THE CHEMICAL NATURE OF IMMUNE SUBSTANCES

              MICHAEL HEIDELBERGER
   Hospital of the Rockefeller Institute for Medical Research, New York
Immunology, one of the youngest of the biological sciences, has
expanded with such rapidity and developed with such intricacy and
complexity that an understanding of its chemical basis has lagged
far behind. Structural organic chemistry, colloid chemistry and
physical chemistry have been freely drawn upon and much data have
been accumulated on the chemical, physical and immunological behavior
of the substances concerned in the phenomena of immunity.  The
interpretation of these data is often rendered difficult by the necessarily
imperfect methods of isolation of the complex and labile substances
involved; hence there is relatively little known of the actual chemical
nature of the material with which the immunologist must work.
The chemical basis of immunity has been so adequately presented in
Professor Wells' splendid monograph (1) that it would be futile for
the writer to attempt a repetition of this task.  He would like, how-
ever, to present a summary of such knowledge as is available in the
narrower field of the actual chemical nature of immune substances,
freely acknowledging his indebtedness to Professor Wells' book, and
attempting chiefly to cover such advances as have been made since
it was written.
Present methods of organic and biological chemistry are such that
the separation of a highly active specific substance from generally
larger amounts of inert accompanying impurities is an exceedingly
difficult matter, and is only possible of realization under particularly
favorable conditions of stability on the part of the specific material.
For this reason a more detailed treatment is given to the section on
Carbohydrates than to those on Proteins and Lipoids.  It is felt that
on the score of thermostability, inertness to the comparatively brutal
reagents often necessary, and a sharply debed specificity which remains
qualitatively the same before and after purification, the carbohydrates
with immunologically specific properties offer perhaps the best avail-
able material for the acquisition of definite knowledge as to the chemi-
cal basis of specificity.
Even in this group it is necessary to proceed with extreme caution,
                           107


108              MICHAEL HEIDELBERGER

since the substances isolated are amorphous, of high molecular weight,
and devoid of the criteria which would stamp them beyond a doubt
as pure substances.  This is even more forcibly the case in the protein
group, with, for example, toxins and antibodies, and in the last analysis
the reviewer of this field must deal with trends and tendencies, rat,her
than with completely established proofs.

In a treatment of the subject such as this many papers of imrnuno-
logical interest will necessarily be omitted and only those chosen for
discussion which contribute in as reasonably clear-cut a fashion as
possible to a broader understanding of the true chemical nat.ure of
immune substances.

I. PROTEINSANDPROTEIN DERIVATIVES. A.Gen~nl. Proteinchem-
istry is at present in a period of transition, and there is a tendency t#o
ascribe increasing importance to diketopiperazine (2), oxazoline t.3 ),
and possibly even pyrrole ring (4) structures as components of the
complex protein molecule, rather than to assume a straight-chain
polypeptide skeleton, as Emil Fischer did. This newer conception is
already being reflected in immunochemical work, and will be discussed
under the head of t,oxins.

In the light of recent work it still seems quite evident that highly
purified proteins may function as antigens. Convincing evidence
has been offered that this is also true in the case of oxyhemo-
globin (5), hitherto the most doubtful antigen of the more easily acces-
sible crystalline and recrystallizable proteins.

Likewise it still seems clear that the presence of protein or the higher
protein degradation products is necessary in order for a substance to
function as an antigen. It would appear, also, that the antigenic
properties of a protein may be profoundly modified, not only by chemi-
cal reaction (as in Landsteiner's classic work), but also in the native
state by combination with a lipoid or with a carbohydrate, so t.hat
changes in specificity are not always to be traced to changes, subtle
or otherwise, in the protein molecule itself.  This will become evident
in subsequent discussions.

Little can be said as to the actual groupings in the complex protein
molecule which determine specificity. Landsteiner has found that,
among the azoproteins the most sharply defined specificity and least
overlapping are shown by those in which the diazotized aromatic
component possesses an acid group, such as in the aminobenzoic acids,
sulfanilic acid and arsanilic acid.  On the basis of this work and the
finding of fatty acids in the specific portion of the heterogenetic antigen
and sugar acids in the polysaccharides discussed below, Landsteinel


CHEMICAL NATURE OF IMMUNE SUBSTANCES       109

ascribes especial significance to acid groups in the determination of
specificity (6).
B. Bacterial toxins.  Recent work has tended to confirm the belief
that the bacterial toxins belong among the higher protein degradation
product,s. Dernby's studies of diphtheria toxin production and de-
struction (7) led him to the view that the toxin is not a lipoid, since
it is rapidly destroyed by proteolytic enzymes, and that it originates
through the action of the proteolytic enzymes of the bacillus on the
organism itself and even on the primary proteoses of the medium, the
proteolysis ultimately continuing until the toxin is dest,royed.  Watson
and Langstaff (8), who applied Watson and Wallace's modifica-
tion (9) of Glenny and Walpole's acetic acid precipitation method (10)
to formaldehyde-treated diphtheria toxin, found this to be very stable
on reprecipitation and dialysis.  The purified toxoid reacted as a
proteose and contained sulfur.

Huntoon (11) has found that in the purification of scarlatinal tosin
addition of 1 per cent of acetic acid and 60 per cent saturation with
ammonium sulfate give rise to a voluminous precipitate which carries
very little toxin.  After removal of this "globulin" fraction a further
precipitate is formed between 70 to 75 per cent saturation. This
carries the toxin, which also appears to be a protein derivative, since
it is not dissolved or inactivated by lipoid solvents, is destroyed by
heating to 90' for one hour, and is rendered inactive by digestion with
trypsin.

A suggestive analogy has been drawn by HaIIauer (12) between the
reversible inactivation of dysentery toxin by dilute acids and the
reversible ring and open chain tautomerism observed by Bergmann and
Miekeley @a) in the case of certain amino acid derivatives. These
changes, taking benzoylserine as an example, may be pictured as
follows:
             CH,-0 &,-N>ac~\
         I           acid
             COOH
        alkali7
       /
       /       `L
     CHzOH                   CH,-0-COCsHs
    I             alkali        I
     CHNHCOCsHs -         CHNH,
    I                     I
      COOH                        COOH


110                 MIGUEL HEIDELBERGER

Hallauer suggested that some such intramolecular rearrangement
occurs in the detoxification and loss of antitoxin-producing power of
dysentery toxin when exposed to the action of dilute hydrochloric acid,
and in the recovery of both functions on addition of alkali.

C. Bacterial proteins. The specific properties of pneumococcus
"nucleoprotein," as contrasted with those of the polysaccharides of this
microkganism, will be discussed under the section on Carbohydrates.

