CHEMO-IMMUNOLOGICAL STUDIES ON CONJUGATED
         CARBOHYDRATE-PROTEINS

III. ACTIVE AND PASSIVE AN- WITH %NTH3ZTIC UGAR-
                PROTEINS  I/'
   BY WIT&TAM S. TIUETT, M.D., OSWALD T. AVERY, M.D.,.
          AND WALTDER F. GOEBEL, PH.D.
(From tie Bos$itd of The Rockefeller Institute fur Medical Research)
      (Received for publication, July l&1929)
In the two preceding papirs of this series Avery and Goebel (1,2)
have reported the results of chemo-immunological studies on con-
jugated carbohydrate-proteins.
In the first of these communications Goebel and Avery (1) described the chemi-
cal methods by means of `which the p-aminophenol ghxosides were synthesized from
glucose and galactose and the dia&aed substances bound to protein.  The two
newly synthesized sugar-proteins d&r from one another chemically only in
specific rotation and molecular co~tion. In the second paper, Avery and
Goebel(2) reported the antigenic properties and serological specifkity of the con-
jugated carbohydrates. They found that the glucosidic radical of the compound
antigen endowed the new complex with specific reactivity, This was demon-
strated in two way& Fit, .when the same glucoside is attached to two d@r&
proteins, as serum' globulin or egg albumin, the serum prepared by immuniaation
with either one of the antigens is spechically reactive with the other.  Second,
when the two d$rtwt glucosides,-glucoside and gaktosideke each com-
bmed with the iawe protein, the newly formed compounds are serologically dis-
tinct. These facts were found to be true even though the individual glucosides
are isomers differing only in the spatial con&ration of a single carbon atom.
On the other hand the kombiued glucosides alone were found to be non-
antigenic. They failed to induce antibody formation in the anhnal body and
caused no visible precipitation. when added, to immune sera +c vitro.  However,
both glucosides possess the capacity of specifically inhibiting the precipitating
action of homologous antisugar?protein serum. When galactoside was mired with
serum prepared by immuniaation with galacto-globulin,l the subsequent addition
l The terms'employed in this paRer to represent the sugar-protein compounds
are the same as those .used by Avery and Goebel(2).
                      551


552   ' CONJUGATED CARBOHYDRATE-PROTEINS. III

of gala&-albumin to the mixture did not cause precipitation.  Furthermore, when
the heterologous glucoside was substituted in the same system, no inhibition of the
precipitin reaction occurred. These results demonstrate the specificity of the
inhibition phenomenon.  Avery and Goebel (2) considered that the uncombined
glucosides possess the immunological properties of haptens.

The r81e of carbohydrate in auaphylaxis has been a subject of
recent experimental investigation.

Tomcsik (3) working with B. lactis aerogenes, and later Tomcsik and Kurotch-
kin (4) employing B. lactis aerogenes, the pneumobacihus, and a yeast, isolated
carbohydrate substances which produced auaphylactic shock in guinea pigs
passively sensitized with homologous immune serum. Lancefield (5) also ob-
tained from streptococci carbohydrate material with which anaphylaxis could be
induced in guinea pigs passively sensitized with antM.reptococcus serum.  Be-
cause of the presence of small amounts of nitrogen in the products, none of these
authors felt justified in concluding that the carbohydrate alone was responsible
for the shock. Avery and Tiiett (6) employing the highly purified polysaccharide
of the type-specific pneumococci showed that guinea pigs passively sensitized with
homologous anti-pneumococcus rabbit serum were thrown into anaphylactic
shock by the subsequent injection of the homologous specific carbohydrate.
Guinea pigs could not, however, be actively sensitized with the purified poly-
saccharides alone.  Since the materials used in those experiments were protein-
free, and in the csse of the Type II and Type III substances also nitrogen free,
the results conclusively demonstrate the capacity of complex;sugars to induce
anaphylactic shock in animals passively sensitized with antibacterial sera.    .

