CHEMO-IMMUNOLOGTCA.L STUDIES ON CONJUGATED
          CARBOHYDRATE-PROTEINS
~11. WOLOGICAL SPECIFICITY OF ANTIGENS hEPARED BY CO&t-
   B-G a- AND ~GLUCOSIDES OP GLUCOSE WITH PROTEINS
BY OSJVALD T. AVERY, M.D., y%THEF. GOEBEL, PH.D., AND FRANK H.
   (From tke Eospitd of The Rockefh In&h& fw Medical Rmarck)
        (Received for publication, February 24,1932)
  The study of antigens prepared by chemically combining sugar
derivatives with protein has revealed the fact that carbohydrates
exert a determinin g inlluence on the immunological specificity of the
compounds of which they form a part.  In preceding papers evidence
has been presented showing that mere differences in the structural
configuration of a single carbon atom may determine the serological
specificity of two sugar derivatives identical save in this one respect.
  As previously pointed out, the p-aminophenol &glucosides of glu-
cose and galactose, differing from each other only in the interchange
h  of the H and OH groups on a single carbon atom, exhibit an individual
specificity irrespective of the protein to which they are attached
(1,2). Moreover, the specific r8Ie of the sugar radical in the reactive
part of the antigen is revealed by the fact that the glucosides alone,
unattached to protein, specifically inhibit the precipitin reaction
between the corresponding antiserum and homologous antigen.
  The inhibition of the antigen-antibody reaction by the intervention of the
specifk sugar hapten was found to occur both in vitro and in Go.  In guinea
pigs passively sensitized with the precipitating serum of immune rabbits, the fatal
anaphylactic shock which invariably f&lows the intravenous administration of
the homologous sugar-protein was completely prevented by a single prophylactic
injection of the speci& glucoside alone. The uncenjugated glucosides, although
&msdvea not capable of inducing shock, specificaJly inhibited the anaphyhxtic
reaction in sensitive animals when given immediately before the introduction of
the shock-producing antigen (3). The specifxity of the &lucosides of glucose
and galactose was demonstrated by the fact that the inhibition of the anaphylactic
reaction, like the inhibition of the precipitin test in vitro, occurred only when the
glumside used was the same as that present in the specifically reacting antigen.
                         769


770     CONJTJGiWZD CARBOHYDRATE-PROTEINS. VII

These earlier studies concerned themselves with the speciiicity of
the fi-glucosides of two different hexoses, glucose and galactose.  The
present paper deals with the specificity of two different glucosides of
the same sugar, namely; the paminofihenol .a- and /3-glucosides of
glucose., :: :,I ;   .:; .!.`.'        1, j.
* The "CY and fl antigens" formed by the union of the diazonium de+
rivatives of these glucosides with protein d$fer from each other only
in the spatial relations of the terminal carbon atom in each sugar
component. Since I the ultimate composition of both antigens is
chemically the same, any observable differences in their immunological
specificity can be referable only to known differences in the molecular
structure of each. The follo+g experiments were carried out to
determine to what extent' two isomeric glucosides of the same sugar,
in combination with a single protein, would influence the specificity
of two antigens otherwise identical. The present paper deals with
the question of the specificity of the precipitins present in the serum
of rabbits immunized with CC- and p-gluco-globulin.

EXPEEIMENTAL.

Methods

The synthesis of the a- and @+-aminophenol glucosides of glucose and the
method of coupling the diazonium derivatives to protein are given in detail in
the preceding paper (4).  The method of the intravenous immunization of rabbits
and the technique of the precipitin and iuhibitioi tests differ in no essential respect
from those described in the earlier studies (2).

The inwnuniaing a?zfigens were prepared by combining each of the glucose deriva-
tives to globulin derived f&m normal horse serum; on the other hand the tGst
a&gm used in the precipitin reactions, contained the respective glucosides bound
to the proteins of chicken serum. The use of proteins of remote biological origin
in the two sets of antigens emludes the possibiity of amunon antiprotein precipi-
tins masking the specificity of the carbohydrate reactions.