As a result of a series of studies on the active principle of tuberculin,
Long and Seibert (13) present new evidence for the view that the
substance producing the skin reaction is a protein (cf. also Mueller
(14)). The active material, isolated after growth of the tubercle
bacillus on a protein-free medium, was completely precipitated by
three-fourths saturation with ammonium sulfate and was separable
into a water-soluble, heat coagulable protein, a water-insoluble, non-
coagulable protein, and a water soluble protein which was not coagu-
lated by heat.  The last fraction had the highest potency and is believed
to be a whole protein, or one hardly degraded as far as the proteose
stage, since it is attacked, with loss of activity, by pepsin and acid, or
trypsin and alkali, but not by trypsin in neutral solution, or by erepsin.
More recently Seibert (15) has isolated a crystalline protein from this
fraction, but was uncertain whether or not this represented the true
active substance of tuberculin.

D. Antibodies. The actual chemical nature of antibodies is still
a matter of doubt.  The question is as yet unanswered whether anti-
bodies are themselves proteins, or whether they are substances of
unknown constitution, so firmly attached to one or another fraction
of the proteins of immune serum that separation from these has been
impossible.

Since Salkowski's brief report on protein-free diphtheria antitoxin
solutions (16) was shown to be in error (17) the work of Huntoon and
his coIlaborators (.18) and of Ottenberg and Stenbuck (19) has re-
mained the chief recent evidence in favor of the non-protein nature of
antibodies.

The former group, in extending the work of earlier investigators,'
absorbed the antibodies from antipneumococcus serum with homolo-
gous pneumococci, washed the agglutinated organisms, and found
a considerable proportion of the antigen-antibody complex to be
dissociated at 42 to 55" by mildly alkaline solutions or those

`For bibliography cf. (18); cf. also Gay and Chickering, Joum. Exper. Med.,
1915, xxi, 389; Chicbering, Ibid., 1915, xxii, 248.


CHEMICAL NATURE OF IMMUNE SUBSTANCES       111

containing sugar. The extracts contained traces of protein, but
since a portion of this could be eliminated by precipitation on
dialysis or by acetic acid without great loss of potency, and since the
antibodies appeared resistant to trypsin and did not correspond in
solubility to typical euglobulin or pseudoglobulin, it was concluded
that they differed from ordinary serum proteins. Ottenberg and
Stenbuck found that the agglutinins in typhoid antibody solutions
obt,ained by Huntoon's method could be largely precipitated at about
pH 6.4 by small amounts of copper ion. After two reprecipitations
by solution in dilute acid and readjustment to the proper pH the anti-
body solutions no longer gave the usual protein reactions.  The final
solution contained about one-twentieth of the agglutinin originally
present in the serum, but only l/3450 of the nitrogen.  Less favorable
results were obtained with the pneumococcus.

In considering this work it must be borne in mind that solutions
obtained by the dissociation of sensitized bacteria are extremely dilute,
and that the ordinary tests for protein are far from sensitive. It
would therefore require the concentration of large amounts of material
and the study of the actually isolated antibody substance, both chemi-
cally and immunoIogically, before it could be stated with certainty
whether or not a protein-free antibody had been attained by any of
these methods.

On the other hand, there are data pointing toward the actual protein
nature of antibodies. Locke and Hirsh (20), later with Main (21),
fractionated anti-sheep rabbit hemolysin both by electrodialysis and
by absorption on sheep cells, dissociation of the antibody-antigen
complex with ether, and extraction of the hemolysin with 0.001 iV
sulfuric acid. The hemolysin which separated on neutralization still
contained some globulin derived from the cells used for absorption,
but appeared to consist entirely of protein of the general behavior of
the globulins. After further purification by electrodialysis and iso-
elect.ric flocculation it still maintained its protein characteristics,
although in the best preparations one hemolytic unit was associated
with as little as 0.000,044 mgm. of protein.

In Felton's extensive work on the purification of pneumococcus
antibodies (22) there is also much to indicate the actual protein nature
of antibodies. Felton found that when antipneumococcus serum is
diluted with 15 to 20 volumes of slightly acidulated water or weak
buffer solution a large proportion of the antibodies is usually precipi-
tated and about 90 per cent of the serum proteins remain in solution.


112                MICHAEL HEIDELBERGER

This method is undoubtedly the simplest yet proposed for effecting
a. rapid and far-reaching initial purification of pneumococcus anti-
bodies. Further fractionation without extensive losses was difficult,
but by removing the small portion separating on dilution with two
volumes of water, and also discarding the relatively inactive first and
last fractions obtained on gradual saturation with ammonium sulfate,
Felton recovered a highly potent so-called pseudoglobulin fraction
with properties differing from the corresponding portion of normal
serum. The chief differences hitherto observed were the shape of
the titration curve of the antibody-containing globulin and its iso-
electric point at about pH 6.8, a reaction considerably more alkaline
than that of the isoelectric points of the ordinary serum globulins.

Felton, of course, does not consider this protein fraction to be pure
antibody.  Indeed, the writer has found as much as 60 to 7.5 per cent
of the protein in the initial solutions obtained by Felton's method to
remain unprecipitated by the appropriate soluble specific substance.'
What is significant, however, is, that on further purification and re-
moval of a portion of the inactive material Felton appears to have
obtained a modified globulin. These modifications could scarcely
have been induced by the cautious handling of the material, and if
they are confirmed by the finding of other chemical changes caused
by the presence of increasing amounts of actual antibody substance,
it would become very difficult to believe that antibodies were other
than proteins appropriately modified.

II. LIPOIDS. A. Helerogenetic antigens. It will be recalled that
Forssman (23) originally showed that following the injection of certain
animal tissues of unrelated species common hemolytic antibodies for
sheep corpuscles appear, and that Landsteiner (24) and Taniguchi
(25) demonstrated such heterogenetic antigens to consist of two com-
ponent parts, one a protein, the other probably a lipoid.  It was also
shown by Landsteiner and Simms (26) that the lipoid fraction of the
heterogenetic antigen, which is itself practically devoid of antigenic
properties, acquires true antigenicity when combined with protein,
and that the a.ntibodies thus induced react with the lipoid fraction.
The protein or serum with which the lipoid was combined made no
difference in the specificity of the resulting antibodies for the hetero-
genetic lipoid fraction, thus showing this fraction to be the actual
specific factor in the formation of the antibodies.  Further work has

? Unpublished results.  Felton found 46 per cent in an apparently more highly
purifird solution.