The immunological specificity of pneumococcus polysaccharides
has a close anaIogue in the serological specificity exhibited by gluco-
protein and galacto-protein.  The immunologic behavior of the syu-
thesized sugar-proteins led to their use in sensitization experiments.
The results, reported in this paper, on anaphylaxis with artifkially
prepared carbohydrate-proteins confkn and extend the serological
tindings previously reported. The production of both active and
passive anaphylaxis was attempted in order to determine the sensitiz-
ing properties of the synthetic antigens and to demonstrate the speci-
ficity of the reactions.

    Gluco-globulin represents phenol &glucoside-azo-globulin.
    Gluco-albumin represents phenol 8-glucoside-azo-albumin.
    Galacto-globulin represents phenol fi-galactoside-azo-globuliri.
    Galacto-albumin represents phenol &galactoside-aso-albumin.
The globulin was prepared from horse serum, and the albumin from egg white.


W. S. TILLETT, 0. T. AVERY, AND W. F. GOEBEL    553

EXPERIMENTAL

Guinea pigs weighing 240 to 275 grams were employed. The sensitiaiig dose,
whether serum or sugar-protein, was always injected hrtraperitoneally. The
&ding dose was unifody injected intravenously into a superficial vein of the
hind leg.

For details concerning the chemical procedures involved in the synthesis of the
materials, the reader is referred to the article by Goebel and Avery (1).
The serological characteristics of the serum employed and the method of prepara-
tion are described by Avery and Goebel(2).

Pas&e Sensitizatio92

A. Results obtained with sugar-pro&h compounds

Eight guinea pigs were injected intraperitoneally with the pooled serum of three
rabbits immunized with gluco-globulin. Five guhxa pigs each received 5 cc. of
serum, one received 3 cc., one received 1 cc., and one 0.5 cc. As previously men-
tioned) serum of this character possesses the capacity to precipitate complex anti-
gens composed of heterologous protein conjugated with the homologous glucoside.
The antigluco-globulin sera used in these experiments had an average titre of
specific precipitins, as determined by tests made with gluco-egg-albumin, of 1 to
80,000. By reason of the fact that horse globulin alone when used as antigen does
not elicit antibodies reactive with egg-albumin, the high precipitm titre of these
sera is obviously dependent upon the conjugated glucoside radical.

Twenty-four hours after the administration of gluco-globulin antiserutqeach
pig received intravenously 1 cc. of gluco-albumin.

From Table I it can be seen that the five pigs sensitized with 5 cc.
of serum all died with typical symptoms of anaphylactic shock.
The animals passively sensitized with 3 cc. and 1 cc., respectively,
of antigluco-globulin sennn had definite and typical symptoms imme-
diately following the injection of 1 cc. of gluco-albumin but recovered.
Pig No. 6, which received 0.5 cc. of serum, exhibited only a slight
reaction.

A similar experiment was carried out using antigalacto-globulin
squm for sensitization and galacto-albumin as the toxigenic antigen.

Sera obtained from three rabbits immuniaed with gab&o-globulin were pooled;
the precipitin titre, as determined with galacto-albumin, averaged 1 to 80,000.
Eight guinea pigs were injected intraperitoneally as follows: 5 animals received
5 cc. of serum each, one received 3 cc., one received 1 cc., and one, 0.5 cc.  The
results given in Table II are equally as definite as those shown in Table I.  All


554

CONJUGATED CARBOHYDRATE-PROTEINS. III

                    4
             TABLE I

   Passive ~naphylaxis with Anti-GElu;o-Glob@~ Serum
Reactiahs induced by the use of the homologous glucoside conjugated with a

deterologous protein-Gluco-Albunk

Guinea pirr No.

5

6

i

Sameanimal
brs. later

8 -'

Same animal 4
hrs. later

~!z!ir
serum i.p.

CG.

5
5
5
3

1

0.5

5

5

hrs.

24
24
24
24
1

24

24

24

24

ihm5?
   . .

`$g/fg
1 cc.
1 cc.
1 cc.
1 cc.

1 cc.