I. Anticarbohydrate Antibodies

(1) Specific Precipitin React&c-The sera prepared by immuniza-
tion of rabbits with a- and B-gluco-globulin were first tested for the
presence of precipitins specifically reactive with an antigen containing
the same carbohydrate radical. The test antigens employed in the
precipitin reactions were in each instance prepared by combining the


0. T. AVERY, W. F. GOEBEL, AND F. H. BABERS    771

rqxctive glucoside with a protein unrelated to that used in the im-
m&&g antigen.  Under these conditions, the results of the tests are
not confused by the presence of a common protein and the specifkity
of the reactions are directly interpretable in terms of known differences
in the chemical structure of sugar radical in the reactive part of each
antigen.

                      TABLE I
@&f.c Pmci@itim in Sera of Rabbits Immunisd with u-lZluco-GloWi~*

                                      J
                     Tat antigen: usltlachurur -*
~UUC r&it -
             1:5,000       1: 10JJoo       1:2o,lm      1:40,000

     1       ++++     +++      ++       +j=
     2                                          f
    3   zi++   E   -ii        +

++A-+ - Complete precipitation with compact d&like precipitate.
* III order to avoid the reactions of a common protein, the irnmvnising antigen
was prepared by combining a-glucoside with the globulin from horse serum, while
the test antigen was similarly prepared by coupling the same glucoside to the pro-
teins of cllickeIl serum.

TABLE II

S#e+ Precipitins in the Sera of Rabbits lmmuniscd with P%4co-Globulin

                   Tat u~tigen: ~&uoJ~~ amum*
Immuuc rabbit aera
             l:S,OOO       1: 10,ooo       1:2o,ooo      ,1:4O,OW

    1 '    +++ . ++       +h
    2      ++++     ++++     +++   +:+
    3       ++++     ++++    +++     +++
+ + + + y Complete precipitation with compact disk formation.
* Unrelated proteins in immunizing and test antigens (see footnote, Table I).

The precipitin reactions between the homologous test antigens and
the antisera of rabbiti immunized with 4~- and &gluco-globulin are
'  given in Tables I and II.                 . .           `.
The results of the prekipitin tests illustrate the capacity of the
immune sera to react with an antigen containing the homologous glu-
coside irrespe&ve of the protein to which it is attached.  They con-
firm the earlier observations concerning the orienting influence of the
                      


772      CONJUGATED CAXBOHYDRATE-PROTEINS. VII

sugar radical on the specificity of the protein with which it is com-
bined.                                              .
(2) Specijit Inhibition Tests.-The selective specificity of the anti-
bodies reactive with the two isomeric derivatives of glucose is clearly
demonstrated by the results of the inhibition tests given in Tables
III and IV.

Analysis of the data presented in Table III shows that the ar-gluco-
side alone, when added in excess to the homologous antiserum specifi-
cally binds the precipitins and thus renders them unable to react subse-

TABLE m

SpGcifiG Inhibition of PrGGipitins in dXm-G~obu&a Ad..wum by Homologous
                   Glucoside

cc.
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2

=
I  Glddrs n/U
I'      B
     .-
        cc.

salt
solution
to volume

cc. '
0.3

     0.3   2
     0.3
         ti
      6

0.3     -
0.3     -  ;
0.3           Pp
      -

.
-

+ + + - Disk-like precipitate
0 - No precipitation, showing complete inhibition.
* See footnote, Table I.

=

I

. _

-

Redt

+++
f++
f++
0
0
0
++*
-I-++
+++

quently with the test antigen containing the same sugar derivative.
The specificity of this inhibition is shown by the fact that the addition
of an equal concentration of the @glucoside to CT immune serum has
little or no appreciable effect on the precipitin reaction with the
Q antigen. Similar relationships are shown to exist in the spe-
cific inhibition of the precipitins in fi antiserum by the B-glucoside
(Table IV).