CHEMICAL NATURE OF IMMUNE SUBSTANCES      113

since been done on this lipoid fraction with a view to separating the
actual specific substance, or "haptene," as Landsteiner has termed
the non-antigenic, specifically reacting portion of complex antigens (24).

After extensive purification by fractional precipitation from various
solvents and by precipitation with basic lead acetate, Sordelli, Wernicke
and Deulofeu (27) concluded that the active material was not in the
cerebroside fraction, contrary to earlier views. Landsteiner and
Levene (28) reached the same conclusions, but found the activity of
their purified preparations to be enhanced by the addition of crude
sphyngomyelin, in itself practically inactive.  By means of a prolonged
series of fractionations from a number of solvents they obtained material
showing [oL]~ + 20" in water.  This water-soluble product was composed
both of lipoid and carbohydrate, since on hydrolysis it yielded soluble
reducing suga.rs which gave purple and green colors with orcin, hydro-
chloric acid and copper sulfate, and also formed osaaones.  An insoluble
portion was precipitated during the hydrolysis, and from this higher
fatty acids and a base resembling sphingosine were obtained.

A product with somewhat similar solubilities and also yielding
sugars and fatty acids on hydrolysis has been isolated by Dienes and
Schonheit (29) by fract.ionation of the lipoids of the tubercle bacillus
after prehminary extraction with acetone. `Ihis product, which
separated from a dry ether solution of a partially purified specific
fraction on long standing, showed complement fixation with rabbit
antiserum to the same strain of tubercle bacillus at a dilution of about
1: 10,000,900. It contained both nitrogen and phosphorus and also
precipitated the immune serum at a dilution of 1:1,000,000 (30) (cf.
also p. 122), so that it was possibly not a single substance.

As far as can be determined from the use of crude alcoholic extracts
it is also possibly a lipoidal fraction of the red blood cells which is
responsible for the specific differentiation of blood groups among
human beings (31).

A new significance is thus given to lipoids as immune substances.
Although Schmidt (32) has reported the lipoids of the typhoid
group to possess no antigenic or specific properties, and although
there is no convincing evidence that pure, isolated lipoids can act
antigenically, it requires apparently but the loosest kind of com-
bination with protein to convert lipoids into antigens capable of
producing a specific antibody response. Whether the production of
specific antibodies is due entirely to the lipoid fraction of the complex
material which has been isolated, for example, from the specific part


114                 MICHAEL HEIDELBERGER

of the het.erogenetic antigen, or is due in part to the carbohydrate
fraction, would seem still to be an open question in view of the findings
discussed in the next section.

The application of the above results to the Wassermann reaction
was a quick development, and has been extensively discussed by Sachs
and his co-workers (33), who believe that spirochete protein is especially
active in converting tissue lipoid into an antigen.  However, the
int,erpretation of their findings has been questioned (34), so that the
true nature of the Wassermann reaction is still a matter of doubt.

III. CARBOHYDRATES. Recent work has shown that bacterial
polysaccharides, as well as bacterial protein, influence largely the
antigenic character of a number of microorganisms although these
sugar derivatives are not in themselves antigenic when isolated in a.
state of comparative purity. The chemistry and immunological
significance of these carbohydrates are therefore discussed below in
considerable detail, since on the one hand many puzzling phenomena
of bacterial specificity and interrelationship have been made clear,
and on the other, a new field of sugar chemistry would appear t,o have
been opened up.

After the demonstration of the existence of three fixed antigenic
types of Pneumococcus (35), Dochez and Avery discovered in fluid
cultures of types I, II and III of this microbe the existence of a sub-
stance which precipitated specifically in antipneumococcus serum of
the homologous type (36).  This "soluble specific substance" was
also found in the body fluids of infected animals and was even demon-
strated in the urine from approximately two-thirds of the cases suffer-
ing from pneumonia due to pneumococci of types I, II and III.  Sub-
sequently, from filtered alkaline extracts of several varieties of bacteria,
including pneumococci, Zinsser and Parker (37) prepared products
which they called `(residue antigens," which appeared free from coagul-
able protein, were specifically precipitable by homologous antisera,
were acid- and heat-resistant, and were considered analogous to the
previously described soluble specific substance of Pneumococcus.

Since Dochez and Avery had found the specific substance to be
thermostable, resistant to the action of trypsin and many reagent.s,
and type specific in the highest degree, it seemed an ideal basis for
the beginning of a study of the relation between bacterial specificit'y
and chemical constitution, and this was accordingly undertaken (38).
The type II pneumococcus was first st,udied and the fractionat,ion
and purification of the specific substance were followed at each step


CHEMICAL NATURE OF IMMUNE SUBSTANCES        115

by means of the precipitin test.  Eight-day cultures of the organism
in meat-infusion phosphate broth were concentrated on the water-
bath to about one-fifteenth volume, precipitated with 1.2 volumes of
alcohol, and centrifuged. Of the three layers formed, the middle
gummy layer contained most of the specific substance, and a large
proportion of the accompanying impurities was thus eliminated in
a single step. Further purification of the middle layers from about
300 liters of culture at a time depended upon repeated precipitation
with alcohol under varying conditions, also by means of saturation
with ammonium sulfate, and final precipitation by pouring into 10 to
15 volumes of acetone.  The yield was usually from 2.5 to 3.5 grams.
As the purification proceeded the material isolated took on more and
more the properties of a polysaccharide, so that it became evident that
a sugar derivative was at least the carrier of whatever might be the
true specific substance itself. Attempts were made to separate this
hypothetical substance from the polysaccharide by precipitation with
basic lead acetate, uranyl nitrate, or safranine, by adsorption on alu-
mina and recovery from this, and even by specific precipitation with
a large quantity of type II antibody solution (39) and recovery of the
specific substance from the immune precipitate, but these failed to
effect a significant change in properties, even when pneumococci them-
selves were used as starting material instead of the broth culture.
Attempts at a separation by means of carbohydrate splitting enzymes
also failed as the sugar derivative proved resistant t,o this type of
hydrolysis.  Moreover, when exposed to the action of 1: 1 hydrochloric
acid in the cold, the substance diminished in specific activity only
after reducing sugars began to appear, so that the specific substance
and the polysaccharide, if not identical, appeared at least to be very
closely associated.  On hydrolysis the specific product yielded about
70 per cent of reducing sugars consisting mainly of glucose, as shown
by the isolation of glucosazone and the formation of saccharic acid
on oxidation, Other possible constituents remained unidentified.