1 cc.

laJJ$S
1 cc.

~~
-0Ti-J
Marked scratcb-
ing, bucking,
coughing, ret@-
story distress
Violent typical
Symptoms

0txasi0na1
scRatchillg

t3f Itlhltes
t31 minute3
t2) minutes
Definite symp-
toms followed
by recovery

Definite symp
toms followed
by recovery
No reaction

None         No reaction

Typical

t4 minutes

None        No reaction

Typical
/
  I

t4+ minutes

t Death of animal.
i.p. = intraperitoneal.
i.v. = intravenous.


     W. S. TILLETT, 0. T. AVERY, AND W. F. GOEBEL    555

pigs sen&ized with homologous immune serum in amounts from 1 to 5 cc. reacted
typically and fatally to the intravpous injection of 1 cc. of galacto-albumin;
0.5 CC. of serum was indicient to sensitize.   a

                       TABLE II

     Passive Amphylak with Ad-GUzcto-GZvb Serum
Reactions induced by the use of the homologous galactoside combined with a

Guinea pis No.

1
2
`3
4
5
6

7

SamCanimdl
hr. later

8       5      24

Sameanima.ll
hr. later

h&rologous 1

cc.

5
5
5
3
1
0.5

5

h.

24
24
24
24
24
24

24

protein-Gal&t
=

"9
   . .

`p&y

1 cc.
1 cc.
1 cc.
1 cc.
1 cc.
1 cc.

IL-g-
1 cc.

`dado-tsg-
dbmir
1 cc.

d$f+@&s-
1 cc.

Ey?g-
1 cc.

Result

gz
~~
Typical
slight scratching
and coughing

t3 minutes
t8 minutes
t3+ minutes
t3 minutes
t3) minutes
Very mild reac-
tion with re-
covery

None         No reaction

Typical

t31 minutes

None        No reaction

p mhlutt?s

tDeath of animal.
i.p. = intraperitoneal.
i.v. = intravenous.

In Tables I apd II it is also &own that the reactions in guinea pigs
induced with sugar-proteins a&l anti-sera are strictly specific.


556      COVJUGATED CAXBOEIYDFL~TE-PROTEINS. In

Animals No. 7 and No. 8 of Table I received antigluco-globulin serum and 24
hours later were injected intravenously with galacto-albumin. No reaction
occurred.  Four hours later, the introduction of gluco-albumin induced typical
fatal shock. Similarly pigs No. 7 and No. 8 of Table II, sensitized with anti-
gslacto-globulin serum, were unharmed by gluco-albumin; the subsequent ad-
ministration of the homologous galacto-albumin antigen caused anaphylactic
death of these animals. The animals also serve to demonstrate the fact that gluco-
albumin and galacto-albumin are not primarily toxic.

B. Results obtaiwd with uncombined glucosides

The specific inhibitory effect exerted by the glucosides on tb
precipitin reaction of sugar-protein and anti-sera has been describe&
in detail by Avery and Goebel'(2) and has been previously commented
upon in this paper.  It, therefore, seemed of interest to determine
whether anaphylactic shock could be elicited by glucosides alone in
passively sensitized guinea pigs, and if not, whether the inhibition
which these substtinces have on the precipitin test would also be
evident in the anaphylactic reaction.

As shown in Table III, two pigs (Nos. 1 and 2), sensitized 24 houm.previously
with antigluco-globulin serum, were injected intravenously with 1 cc. of the un-
combined homologous glucoside.  No reaction occurred.  Two hours later 1 cc. of
gluco-albumin injected into the same animal caused prompt anaphylactic death.
In guinea pigs Nos. 3,4, and 5, the introduction of glucoside alone was followed
immediately by an injection of gluco-albumin.  Except for slight scratching, no
response was elicited.

From these results it may be seen that the injection of glucoside
into a sensitized animal exerts a definite but transitory protection
against the shocking capacity of material which otherwise would be
fatal. That the protective action of the glucoside is specific is demon-
strated by guinea pigs Nos. 6, 7, and 8.  These animals were injected
with the heterologous galactoside; when, immediately thereafter they
were given gluco-albumin no protection resulted and they died
promptly with typical anaphylactic shock.