The results of the inhibition tests reveal the specikity of the anti-
carbohydrate reactions, since in the case of both (Y and B antisera the
precipitins for the homologous antigen are inhibited only by the corre-


   0. T. AVERY, W. F. GOEiEL, AND F. H. BABERS    773

spending glucoside. Although the glucosides by themselves, un-
attached to protein, fail to produce precipitation in immune sera,
each by binding the specific antibodies in its homologous antiserum
prevents them from again reacting with a test antigen containing the
same glucoside. In this sense the glucosides unattached to protein
function as haptens, the chemo-specific groups of which unite with
the corresponding antibodies without causing visible change in the
reaction mixture. Marrack and Smith (5) using a direct method have

                     TABLE IV

,Qecifi Inhibitian b Homologw Gk~osidk of PrecilUins in &Gk&obtdin

=

Ghoslda ~113

      cc.
    0.2
    0.2
    0.2
    0.2
    0.2
    0.2
    0.2
    0.2.
    0.2
+++T- = Complete precipitation with disk-like precipitate.
0 = No precipitation, showing complete inhibition.
* See footnote, Table I.    f         `,

(z

cc.

0.3
0.3
0.3

=
I

B

cc.

0.3
0.3
0.3

salt
aokloa
0 volrunc

cc.
0.3
0.3
0.3

A

=
,
I,

1
--
~
-

recently brought experimental proof of Landsteiner's original view
that the union between hapten andantibody is a specific combination
between the chemo-spe& groups of the rkcting~agents.  As Land-
Steiner has pointed out in the case of non-protein radicals which func-
tion as haptens, the union of these glucosides with antibody does not
lead to precipitation unless the reacting groups are attached to protein.

The preceding experiments have dealt with the homologous immune
reactions of two antigens of. knoti chemical constitution which differ
one from the other only in the CY and @ type of glucosidic union.  The
results show. that mere dEerences in the spatial arrangement of the


774      CONJUGATED CARBOHYDRATE-PROTEINS. M

groups on the terminal carbon atom in these two derivatives of. glucose
sufKce to confer on each antigen a marked degree of difbzrential
spec&ity. For, as shown by the specific inhibition reactions, it is
possible to differentiate selectively between the two isomeric glucosides
of the same sugar by the use of immune sera.

Despite the reflection in serological specificity of differences in the
stereochemical relationships of the first carbon atom bearing the non-
sugar constituent of each glucoside, it must be borne in mind that
in both the a! and ,9 derivatives the spatial arrangement of the polar
groups on the r emaining five carbon atoms is identical.  Quite dif-
ferent, however, are the structural relations existing in the case of
the @-glucosides of glucose and galactose, the immunological specificity
of which has previously been shown to be absolute (2).  In these latter
glucosides the stereochemical arrangement of the groupings on the
terminal carbon atom are both in the @ position, but the symmetry of

the polar groups on the remaining five carbon atoms is not identical,
for on the fourth carbon atom of galactose the H and OH groups are
rotated through an angle of 180o.  This difIerence in molecular con-
figuration, with its consequent change in the spatial relationship of
the polar groups, is the important if not the sole factor determining
the individual specificity of the @-glucosides of glucose and galactose.
On the basis of these facts, one might anticipate an absolute specificity
in the case of the similar glucosides of two different hexoses, but an
overlapping specificity in isomeric glucosides of the same hexose.

(3) Cross-Precipitin Reactions.-It is therefore not surprising to
find that this partial similarity in the chemical structure of the (r and
@ derivatives of glucose is reflected in the serological relationships of
both substances. The results of the cross-precipitin tests presented
in Table V show that antibodies present in a and B antisera cross-react
in each instance with the heterologous test antigen.
The cross-precipitin reactions are sharply defined and are quantita-
tively only slightly less than the reactions between each antiserum and
its homologous antigen.

Interesting relationships in the overlapping speciflcity of both sugar
derivatives are brought out in the reciprocal inhibition tests, the results
of which are given in Table VI.