Little work was necessary on the soluble specific substance of type
III pneumococcus to show that marked chemical differences existed
between it and the corresponding derivative of type II. The type
III substance proved to be the soluble salt of an insoluble acid, far
stronger than the type II substance, and capable of being thrown
out of solution by an excess of strong hydrochloric acid.  This property
was of great use, not only in separating the specific substance from
accompanying glycogen or erythrodextrin, but also in effecting a


116                 MICHAEL HEIDELBERGER

rapid purification without the use of ammonium sulfate and wibh
fewer fractionations by alcohol. Successive lots also agreed very
closely in their physical and chemical properties, indicating t.hat one
was apparently dealing in this case with a much more definite chemical
entity than was the type II product.

The type III soluble specific substance was thus also isolated as a
nitrogen-free polysaccharide.  No further purification could be realized
either by precipitation with barium hydroxide in excess, or by adsorp-
tion on highly active alumina (40).

Once the properties of the type III specific substance had been
established it was considered proper to add glucose to the usual Pneu-
mococcus broth, thus securing greatly increased growth and improving
the yields of specific substance from 6 to 9 grams per 300 liters of
broth to 35 to 40 grams.- Wit,h these increased amounts of material
a more detailed chemical study of the substance was made possible.

When the type III specific substance is treated in the cold with
75 per cent (by weight) sulfuric acid until it no longer precipitates
on dilution, and is then further hydrolyzed with boiling normal sulfuric
acid until the maximum reducing power is attained, a small amount
of glucose is formed.  This was identified through the osa.zone and
by oxidation to saccharic acid.  85 per cent of the hydrolysis products
are, however, precipitated by basic lead acetate and yield a crystalline
morphine salt, from which is obtained a sugar acid differing little from
the crude acid before purificat,ion.  Its reducing power is 50 per cent
that of gIucose; its acid equivalent is 363, while the value caMsted
for a disaccharide acid, CliHISO1O.COOH, is 356; on prolonged hy-
drolysis only a small amount of glucose, in addition to unhydrolyzed
material could be isolated, the acid half of the portion hydrolyzed
apparently decomposing similarly to glucuronic acid and yielding
approximately the expected amount of furfural; the reducing group of
this disaccharide acid is aldehydic, as shown by its quantitative de-
termina'tion by fhe WillstBtter-Schudel method (41); the acid also
gives the color reaction with naphthoresorcinol characteristic of the
glucuronic acid type.  Whether t.he union of the glucose to the sugar
acid is through the reducing group of the glucose, or through that of
the sugar acid is as yet unknown.

Since only about 9.5 per cent of the hydrolysis products of the original
polysaccharide appear as glucose, and since the above disaccharide
or aldobionic acid has been shown to yield glucose slowly on further
hydrolysis, it is not illogical to assume that the glucose set free during


CHEMICAL NATURE OF IMMUNE SUBSTANCES      117

the hydrolysis of the specific polysaccharide owes it.s origin, not to a
separate part of the carbohydrate molecule, but chiefly to a secondary
reaction involving the aldobionic acid.  This is also supported by the
fact that no glucose is split off during the preliminary hydrolysis by
75 per cent sulfuric acid in the cold. Since also, the actual partial
hydrolysis products obtained in this way show, by their acid equiva-
lents, one carboxyl group for every two sugar nuclei, it would seem
t,hat the polysaccharide as a whole is built up of units of the disac-
charide acid.  A substance of this composition should have the formula
(Ci2Hi,Oii),, with an acid equivalent of 338 and a carbon and hydrogen
content of 42.6 per cent and 5.4 per cent, respectively.  These figures
are practicaly identical with the values obtained on analysis of the
substance (cf. table 1).

The soluble specific substance of type I pneumococcus was present
in the culture fluid in relatively smaller amount than those of the type
II and type III organisms and therefore required 1.3 to 1.4 volumes
of alcohol for its precipitation from the culture concentrate. Other
modifications were necessitated by the insolubility of the substance
at its isoelectric point, about pH 4, and advanta.ge was taken of its
ability to form a precipitate with barium hydroxide in excess.  Finally
the specific substance was reprecipitated by alcohol in the presence
of hydrochloric acid, and dialyzed. Being a weak base, it precipitated
as the excess of hydrochloric acid was removed.  The yield was 2 to 3
grams. A preparation from type I pneumococci which had been
separated from the accompanying broth, and one subjected to additional
purification by adsorption on alumina failed to show differences from
those isolated as above.

The type I soluble specific substance also appears to be a sugar
derivative, but differs sharply from the other two substances in the
lower percentage of sugar liberated on hydrolysis and in containing
nitrogen as an apparently essential component.

The following data and table 1 are given as a brief summary of the
properties of the soluble specific substances of the three fixed types of
pneumococcus in the state of purity thus far attained.

The type I soluble specific substance rotates the plane of polarized
light about 300" to the right, is a strong acid and a weak base, and is
very sparingly soluble at its isoelectric point, which lies at about pH 4.
In spite of a nitrogen content of 5.0 per cent the substance gives none
of the usual protein color tests.  One-half of the nitrogen is liberated
on treatment with nitrous acid and reducing sugars appear at the


118                MICHAEL REIDELBERGER

same time, while the specific reaction vanishes.  Under the same
conditions the type II and type III substances are unaffected. It
would thus appear that this portion of the nitrogen, at least, is an
int,egral part of the specific substance, and is possibly linked to the
reducing group of a sugar derivative, as Karrer believes is the case in
the polyglucosamines (42).  The substance gives the color react,ion for
glucuronic acid with naphthoresorcino1, but yieIds mucic acid on
oxidation, indicating a relationship to galact.ose. Since the carbon
and hydrogen contents of the substance are close to the theoretical
values for polysaccharides it appears possible that in it a nitrogenous
sugar derivative is linked to galacturonic acid through the reducing
group of the latter.  The type I substance is precipitated by barium
hydroxide in excess, by heavy metal salts, and by phosphotungsbic
acid. In the specific precipitin reaction with type I antipneumococ-
cus serum it can be detected in dilutions as great as 1:6,000,000,
while at a concentration of 1:400 it gives a faint cloud with type III
antiserum.

The soluble specific substance of type II pneumococcus is apparent'ly
a weakly acidic, nitrogen-free polysaccharide made up chiefly of glu-
cose units.  Its acid equivalent is about 1250 and the specific optical
rotation is about +74".  It is not precipitated by barium hydroxide
or heavy metal salts with the exception of basic lead acetate and uranyl
compounds.  It reacts at a dilution of 1: 5,000,OOO with type II anti-
pneumococcus serum but does not precipitate type I and type III
antisera at a concentration of 1:400.  The substance is converted by
acetic anhydride and pyridine into a very sparingly soluble t,riacetyl
derivative.