Table IV presents the results obtained with guinea pigs, which,
after having been sensitized with antigalacto-globulin serum, were
protected by the galactoside from the toxigenic effect of galacto-
albumin.


Guines
Pig No.

      Interval
hti-   between
lUCO-
d     injection
obulin  of Serum
acrum   and ma-
i.p.   phylactk
       test
--

G.     h.

s     24
      24
; 24

!I     24

5     24

--

s     24

5     24

5     24

TABLE III

Passive Anafihylads with Anti-Gluco-Globdin Serum
     Effect of Uncombined Glucoside

3y$Yf
  . .

gfwcosi;ir

1 cc.
1 cc.
1 cc.

1 cc.

1 cc.

ga&dosfde
1 cc.

1 cc.

1 cc.

Result

No reaction
No reaction
No reaction

No reaction

No reaction

`No reaction

No reaction

No reaction

xaterpal betwcon'
injection of
&os& and sugar-
  protein

hn.

    2
    2
Followed inune-
diately by
Followed bnrne-
diately by
Followed imroe-
diat&ly by

Follow@ imme-
diately by
Followed inune-
diatoly by
Followed imme-
diately by

rf+J:
1 cc.
1 cc.
1 cc.

1 cc.

1 cc.

1 cc.

1 cc.

1 cc.

symp-

Scratches slightly, no
other symptoms
Scritches slightly, no
other symptoms
Scratches slightly, no
other symptoms

Typical

Typical

Severe symptoms, falh
on side.  Apn-,
on feet

Result

t4 minutes.
14 minutes
No reaction

No reaction

No reaction

t7) minutes

t3) minutes

Severe shock followed
by recovery

t De& of auimal.
i.p. = intqeritoneal.
i.v. = intravenous.


      Allti-
Guinea  galacto-
Pll No.  elobulin
      sylm
       `.h

--

        cc.

7' 5

8     5

Passive Anaphylaxis vith A~i-Gdacto-Gl&&n Scwm
    Effect of Uncombined Galactoside

hts.   &tur0~~

24 1 cc.

24    1 cc.
24    1 cc.
24    1 cc.

24    1 cc.

24    1 cc.

--
     glum*
24    1 cc.

24    1 cc.

Result

No reaction

No reaction
No reaction
No reaction

No reaction

No reaction

No reaction

No reaction

Interval between
injcctlon of slucoEfdc
and sugar-protein

h.

2

   4
   23
Followed immedi-
ately by
Followed immedi-
ately by
Followed immedi-
ately by.

Followed immedi-
ately by
Followed immedi-
ately by

`*F
1 cc.

1 cc.
1 cc.
1 cc.

1 cc.

1 cc.

1 cc.

1 cc.

SymPto=J

Marked scratching,
wughing, baclc-
ing, respiratory
distress
Typical
Typical
None

Typical

w-1

    Resulr    . 8
   4
Definite symptoms  8
followed by re- c)
covery    is
t4 minutes          0
t31 minutes
No reaction   8

No reaction

No reaction   g
        8
      3
                .
f2f minutes     Ef
t3mitultes

t Death of animal.
i.p. = intraperitoneal.
i.v. = intravenous.


TABLE V

Desmsitithon Induced in Passively Sensitized Cuinecr Pigs by Injedim oi Glucoside and Homologous Sugar-Prokin

.-

Guiaea
Pi No.

. 1

2

3

5   cc.  anti-gluco-
, globulin serum

5 cc. anti-gluco-
globulin serum

5 cc. anti-gluco-
globulin serum

5 cc. anti-galacto-
globulin serum ,

5 cc. anti-galacto-
globulin serum

5 cc. anti-galacta-
globulin serum

i.p. - intraperitoneal.
i.v. = intravenous.

   prevhs teats.
Injectiona made 24 ha. after
   7?
       . .