Analysis of the data presented in Table VI shows that the addition
of cr-glucoside to Q! antiserum ,inhibits the precipitins from reacting


0. T. AVERY, W. F. GOEBEk, AND F. H. BABERS    775

witi both a and @ tat antigens. However, the addition of an equal
concentration of &glucoside to a antiserum inhibits &e precipitin

                      TABLR V

CroJs-Preci#t*tin RediofzS of a- and &%U&~obdin Antisera with Hete&og~
               Antigens

                            Test atuigam*

      Immuoo-         a-glucoshIcLn -       b-g- serum

                  1:5,000  1: 10,Ow 1:2o,om   l:S.am  1: 10,ocnJ l:2O,Ow
                    ------.
a-gluco-globulin. . . , . . . . . . . . . ++++ +++ ++ ++* ++
&glum-globulin. . . . . , . . . , . . . .  ++   ++  + ++++++++ +:+

`++++ = Complete precipitation with compact disk-like precipitate.
* See footnote, Table I.

                     TABLE VI

Inhibitiun OJ

' Cross-Precipitin Reucticma I

I Homologous and Huterotogm

us
=
I

GZucosi&

Glued

   s.
o! 0.2
P 0.2
a0.2

uo.2
a0.2
a0.2

@ 0.2
80.2
8 0.2

6 0.2.
8 0.2
80.2

GlUdo x/l.5 '

  a     8
--
  cc.      cc.

0.3 -
       0.3

0.3 -
       0.3
--

       0.3
0.3 -

       0.3
0.3 -

=
I

2A
to volume
04;

0.3

0.3

0.3

,-

I Tat mti,gcn' 1: 10,000

8

cc.

0.5
0.5
0.5

0.5
0.5
0.5

A

Basulta

+++
+L

++
  0
  0

++++
  0
+++*

++
  0
  0

Plus signs indicate positive reaction with gradation in amount of precipitate.
0 = no precipitation, showing complete inhibition.
- = reagent not used.
o See footnote, Table L  ,.

reaction with the /3 test antigen without appreciably diminishing its
capacity to precipitate the a antigen. Similar relationships hold in


776      CONJUGATED CARBOHYDRATE-PROTEINS. VII

the case of the @ antiserum with respect to the inhibition of precipitins
by the homologous and heterologous glucosides.

It is evident from these results, that the addition of the homologous
glucoside to its antiserum completely inhibits the precipitins for both
the homologous and heterologous test antigens.  On the other hand,
the addition of the heterologous glucoside to immune serum inhibits
only the precipitins for the heterologous test antigen and has but
slight effect upon the antibodies reactive with the homologous antigen.
The results of the cross-inhibition tests with the a- and @-glucosides
show that the reaction of an immune serum with its homologous anti-
gen is specifically inhibited only by the homologous glucoside; while
the cross-reaction between this serum and the heterologous antigen is

TABJaE VU

Precipilins for Globulin in Sera oj Rabbits Immun&ed with cy- and &GkuoGlobdin*

Immune ma

I       Globulin from horse -

1:s.ooo I 1:10.ooo I 1:20*wm I 1:4o.o00

cc-&o-globulin. . .,. . . . . . . . . . . . . . . . . . . . . . .
8-gluco-globulin . . . . . . . . . . . . . . . . . . . . . . . . . . .

+ + + - flocculent precipitate, not compact.
* Both immunizing antigens were prepared by combining each glucoside with
globulin from horse serum.

completely inhibited by either glucoside. This lack of reciprocal
inhibition of the precipitins in LY and @ antisera may be interpreted as
further evidence of the lack of the immunological identity of the two
isomeric glucosides.

II. Antijwotein Antibodies

As previously described the immunizing antigens were prepared by combining
the cx and 0 derivatives of glucose with globulin obtained from normal horse serum.
The sugar-protein antigens were standardized on the basis of their nitrogen con-
tent so as to contain 5 mg. of protein per cc.  Each rabbit received during the
course of immunization a total of approximately 160 mg, of protein antigen.

In addition to the antibodies reactive with the specific glucosides,
there are also present in both immune sera precipitins for the globulin
used in preparing the immunizing antigens (Table VII).