The type III soluble substance, while also isolated as a nitrogen-
free polysaccharide, is a st.rong acid with an acid equivalent of about
340.  It rotates the plane of polarized light about 33" to the left.  It
is precipitated by barium hydroxide in excess and by heavy metal
salts, and is also rendered insoluble by the addition of strong hydro-
chloric acid.  In as high a dilution as l:G,OOO,OOO it still reacts wit,h
type III antipneumococcus serum.  It appears to be a definite chemi-
cal individual, built up by condensation of an aldobionic acid in such
a way that the reducing groups disappear and the carboxyl groups
remain free.  The evidence so far collected indicates that in the aldo-
bionic acid, CnH200ti, glucose and a hexose-uranic acid are combined
in such a way that one aldehydic group and the carboxyl remain free.
The polysaccharide is thus unusual not only in its possession of the


        CHEMICAL NATURE OF IMMUNE SUBSTANCES       119

property of immunological specificity, but in its chemical constitution
as well.

The three polysaccharides contain no sulfur or phosphorus and
differ from the starch-glycogen group of carbohydrates in their acid
properties, in giving no color with iodine, and in their resistance to
the ordinary carbohydrate-splitting enzymes.  Each substance breaks


                          TABLE 1

Soluble specific substances of the three fixed antigenic types of pneumococcus and of

kiedl&tde-r~ bacillus (type B)

TYPlD

_I

I





II
III

b1L.l

i-300




+74
-33

Friedlander
bacillus B +lOO

-









_-




0






0
0



--


0

-

1250 45.8
340 42.7

--

685 44.6  6.10.0

H N

--
WPQ
06nt cd

5.85.0.

6.40.0
5.30.0

--

-

-
!c7,&w
f&Z
28

_-
I:
P
t









-

-

I
,









. .





-

70
75


-


73
-

(Galacturonic
acid)
(Amino sugar
derivative)
Glucose
Aldobionic
acid, glucose

Glucose

1:6,000,000

1:5,000,000
1:6,000,000

1:2,000,000$

* Theory for (C$HI~O&,,C. 44.4 per cent; H, 6.2 per cent.
i Amino N, 2.5 per cent.
1 Rabbit antiserum.

down on hydrolysis into reducing sugars, a part of which, at least, is
peculiar to itself. The type I substance differs sharply from the other
two in containing nitrogen and in possessing basic as well as acid
properties, while of the other two substances, the type II is a dextro-
rotatory weak acid and the type III a levorotatory strong acid.  Es-
pecially striking is the occurrence of specific substances of such widely
differing properties in microorganisms as closely related as the three
fixed types of Pneumococcus.

With the possible exception of the type III polysaccharide, it is


120                 MICHAEL HEIDELBERGER

doubtful whether each of the specific substances as at present isolated
represents a definite chemical compound. However, in the case of
the three fixed types of Pneumococcus three totally distinct carbohy-
drates have been isolated from cultures grown in the same medium.
Successive preparations of each specific substance have been quite
uniform regardless of the widely different methods employed in the
process of purification, and whether derived from the microorganisms
themselves or from autolyzed broth cultures.  Furthermore, the only
one of these substances as yet investigated in detail appears to differ
in its structure from that of any other known non-nitrogenous poly-
saccharide.

It is thought that these and other considerations based on the data
presented warrant the belief that the three polysaccharides isolated
represent the actual specific substances, stripped of at least a large
portion of accompanying impurities, and that they do not merely
represent inert material carrying an extremely minute amount of the
true specific compounds.  This, if admitted, would afford a chemical
corroboration of the fact of type specificity in Pneumococcus, since
only biological methods had been used in the original investigation (35j.

The extension of the above methods to the Friedlander group of
bacilli resulted in the preparation of another polysaccharide with
specific properties (43), from agar cultures of the so-called "13" strain
of t,his bacillus (now known as type B (44)).  After repeated precipi-
tation with alcohol, then with barium hydroxide, and finally with
alcohol in the presence of hydrochloric acid, it was obtained as a nitro-
gen-free strong acid with an equivalent value of about 685. The
specific optical rotation was +lOO". It did not precipitate with
silver or copper ions, but was thrown down by barium hydroxide in
excess and by both neutral and basic lead acetates.  It gave no color
with iodine. The polysaccharide itself was non-reducing but on
hydrolysis with mineral acid yielded reducing sugars, among which
glucose was shown to be present by isolation of its osazone and by
oxidation to saccharic acid.

From table 1 it will be seen that this substance greatly resembles
that, of the type II pneumococcus, and the similarity was found to
extend even to precipitation of type II antipneumococcus serum.  The
type II substance in its present state of purity is, however, a weaker
acid and is not precipitated by barium hydroxide or neutral lead acetate.
It is possible that these differences depend only on the presence of
different impurities in the two cases, but opposed to this view are the


CHEMICAL NATURE OF IMMUNE SUBSTANCES      121

findings that different preparations of each substance, purified by
widely different methods, showed great constancy in each case, and
that the absorption of agglutinins and precipitins is not reciprocal
with the two organisms.  If the fact that bacteria possess mutual
absorptive capacity be accepted as the criterion of their antigenic
identity, then the failure of the organisms in question to exhibit this
property may be taken as further evidence of the lack of identity of
the substances involved.

However, granted a chemical difference between the two specific
substances, it becomes necessary to account for their marked immuno-
logical similarity.  In the absence of further evidence as to the struc-
tural 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 configuration of atoms. This essential similarity in
molecular grouping would then determine the immunological similarity
of the two substances.

In the case of Pneumococcus it has been shown that the polysaccha-
rides by themselves are not amigenic, and it is believed that they become
antigenic only when attached to some other substance, possibly the
protein of the cell. The type-specific character of the antigenic re-
sponse, however, is dependent almost entirely upon the nature of the
polysaccharide and not upon the substance to which it is attached.
Therefore, since the specific carbohydrate of the Friedlander bacillus
(type B) and that of type II pneumococcus exhibit similar chemical
properties the antigenic response to each may also be similar even
though the proteins or other substances with which they are combined
be quite dissimilar.

A striking and probably analogous example of common antigenic
properties in substances of remote biological origin is furnished by the
phenomenon of heterogenetic specificity originally described by Forss-
man (23; cf. also p. 112).