1 cc. glucoside followed im-
mediately by 1 cc. gluco-
egg-albumin
1 cc. glucoside followed im-
mediately by 1 cc. gluco-
egg-albumin
1 cc. glucoside followed fm-
mediately by 1 cc. gluco-
egg-albumin

1 cc. galactoside followed
lmmediate~y by 1 cc.
galac~egg-albumin
1 cc. gahctoside followed
immediately by 1 cc.
gala&-egg-albumin
1 cc. galactide followed
immediately by 1 cc.
galacto-egg-albumin

Reaalt

No reaction

No reaction

No reaction

No reaction

No reaction

No reaction

1 cc. gluco-egg-
albumfn

1 cc. gluco-egg-
albumin

1 cc. gluco-egg-
albumin

1 cc. gala&o-egg
albumin

1 cc. `&W-egg-
albumin

1 cc. gelactQ-@g-
albumin

None   No reaction

None   No reaction

None   No reaction

None   No reaction

None   No reaction

None   No reaction

Result

4
"

i
P


560 *     CONJUGATED CARJ3OBXDRATE-PROTEW. m

The results are, in every respect, identical with those given in
Table III both with regard to the transitory nature of the phenomenon
and to its specificity.

The mechanism of the protection afforded by the glucosides is not
as yet understood. The fact that the protective effect is no longer
demonstrable after two hours indicates that "desensitization,"-if such
has occurred-is transitory. Instances of what appears to be true
desensitization have been observed in these experiments and are
recorded in Table V.

Guinea pigs Nos. 1, 2, and 3, passively sensitized with antigluco-globulin
serum, were protected from the shocking e&t of 1 cc. of gluco-albumin by a pre-
vious injection of homQlogous glucoside.  Four and one-half hours later the same
pigs received a second injection of 1 cc. of &co-albumin.  No reaction occurred.
The absence of shock following the second injection of khole antigen seems to be
dependent upon the first dose of gluco-albumin. That the glucoside alone plays no
direct part in the refr~tory state is demonstrated by its ineffectiveness when in-
jected singly, two hours prior to the shocking dose (Table III). Pigs Nos. 4,5, and
6 of Table V demonstrate the same principle, the difference being that antigalacto-
globnlin serum was used for sensitization, and galactoside and galacto-albumin
were empIoyed to complete the test.

Avery and Goebel(2) have shown that the serum of rabbits immu-
nized with synthetic sugar-proteins (gluco- or galacto-globulin) pos-
sesses two distinct antibodies; 1) the specifk precipitin so intimately
associated with the carbohydrate radical of the compound; 2) the
"common" precipitin, reactive with globulin alone. Passive ana-
phylaxis experiments were, therefore, carried out, using pure horse
globulin as the toxigenic material.

For sensitization, one pig received intraperitoneally 1 cc. of senmr prepared by
immunization with gluco-globulin; a second pig received 1 cc. of serum derived
from a rabbit immunized with galacto-globulin.  Twenty-four hours later each
received 12 mgms. of globulin. Both animals diedin typical anaphylactic shock
(Table VI). A normal pig receiving the same dose of globulin gave no reaction.


W. S. TILLETT, 0. T. AVERY, AND W. F. GOEBEL    561

TABLE VI

Passive Ana#tylaxis with A&Glum-Globulin ad Atrti-Cahcto-Globuh SW~
       Reactions induced by the use of horse globulin

Guinea
pig No.

1   1 cc. anti-gluco-
   globulin serum
2   1 cc. anti-galacto-
   globulin serum
3   Normal control

Time
interval
.-
  h.
  24

24

-

Sho$iy dose
   . .

1 cc. horse-globulin
(12 mpms.)
1 cc. horse-globulin

1 cc. horse-globulin

Typical

Typical

None

Result

t3 minutes

t2* minutes

No reaction

t Death of animal.
i.p. = intraperitoneal,
i.v. = intravenous.