0. T. AVERY, W. P. GOEBEL, AND I?. H. BABERS    777

The precipitating antibodies for horse serum globulin may arise in
response to the presence in both antigens of free protein unbound by
the &a~~t.id glucosides.  However, it is also possible that even in
the absence of free protein, the sugar-protein antigen as a whole may
stimulate the formation of two qualitatively different antibodies, each
specifically related to the corresponding component of the antigenic
complex.  This concept of the dual antigen+ of a single complex
antigen involves the assumption that the coupling of the glucoside
to the protein has not masked the groups essential to the specificity
of the protein, and that while the sugar radical through conjugation
acquires specific antigenicity, the protein molecule retains, in part at
least, its original antigenic properties.

DISCUSSION

Landsteiner and Lamp1 (6), working with ortho, meta, and para
substituted aromatic compounds, have pointed out the significance
of the spatial arrangement of the groupings upon which the speticity
of these compounds depends.  In more resent studies on the serologi-
cal differentiation of steric isomers of p-aminobenzoylamino acetic
acid and tartaric acid, Landsteiner and van der Scheer (7) have further
emphasized the dependence of immunological specificity upon chemical
structure.

The results of the present study add further evidence in support   .
of the view that the immunological specificity of carbohydrates is
determined by their chemical constitution. Di&rences in the specific
behavior of the a- and /?-glucosides of glucose may be accounted for
by known differences in the spatial position of the groups on the first
carbon atom of each glucoside. These structural changes are so
sharply reflected in serological specificity that it is possible by means
of immune sera to diiIerentiate selectively between the two isomeric
glucosides of the same sugar.  However, granting the difference in
the chemical structure of the two glucosides it becomes necessary to
account for their overlapping specificity. The chemical basis for
this immunological crossing may lie in the fact that the spatial arrange
ment of the polar groups on the mmaining five carbon atoms is identi-
cal in both glucosides. This partial similarity of molecular grouping
might then account for the degree of immunological likeness exhibited


778      CONJUGATED CARBOHYDRATE-PROTEINS. VII

by the two substances. Although in a portion of the molecule of both
glucosides the structural relationship is identical, nevertheless they
`behave chemically as separate entities and possess serologically a
separate and distinct specificity.  If Complete reciprocal inhibition of
precipitins is accepted as the criterion of s&ological identity then the
failure of both glucosides to exhibit ,this capacity may be taken as
flirther evidence of differences in the immunological specificity of each.

The lack of reciprocal absorption of aggltitinins by two organisms
mutually agglutinable in the immune serum of each is generally con-
ceded to indicate a lack of immunological identity. `Relationships of
this order are known to exist between Pneumococcus Type II and
Friedlgnder bacillus Type 13.  In both instances the reactive sub-
stance has been identified chemicallly as the specific poIysaccharide
peculiar to the capsule of each organism (8). While the structural
constitution of these complex sugars is not as yet fully known, con-
siderable knowledge has been gained concerning their chemical prop-
erties. For example, it is known that the Pneumococcus Type II
polysaccharide is built up of glucose units and that chemically it bears
a close resemblance to the polysaccharide recovered from the Type B
FriedEnder bacillus. However; the two substances are not chemi-
cally identical, although the similarity between them is sufficient to
result in a certain likeness in immunological speciiicity (9). In the
absence of precise knowledge of the structural relations of the two
.polysaccharides, it seems reasonable to assume that both contain in a
portion of the complex molecule the same or a closely similar configura-
tion of atoms. This &nilarity of molecular grouping might then
account for the immunological similarity of the two substances.

Considerable evidence for this point of view is found`in the results
of the present study concerning the specificity of the cy- and /3-gluco-
sides of glucose. A, comparison of the serological relatiohships be-
tween the two isomeric. derivatives of glucose and the capsular poly-
saccharides of the two organisms in question is presented inTable VIII.

Analogous relationships ex,tending even to the cross-immunity?eac-
tions and the lack of reciprocal absoi-ption and inhibition of antibodies
are evident-in both the groups of simple and complex carbohydrates.
While the comparison is instructive it is not necessarily, valid in .the
case of the capsular polysaccharides, for the final intekpretation of


0. T. AVERY, W. F. GOEBEL, AND F. H. BABERS    `779

these relationships must await further knowledge of the structural
relations of the more complex bacterial sugars.