The fact t.hat two biologically unrelated organisms, pneumococcus
type II and Friedlander's bacillus (type B), possess certain similar
serological and antigenic properties suggests that examples of hetero-
genetic specificity likewise occur among bacteria.  In the case of the
bacteria hitherto studied, however, the specific substance appears to
be a poIysaccharide, instead of a lipoid, and it further appears probable
that when the analogous specific polysaccharides of otherwise totally
unrelated microorganisms correspond sufficiently in chemical con-
stitution an immunological correspondence also results.


122                 MICHAEL HEIDELBERGER

From another strain of FriedlSinder's bacillus, the type of which
has not yet been identified, Mueller, Smith and Litarczek (45) isolated
carbohydrate-containing material with a nitrogen content of 1.3 per
cent and showed that at high dilutions it precipitated homologous
immune serum.

An apparent polysaccharide with specific properties has also been
discovered in the tubercle bacillus, and somewhat similar products
have been obtained by Laidlaw and Dudley (46) from the defatted
bacilli, and by Mueller (47) from the culture itself, by widely differing
methods. Laidlaw and Dudley heated the bacilli with weak alka.li,
and the portion of the extract not precipitated by acetic acid or copper
sulfate was adsorbed with uranium hydroxide and the specific material
carried by the adsorbent freed from this and separated from accompany-
ing glycogen by repeated fractionation with alcohol. Mueller freed
the culture concentrate from nucleic acid derivatives, treated it wit,h
"anfiformin," and fractionated the specific material under varying
conditions from alcohol.  In both instances products giving the reac-
tions of a pentose-containing polysaccharide were obtained, dextro-
rotatory, practically nitrogen free, and reacting at high dilutions wil;h
the sera of animals immunized against the tubercle bacillus. The
sugars obtained on hydrolysis were not identified. Laidlaw and
Dudley `s mat.erial showed [a]ng green + 79' and 50 per cent of reducing
sugars on hydrolysis, while the figures for Mueller's product were
[a], +24" and 99 per cent, respectively.  The polysaccharide appears
responsible for the precipitin reaction given by tuberculin.

Specifically reacting material, also apparently carbohydrate in nature,
has been isolated from yeast by fractionation with alcohol. The
product, closely associated with, or identical with the yeast gum,
precipitated the sera of rabbits immunized against the yeast (48).

The wide distribution of specific polysaccharides among micro-
organisms of the most diverse types made it seem not improbable
that there might occur among the higher plant forms other carbohy-
drates with specific properties.  In a series of tests on water-soluble
plant gums samples of gum arabic (gum acacia) were actually found
which showed some degree of specific activity.  By fractional hydroly-
sis with 1: 1 hydrochloric acid it was found possible to remove about
one-half of the pentose present.  [LY]~ of the recovered gum was about
- 10" and its specific activity had increased lOO- to 150-fold (49).  The
interpretation of these findings is withheld until further data are
available.


CHEMICAL NATURE OF IMMUNE SUBSTANCES       123

Initial studies of the relationship of the soluble specific substances
of bacterial origin to other cell constituents have been made in the
case of Pneumococcus (38b, 50, 51, 38e). When these microbes are
dissolved, either with the aid of bile, or by repeated freezing and thaw-
ing, the resulting solution yields a precipitate of so-called "nucleo-
protein" on acidification with acetic acid.  While probably a mixture
consisting largely of nucleoprotein and mucoid, it still possesses im-
munological properties which differ sharply from those of the soluble
specific substance. In the first place, the protein is antigenic, while
the soluble specific substance, though reacting specifically with anti-
bodies to the highest degree, is non-antigenic and unable by itself to
stimulate the production of antibodies when injected into animals.
Moreover, the protein isolated from any one of the three fixed types
of Pneumococcus, or from a strain of the heterogeneous group IV,
appears serologically the same as that from any of the other types.
Thus this portion of the pneumococcus protein is not type-specific,
like the soluble specific substance, but is, rather, species-specific.
Since many of the immune reactions participated in by bacteria
are presumably surface phenomena, the nature of the reactive material
at the periphery of the cell may determine the readiness of response
and even the specificity of reaction.  Pneumococcus is an encapsulated
organism, and there are grounds for the belief that the ectoplasmic
layer of the cell is composed of carbohydrate material which is identical
in all its biological characters with the type-specific substance. On
the other hand, the endoplasm, or somatic substance, consists largely
of protein which, as previously pointed out, is species- and not type-
specific. This protein is possessed in common by all pneumococci,
while the carbohydrate is chemically distinct and serologically specific
for each of the three fixed types.  The cell, therefore, may be con-
ceived of as so constituted that there is disposed at its periphery a
highly reactive substance upon which type specificity depends.
The fact that this specifically reactive carbohydrate is non-antigenic
when separated from the other cellular constituents and is capable of
inciting antibody formation only in the form in which it is present in
the intact cell, forces the conclusion that in the latter instance it exists
not merely as free carbohydrate but also in combination with some
other substance which confers upon it specific antigenic properties.
Immunization with intact bacteria containing this carbohydrate
complex elicits antibodies which not only agglutinate the formed cells
but precipitate solutions of the carbohydrate isolated from pneumococci


124                MICHAEL HEIDELBERGER

of the homologous type.  How the specific polysaccharide is combined
in the cell, whether with protein or some other constituent is not yet
clear, but it is evident that the compound thus formed is the dominant
and essential antigen of the cell, and the one responsible for type
specificity. Immunization with dissolved pneumococci, on the other
hand, results in the formation of antibodies which precipibate the
pneumococcus protein but not the soluble specific substance.

The immunoIogica1 relationships of the protein and carbohydrate

TABLE 2

Material used for immunization

Intact cells (SP)t . . . . .
Carbohydrate St. . .
Protein Pg. . . . . . . . _.
Solutions, extracts containing free
S and free P . . . . . . . .
Suspension of intact cell8 and dis-
sociated cell constituents (SP),
free S, free P.. . . . . . . . _.

-

Effective
antigen

em
None
P


P

W), p

-




I .-
.c
2
P
=z
T
-
-


-



+

-

-




. -

I%-
cipitins

- -



S
- -
+
-
-


-



t

-



P
-
-
-

+


+



+

-

Comple-
ment
fixation

Tl
I'

-



S'
-
+
-
-


-



+

-




_-

_-



I'
-
-
-
+


+



+

--

3Pecilicity

I
`5
d
P

-
-

+


+



+
_-

* = Free S, as antigen, does not fix complement with immune horse serum;
is active with immune rabbit serum.J
t (SP) = Carbohydrate and protein, combined antigen of cell.
$ S = Free carbohydrate, the soluble specific substance of cell.
9 P = Free protein of cell.

fractions of the cell are graphically presented in table 2, in which S
represents the soluble specific substance (carbohydrate) and P the
protein of Pneumococcus.