Active Amzphylaxis

For purposes of testing the capacity of the synthetic sugar-proteins
to produce active sensitization, 10 guinea pigs were injected intra-
peritoneally with 5 cc. of glum-globulin and 10 other animals were
similarly inoculated with 5 cc. of galacto-globulin.

In the preparations empIoyed, 5 cc. of sugar-globulin contained 50 mgms. of
protein. It is estimated (1) that 15 per cent by weight of this complex represents
chemically combined giucoside.  Consequently each pig received approximately
7.5 mgms. of the synthesized sugar-protein. The 20 pigs were tested 21 days later
for active sensitization. Asin the experiments on passive anaphyiaxis, the sensi-
tizing dose consisted of materiai in which the giucoside was joined to a protein
heterologous to that used for sensitiaation. As previously mentioned, this pre-
caution was taken in order to ehminate the possibility of protein-antiprotein
reactions entering into the ~izsults.

In Table VII, the results of active sensitization obtained by the use
of gluco-proteins are given.

PigsNos. 1, and 2, previously sensitised with gluco-globulin, were injected in-
travenously with 1 cc. of giuco-egg-albumin. Each promptly succumbed with
typicai symptoms. Pigs Nos. 3 and 4 of Table VII demonstrate the specitkity
of the sensitization; both of the animals when tested with 1 cc. of gala&o-albumin
showed no reaction. However, tihen, 3 hours later, 1 cc. of giuco-albumin was
introduced, they reacted fatally. Pigs Nos. 5,6,7, and 8 were used to determine
the influence of homologous uncombined glucoside on the reaction.  In these tests
the same relations were found to exist as described in the experiments on passive


TABLE VII

Active Anaphylaxis in Guinea Pigs Smitid with Gluco-Globulin

=
I

1st Injection

Interval between
1st and 2nd
injection

2nd Injection

3rd Injection

       IntWWl
Se%2tw   between
      sensitizing
i.p.    injection
       and test

GUlIlCa
Pi No.

1
2

Material
i.v.

Result

Result

ah.

-

  t3
Severe symp-
toms. Re
covery

  t3
  t24
  t44
No reaction

No reaction

--

_-

--

--

-

`iKz-
1 cc.
1 cc.

min.
::1

gf%"    days

5 cc.     21
5 cc.     21
~-

5 cc.     21
5 cc.     21

--

5 cc.     21
5 cc.     21
5 cc.     21
5 cc.     21

--

5 cc.     21

3
4

No reaction      3
No reaction      3

duco-w-
dbnmim
1 cc.
1 cc.

gkumida
1 cc.
1 cc.
1 cc.
1 cc.

l-

1 cc.

No reaction
No reaction
No reaction
No reaction

  13
   2
   2
Followed im-
mediately

No reaction      .4

1 cc.
1 cc.
1 cc.
1 cc.

"yy+;~-
1 cc.

9

t44

t Death of animal.
i.p. = iutraperitoneal.
i.v. - intravenous.


TABLE VIII

Active Anaphylazis in Guiw Pigs Sensitized with Gala&o-Globulin

-
,I

=

P

Sd.&S
i.p.