TAtiLE VIII

Comparison of the Serological Re&ztimship between I$omeric Derivatives of Glucose
  and the' Capstitar Polysaccharides of Two Unrelated Species of Bacteria
                    Antipneumococcus serum & II

.    a&co-globulin antiserum

By addition of

   Precipitios are

inhibited for I lot inhibited fool
         _ -

a-glucoside

a and fl test
antigens

&gIucoside

@ test anti-  a test anti-
gen     gen

   ,.

@-gluco-globulin antiserum

/3-glucoside

                      Anti-jriedlaendcri serum Type B
-
     Precipitins are                    Agglutinins `are
: .                    Absorbed with
  inhibited for 1 not inhibited lo]             removed for   not removed for
- .
a andi test       *.    B.. frkdt&z- Pneumococ-
  antigens        deri Type   cus Type
             B." `.'     II
                 B. fried&n-
                     deri Type
                            B

a-glucoside

                           
`a test anti-  B test anti-  Pneumococ- Pneumococ- B. friedlam-
   gen       en      cus Type   cus' Type   deri Type
                    II        II ,B,
-

Absorbed with

Pneumococ-

cus Type
II

i.. ffiedaen-
deri' Type
B

Agglutinins are

removed for I not removed for

Pneumococ-
cus Type
II
B. fried&z-
deri Type
B,

B. f&d&m  Pneumococ-
deri Type   cus Type
B       II

The test antigens in all instances were prepared by cdmbinhg tie respective
glucoside with a protein biologically unrelated to that in the imm&ing antigen;
see footnote, Table I.

Enders (10) has ,recently suggested that crdss-relationships between
Type II Pneumococcus and' Type B Friedlgnder bacillus `may be.


`180     CONJKfGATED C!ARBOE!i!D~TE-PXOTEINS. VII _

due to the presence in pneumococci of a type-specific agglutinogen
unrelated to the specific carbohydrate, ;.., `.`i -;.   . .
In the case of the synthetic antigens containing the a and fl com-

pounds of glucose alone, the evidence indicates that the immunological
relationships of the reactive glucosides are determined by known
variations in their chemical constitution and are independent of the
protein to which they are attached. In view of these findings it seems
not unlikely that in the case of the polysaccharides, because of their
more complicated structure and the greater possibility for variation
in molecular configuration, there may be found many examples of a
sin&r overlapping specificity among carbohydrates of unrelated
OIigiIL

S-Y

The chemical and immuno1ogica.l properties of the p-aminophenol
a- and @-glucosides of glucose are described and correlated. The
results are discussed with reference to their possible bearing on the
chemo-immunologicad nature of the speciftc polysaccharides of bac-
terial origin.

BIBLIOGIUPHY

1. Goebel, W. F., and Avery, 0. T., J. Exp. dbed., 1929,50,521.
2. Avery, 0. T., and Goebel, W. F., J. Exfi. Med., 1929,50,533.
3. Tillett, W. S., Avery, 0. T., and Goebel, W. F., J. Exfi. Med., 1929,50,551.
4. Goebel, W. F., Babers, F. H., and Avery, 0. T., J. Ex$. Med., 1932,55,761.
5. Marrack, J. R., andsmith, F. C.,Nawe, 1931,128,1077.
6. Landsteiner, K., and Lamp& H., Biochem. Z., 1918,86,388.
7. Landsteiner, K., and van der Scheer, J., J. a?&#. Med., 1928,48,315; 1929,
  50, 407.

8. Heidelberger, M., Goebel, W. F., and Avery, 0. T., J. l&j. Med., 1925,42,
  701.

9. Avery, 0. T., Heidelberger, hf., and Goebel, W. F., J. E.@. Med., 1925,42,
  709.

10. Enders, J. F., J. Ex#. Med., 1932,55, 191.