It is evident that morphological dissolution of pneumococci is accom-
panied by antigenic dissociation, for sera prepared from filtered solutions
of disintegrated cells free of formed elements fail to exhibit any of the
dominant type-specific properties which characterize sera obtained
by immunization with whole bacteria. The injection of suspensions
of pneumococci into animals induces the formation of antibodies

8 Unpublished data obtained by E. Vollmond.


CHEMICAL NATURE OF' IMMUNE SUBSTANCES       125

against S alone or against both S and P separately, depending upon
whether or not these suspensions contain only intact cells or a mixture
of both intact and dissolved cell bodies.  Since pneumococci readily
undergo autolysis and dissolution, suspensions and indeed cultures of
these organisms almost invariably contain not only formed elements,
but also more or less of dissociated cell constituents in solution.  There-
fore, use of suspensions of pneumococci containing both intact cells
and the soluble products of cell disintegration yields on immunization
not only type-specific antibodies but antibodies reacting with the
protein substance which is common to all pneumococci. While the
former generally predominate it is the presence of this protein antibody
with its broader zone of activity which is responsible for the confusing
cross-immunity reactions occasionally encountered in supposedly
type-specific sera and which has in some instances led workers even
to deny the existence of three distinct antigenic types of pneumococci.
That the two sets of antibodies involved are separate and distinct is
shown by absorption test's; the antiprotein reacting bodies in such
sera can be removed by absorption with the protein of a heterologous
type without diminishing the titer of specific agglutinins for the homolo-
gous culture or the precipitins for the specific polysaccharide of t,he
corresponding type.

The possible relation of the specific polysaccharides to virulence and
disease has been discussed elsewhere (lot. cit., also 52,53), and is outside
the scope of the present paper, as is also a discussion of advances which
the above type of analysis has already permitted in the study of the
Encapsulating group (54) and the non-hemolytic streptococci (55).

The specifically reacting polysaccharides offer much of chemical
interest.  While it had generally been assumed that only the proteins
and their derivatives provided the innumerable opportunities for iso-
merism and subtle changes requisite for the substances exhibiting the
phenomena of specificity, the discovery of carbohydrates with specific
properties is not as surprising as might appear on first thought.  When
one considers the number of asymmetric carbon atoms in the hexoses
and pentoses, the different possible points of attachment of the lactone
bridge, the possibility of a- and @- glucosidic unions at various positibns
in the molecule, and the addition of sugar acids, the analogs of amino
acids, to the large number of sugars theoretically capable of entering
into the composition of such polysaccharides, it becomes clear that
perhaps only among the carbohydrates could another sufficiently large
and protean group of substances be found to afford the possibility of
specific manifestations.


126                 MICHAEL HEIDELBERGER

At the present early stage of the development of the field thus opened
up it would be idle to speculate on the relationship within this group
of specificity to chemical constitution.  That some such relation
exists, however, and is reflected by marked chemical changes has been
fully demonstrated in the case of the soluble specific substances of the
three antigenic types of Pneumococcus.  Also significant is the finding
that, even in the case of such widely different microorganisms as
Pneumococcus type II and the type B Friedlander bacillus, a certain
similarity in the chemical nature of their specific polysaccharides is
accompanied by a corresponding similarity in the immunological
properties of the organisms themselves.

So far only the specific polysaccharide of type III pneumococcus has
been investigated in detail, and it is too early to render judgment as
to whether or not the polysaccharides with specific properties form a
chemically distinct group of carbohydrates.  In this one instance the
structure certainly appears different from that of any known non-
nitrogenous sugar derivative, although an analogy to the nitrogen-
containing chondroitin sulfuric acid (56) is discernible, and perhaps
even a distant relationship to the pectins (57).  In this connection the
finding of a specifically reacting polysaccharide as a constit,uent of
gum arabic is of interest, and it is not impossible that sugar derivatives
with specific properties may occupy a position of biological significance
among higher forms of life as well as among bacteria.

Under the three main headings which have been taken up, the list
of immune substances of interest and importance has by no means
been exhausted. Complement, for example, has not been discussed,
since recent work has failed to establish whether complement is merely
a colloidal state, or an actual chemical entity or group. The study
of bacteriophage, as well, is still in too early and controversial a state
for discussion in a review of such limited scope as the present one.

The fragmentary and incomplete character of these gropings toward
the actual chemical nature of immune substances is obvious.  Future
progress is dependent upon new methods as well as upon the applica-
tion of those used with success in other fields of organic chemistry.
However, the little that has already been accomplished has not been
without theoretical and practical implications sufficient to stimulate
renewed endeavor.


CHEMICAL NATURE OF IMMUNE SUBSTANCES       127

BIBLIOGRAPHY

(1) WELLS, H. G. The chemical aspects of immunity. New York, 1925.
(2) ABDERHALDEN, E. ET AL. Zeitschr. physiol. Chem., 1924, cxxxix, 169, 181,
    and numerous other papers.

(3s) BERQMANN, M. AND A. MIEKELEY.  Ibid., 1924, cxl, 128.
(3b) BERWANN, MIEHELEY, F. WEINMANN AND E. KANNS. Ibid., 1925, cxliii,
     108.

(4) TROENSQAARD, N. AND Eug. FISCHER. Ibid., 1925, cxlii, 35; cxliii, 304,
    and other papers.

(5) HEIDELBERQER, M. AND K. LANDSTEINER. Journ. Exper. Med., 1923,
    xxxviii, 561.

(6) LANDSTEINER. Klin. Wocheuschr. (in press).
(7) DERNBY, K. G.  Journ. Gen. Physiol., 1926, viii, 311.
(8) WATSON, 0. F. AND E. LANQSTAFF. Biochem. Journ., 1926, xx, 763.
(9) WATSON AND U. WALLACE.  Journ. Path. Bact., 1924, xxvii, 289.
(10) GLENNY, A. T. AND G. S. WALPOLE.  Biochem. Journ., 1915, ix, 298.
(11) HUNTOON, F. M.  Proc. Sot. Exper. Biol. and Med., 19234, xxi, 513.
(12) HALLAUER, C. Zeitschr. Hyg., 1925, cv, 138.
(13) LONG, E. R. AND F. B. SEIBERT.  Amer. Rev. Tuberc., 1926, xiii, 393, 398,
    404,408,431, 441, 448.
(14) MUELLER, J. H. Journ. Exper. Med., 1926, xliii, 1.
(15) SEIBERT. Science, 1926, lxiii, 619.
(16) SALKOWSKI, E. Biochem. Zeitschr., 1922, cxxxii, 84.
(17) DOLD, H. AND E. FREUDENBERQ. Ibid., 1925, clxii, 169.
(18a). HUNTOON, F. M. Journ. Immunol., 1921, vi, 117.
(18b) HUNTOON AND S.ETRIS. Ibid., vi, 123.
(18~) HUNTOON, P. MMUCCI AND E. HANNUM.  Ibid., vi, 185.
(19) OTTENBER(;I, R. Proc. Sot. Exper. Biol. and Med., 19234, xxi, 14; OTTEN-
     BERG AND F. STENBUCK. Ibid., xxi, 303; 1924-5, xxii, 211,215; 1925-6,
    xxiii, 23,

(20) LOCKE, A. AND E. F. Hmsca. Journ. Inf. Dia., 1924, xxxv, 519; 1925, xxxvii,
      449.