2nd Injection

~~~~       1st Injection
ensitiaiig
injection
and test  M;`:M
        . .   - .

Result

3rd Injection

Interval between
1st and 2nd
injection

min.

t34
t4

ha.

No reaction    24
No reaction      2

No rea$ion
No reaction
No reaction
No reaction

  14
   3
   3
Followed im-
medidy
by

No reaction
No rektion
-

4
4

Yti"
. .

Result

min.

t4
t3

  t4
  t3
  t2
Very   slight
symptoms.
Recovery

No reaction     1
No reaction     1

.-

  1
  2
A

  3
  4

rdodo-
ghflin
5 cc.
5 cc.

aove  `"p&?[t-
21     1 cc.
21     1 cc.
--
   `g+~-
21    -_ 1 cc.

@$&--$-
1 cc.
1 cc.

5 cc.
5 cc.

21     1 cc.
m-
     mLxhi&
21     1 cc.
21     1 cc.
21     1 cc.

5 cc.
see.
see.
5 cc.

1 dc.
1 cc.
1 cc.
1 cc.

21     1 cc.

-l-

CJw-rtTr-
albumin
1 cc.
1 cc.

    grvcotida

21     1 ck.
21     1 cc.

5 cc.
5 cc.

t Death of animal.
i.p. = intraperitoneal.
i.v. = intravenous.


564     CON JrtGATED CARBOHkDRATE-PROTEINS. III

anaphylaxis. When glucoside was injected immediately before gluco-albumin,
complete inhibition of anaphylaxis resulted.  However, when glucoside was in-
jected one and one-half to two hours before the shocking dose (Pigs Nos. $6. and
7), no protection occurred.  Pig No. 9 of Table VII is further evidence of the
specificity of active sensitization; in this a&al attempts to inhibit shock with
heterologous galactoside and to desensitize with gakto-albumin were ineffectual
since the subsequent injection of gluco-albumin produced characteristic death.

TABLE IX

Active Anaphylaxis ia Guinea Pigs Sen.sitizd with Glum-GlobuJin and
             Galacto-GloWin

Reactions induced by the use of horse globulin-the protein common to both

Guinen
pip No.

scnsi* -
   i.p

5 cc. gluco-globulin

5 cc. gluco-globulin

5cc. galacto-
globulin
5 cc. galacto-
globulin

Normal control

Time
lt.ervB

davr
21

21

21

21

antigens

Shocking dose
  i.v.

1 cc. horse-globulin
(18 mgms.>
0.5 cc. horse-glob-
ulin (9 mgmS.)
1 cc. horse-globulin
(18 mgms.)
0.5 cc. horse-
globulin
(9 mgms.)
1 cc. horse-globulin
(18 mgms.)

symptoms      Result

Typical   t33 minutes

Typical   t14 minutes

Typical   t3 minutes

Typical   t73 minutes

None

No reaction

t Death of animal.
i.p. = intraperitoneal.
i.v. = intravenous.

In Table VIII, a similar group ' of experiments was carried out
employing galacto-globulin for senkitization instead of gluco-globulin.
Galacto-albumin was the toxigenic agent; galactoside was injected
for inhibition tests. Results comparable in every respect to those
recorded in Table VII were obtained. Consequently a detailed
description need not be given.

Table IX presents t&e results obtained in guinea pigs actively
sensitized with gluco-globulin (animals Nos. 1 s&d 2) or with galacto-
globulin (Nos. 3 and 4) and subsequently injected with horse globulin.


W. S. TILLkTT, 0. T. AVERY, ANI3 W; F. GOEBEL    565

All the animals gave typical reactions. Active sensitivity in these
pigs was in all probability induced by the uncombined globulin present
in the sensitizing material.

DISCUSSION

The experiments reported in this paper demonstrate the capacity
of artificially prepared sugar-proteins to produce both active and
passive anaphylaxis.. The tests were devised and carried out in such
a manner as to emphasize the significance of the carbohydrate radical.
The fact that guinea pigs, passively sensitized with antigluco-globulin
serum, or actively sensitized with gluco-globulin, can be subsequently
shocked with gluco-albumin, demonstrates that the antigen-antibody
specificity in these instances is directly dependent upon the carbo-
hydrate fraction of the antigenic compounds.

The introduction of the sugar radical into the protein molecule
endows the new complex with a sharply defined specific antigenicity.
This fact is brought out by experiments in which galactoside was
substituted for glucoside in the preparation of sugar-proteins used
for sensitization. The same specific relations hold in the production
of anaphylaxis with galacto-proteins as that described for gluco-
proteins. Attempts to incite anaphylactic shock with heterologous
material were ineffectual. The results of the anaphylactic experi-
ments conform to the results anticipated by the serological findings
of Avery and Goebel (2).

Landsteiner (7), employing complex antigens, has reported experi-
ments on anaphylaxis of a similar character to those presented in this
