.2 c.c
NaCl (0.9%) 0.2 cc
1
^
Complement
(1:10) 0.2 cc
Mixture A-B 0.4 cc.
NaCl 0.9% 0.2 cc
Corpuscles
(5%) 0.2 cc
1
§
1
CO
0.9% NaCl 0.2 cc
Mixture A-B 0.4 c.c.
Amboceptor
(20 units) 0.2 cc
Corpuscles
(S%) 0.2 cc
t
1
c«
Heated comple-
ment (1:10) 0.2 c.c.
Mixture A-B 0.4 c.c
Amboceptor
(20 units) 0.2 cc
Corpuscles
(5%) 0.2 cc
G
0)
i
•a
1
H
Complement
(I :io) 0.2 cc
Mixture A-B 0.4 cc.
Amboceptor
(20 units) 0.2 cc
Corpuscles
(5%) 0.2 cc
1
1
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Jacob Bronfenbrenner and Hideyo Nogv^Kx.
683
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Bio^emical Study of Complemeni-SpUUing.
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Jacob Bronfenbrenner and Hideyo Nogicchi. 68&
COMPLEMENT-SPLITTING IN COMPLEMENT DEVIATION PHENOMENA.
Much more complicated is the phenomenon first described by
Skwirsky, who found that the supernatant fluid of the mixture con*
taining a positive syphilitic serum, syphilitic antigen, complement,
sheep's corpuscles, and antisheep amboceptor, has the power to cause
hemolysis upon a fresh lot of sheep corpuscles when the latter are
previously persensitized. By the term of persensitization is meant
the digestion of washed sheep corpuscles in a mixture of sixteen
TABLE VIII.
Action of Mixture A-B upon the Substances Concerned in the Wc^sermann
Reaction,
'Syphilitic serum
(heated at 56^ C.) 0.1 c.c.
Antigen (o,^ %) a5 cc
(lipoids)
Complement (i:io) 0.5 cc.
NaCI(o.9j|^) 0.4 cc
>
Each of the tubes contained this mixture ( 1.5 cc ) The-
tubes were incubated at 37^ C. for 30 min. then vary-
ing amounts of A-B mixture were added and the volume
was brought up to 2*5 c.c. with NaCl (0.9 %). After
the second incubation for 30 min. at 37^ C., 0.5 cc. of
5 ^ sheep corpuscles and 50 units of amboceptor were
I ^ added and the whole was again incubated for 30 min.
1.5 cc J at 370 C.
Varying amounts of
the mixture A-B. I cc a7c.c. 0.5C.C 0.3C.C. 0.2c. c o.icc 0.07c c 0.05c. c o
Result of the hemo-
lytic test No H. No H. No H. No H. No H. No H. No H. No H. No H.