(21) LOCKE, E. R. MAIN AND HIRSCH.  Ibid., 1926, xxxviii, 126.
(22a) FELTON, L. D.  Boston Med. and Surg. Journ., 1924, cxc, 819.
(22b) FELTON. Journ. lnf. Dia., 1925, xxxvii, 199, 309; Bull. Johns Hopkins
    Hosp., 1926, xxxviii, 33.
(22~) FELTON AND G. H. BAILEY.  Journ. Immunol., 1926, xi, 197.
(22d) Paper read before American Chemical Society, Philadelphia, September 9,
     1926.

(23) FORSSMAN, J. Biochem. Zeitschr., 1911, xxxvii, 78.
(24) LANDSTEINER. Ibid., 1921, cxix, 296.
(25) TANIQUCHI, T. Journ. Path. and Bact., 1921, xxiv, 253, 254.
(26) LANDST~INER AND H. S. SIMMS. Journ. Exper. Med., 1923, xxxviii, 127.
(27) SORDELLI, A., R. WERNICKE AND V. DEULOFEU. Compt. rend. BOC. biol.,
     1925, xcii, 898.

(28) LANDSTEINER AND P. A. LEVENE. Journ. Immunol., 1925, x, 731; Proc.
    Sot. Exper. Biol. and Med., 1925-6, xxiii, 343.


128                 MICHAEL HEIDELBERGER

(29) DIENES, L. AND E. W. SCHOENHEIT. Journ. Immunol., 1925, x, 631; Proc.

    Sot. Exper. Biol. and Med., 1925-6, xxiii, 106.
(30) DIENES AND J. FREUND.  Journ. Immunol., 1926, xii, 137.
(31) LANDSTEINER, J. VAN DIR SCHEER AND D. H. WITS. Proc. Sot. Exper.

Biol. and Med., 1924-5, xxii, 289.

(32) SCHMIDT, H. Zeitschr. Immunitiitsf., 1924, xxxviii, 511.
(33) SACHS, H., A. KLOPSTOCK AND A. J. WEILL. Deutsch. Med. Wochschr.,

1925, Ii, 589, 1017.

(34a) KLOPSTOCIC, F. Ibid., 1926, lii, 226.
(34b) LANDSTEINER AND VAN DER SCHEER.  Proc. Sot. Exper. Biol. and Med.,
    1925-6, xxiii, 641.

(35) DOCHEZ, A. R. AND L. J. GILLESPIE. Journ. Amer. Med. Assoc., 1913, lxi,

      727.
(36) DOCHEZ AND 0. T. AVERY.  Journ. Exper. Med., 1917, xxvi, 477.
(37) ZINSSER, H. AND J. T. PARKER.  Ibid., 1923, xxxvii, 275.
(38a) HEIDELBERGER AND AVERY. Ibid., 1923, xxxviii, 73.
(38b) AVERY AND HEIDELBERGER. Ibid., 1923, xxxviii, 81.
(38~) HEIDELBER~ER AND AVERY. Ibid., 1924, xl, 301.
(38d) AVERY AND HEIDELBERQER. Ibid., 1925, xlii, 367.
(38e) HEIDELBERQER, W. F. GOEBEL AND AVERY.  Ibid., 1925, xlii, 727.
(38f) HEIDELBERQER AND GOEBEL.  Journ. Biol. Chem., 1926, lxx, 613.
(39) Prepared essentially according to FELTON (22a).
(40) Prepared according to R. WILLSTHTTER AND H. KRAUT.  Ber., 1923, lvi, 149.
(41) WILLSTXTTER AND C. SCHUDEL.  Ber., 1918, li, 780.
(42) KARRER, P., 0. SCHNIDER AND A. P. SYIRNOFF. Helvetica Chim. Acta,

1924, vii, 1039.

(43a) HEIDELBERQER, GOEBEL AND AVERY. Journ. Expor. Med., 1925, xlii, 701.
(43b) AVERY, HEIDELBERGER AND GOEBEL.  Ibid., 1925, xlii, 709.
(44) JULIANELLE, L. A.  Ibid., 1926, xliv, 113.
(45) MUELLER, D. E. SMITH AND S. LITARCZEK. Proc. Sot. Exper. Biol. and

Med., 1924-5, xxii, 373.

(46) LAIDLAW, P. P. AND H. W. DVDLEY.  Brit. Journ. Exper. Path., 1925, vi,
      197.

(47) MUELLER. Journ. Exper. Med., 1926, xliii, 9.
(48) MUELLER AND J. TOMCSIK.  Ibid., 1924, xl, 343.
(49) HEIDELBERQER, AVERY AND GOEBEL. Paper read before Annual Meeting,
    American Chemical Society, Philadelphia, September 9, 1926.

(50) ZINSSER AXD T. TAMIYA.  Journ. Exper. Med., 1925, xlii, 311.
(51a) AVERY AND H. J. MORQAN. Ibid., 1925, xlii, 347.
(51b) AVERY AND J. M. NEILL.  Ibid., 1925, xlii, 355.
(52) SIA, R. H. P.  Ibid., 1926, xliii, 633.
(53) FELTON AND BAILEY.  Journ. Inf. Dis., 1926, xxxviii, 131.
(54) See (4) and Journ. Exper. Med., 1926, xliv, 683, 735.
(55) LANCEFIELD, R. C. Ibid., 1925, lxii, 377.
(56 LEVENE. Hexosamines and mucoproteins. Longmans, Green & Co.,
    London, 1925.

(57) EHRLICH, F. AND R. VON SOMMERFELD.  Biochem. Zeitschr., 1926, clxviii,
      263.