report. He found that guinea pigs sensitized with one azoprotein
could be shocked by the `injection of a second compound containing
the same azo-groups attached to a different protein.

In addition to the new specificity which the carbohydrate radical
confers upon the conjugated proteins, the uncombined glucosides by
themselves also exert a detite infIuence on the reactivity of sensitized
animals. When sensitized pigs are injected with the homologous
glucoside immediately before the introduction of the toxigenic sugar-
pro;tein, they are completely protected from shock. However, the
protection afforded by the glucoside alone is apparently only transi-
tory; for, when the interval between introduction of glucoside and


566      CONPGATED CARBOHYDRATE-PROTEINS. III

shocking agent was as long as two hours, the injection of homologous
sugar-protein produced prompt and typical anaphylactic death.
That the temporary protection just mentioned is specific, was
demonstrated by the experiments in which uncombined carbohydrate
of heterologous type was shown to exert no such protective action.

The transitory, specific protection aEorded by the glucosides alone
is not yet understood. Landsteiner (7) found in experimtits on ana-
phylaxis with axoproteins that the azo-component, when injected one
hour before the conjugated azoprotein, inhibited shock. He con-
sidered that a state of anti-anaphylaxis had been induced.  In the
tests with glucosides and sugar-proteins, sufbcient evidence has not
been obtained to interpret the mechanism other than to say that the
inhibitory effect of the glucosides disappears in at least two hours.

Active and passive anaphylaxis has also been elicited with uncom-
bined globulin. Whether animals were passively sensitized with anti-
gluco-globulin or antigalacto-globulin serum, the toxigenic action of i
globulin was equally effective. Since the sera employed contained .
anti-globulin antibodies, these results were to be expected.  Guinea
pigs actively sensitized with either gluco-globulin or gala&o-globulin,
were found to be equally sensitive to uncombined globulin.  Since the
mated- used to produce active sensitization contained free globulin,
the subsequent intoxication with horse globulin is obviously based on
a simple protein- anti-protein reaction.

CONCLUSIONS

1. Guinea pigs passively .sensitized with the serum of rabbits
immunized with an artificially prepared sugar-protein (gluco-globulin)
exhibit typical anaphylactic shock when subsequently inoculated with
gluco-albumin; the serum of rabbits immunized with a second syn-
thetic sugar-protein (galacto-globulin) similarly sensitizes guinea pigs
to galacto-albumin. The reactions, in each instance, are specific and
depend for their specificity on the carbohydrate component, and not
on the protein fraction of the synthesized sugar-protein.

2. Guinea pigs actively sensitized with gluco-globulin or galacto-
globulin are similarly subject to anaphylactic shock, when injected,
after 21 days, with sugar-proteins containing carbohydrate identical
with that present in the sensitizing antigen, regardless of the kind of
protein with which it is combined.


W. S. TILLETT, 0. T. AVERY, AND W. E. GOEBEL    567

3. `Jhe unconjugated glucosides, although themselves not capable
of inducing shock, inhibit the anaphylactic reaction when injected
immediately prior to the introduction of the toxigenic sugar-protein.
The protective action of the glucosides disappears within ,two hours
after injection. In order to elicit the phenomenon, the carbohydrate
must be the same as that combined in the sugar-protein complex.

4. Anaphylactic shock may be induced by uncombined globulin in
guinea pigs passively sensitized with either antigluco-globulin serum
or antigalacto-globulm serum; globulin is similarly &ective in animals
actively sensitized with gluco-globulin or galacto-globulin.  The reac-
tions elicited by globulin alone are dependent upon the common pro-
tein present in the antigens, and exhibit only species specificity.

                   L   BIBLIOGRAPHY
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2. Avery, O.T., and Goebel, W. F., J. Exp. i&d., 1929,50, 533.
3. Tomcsik, K., &UC. Sac. Exe. Biot. and Med., 1927,24,812.
4. Tom&, K., and Kurotchkin, T. K, J. ExQ. &fed., 1928,47,379.
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