IMMUNOCHEMISTRY*
        BY MICHAEL HEIDELBERGER
    Department of Medicine, College of Physicians and Surgeons
      Columbia University, and the Presbyterian Ho&al
               New York City

The division of the subject is as in Volume II. Space requirements
have again made it necessary to eliminate mention of many papers.
The period covered is essentially that from December, 1932, to No-
vember, 1934.
During this time the literature of immunochemistry has been
enriched by three important books. The first, by Landsteiner (l),
which is to appear in English,l gives impartially and fairly all possible
interpretations of the phenomena of specificity with amazingly com-
plete references to the original literature. The second, by Topley (2))
provides a stimulating discussion of modern work and theories, em-
.phasizes the importance of the bacterial surface in reactions with
antibodies, and the necessity of the evaluation of the probable error
before a conclusion is drawn, but abandons the last principle in ac-
cepting the "optimal proportions" method as a standard for precipitins
and agglutinins. The third, a monograph by Marrack (3), gives
clearly and with graphs, which in some cases improve on the original
papers, a critical survey of the physics and chemistry of antigens,
antibodies, and their interaction. To the benefit of his reader, Mar-
rack does not hesitate to speculate, and is thereby enabled to provide
a most plausible and suggestive picture of specific combination, pre-
cipitation, and agglutination, with which, however, the reviewer is not
wholly in accord. In a monograph by Abramson (4) there is also a
chapter on "bacteria, antibodies, viruses, and related systems." For
a review on antigens and a lecture on immunochemistry see 4a and 4b
respectively.
      THE CHEMISTRY OF IMMUNE SUBSTANCES

              NATURALLY OCCURRING ANTIGENS
From precipitin tests with native serum albumin (A), heat-dena-
tured or acetone-denatured serum albumin (B), and B in which the
denaturation had been reversed (C), and the corresponding antisera,
* Received Jan&y 16, 1935.
  1 Private communication from the author.

                     569


  570       ANNUAL REVIEW OF BIOCHEMISTRY
Miller (5) concludes that B is markedly changed in its antigenic prop-
erties, although anti-C serum reacted almost equally well with A, B,
and C. A and C were indistinguishable immunologically. The differ-
ences in the case of B were accentuated by a long-continued denatur-
ation process. IIcwitt (6)' however, considers evidence lacking that
the "reversed" albumin had ever been denatured. It is to be hoped that
Anson and Mirshy, who have worked so much with the reversal of
denaturation, will clear up these difficulties. Hektoen & Welker (7)
have shown that plasma, adsorbed on AI(O stimulates a lasting
production of antibodies difiering by the presence of anti-fibrinogen
from those produced by serum. These workers (8) have also studied
the antigenic properties of "Arndt" and Bence-Jones urinary protein
fractions. Ronse (9) has shown that. the antibody to human casein.
does not precipitate the casein of cow's milk, while Lewis (9a) found
goat casein to be isoantigenic by the complement-fixation reaction.
Precipitin formation was not mentioned. Ratner & Gruehl (10) have
given evidence that intact protein could pass. the intestinal walIs of
some of the guinea pigs tested. Block & Brand (11) have shown that
90 per cent of brain protein has the properties of nucleoprotein, and
that these fractions of beef and pig-brain proteins are antigenically
distinct. Schwentker & Rivers (12) have found that fresh emulsions
of homologous brain are scarcely antigenic, but autolyzed emulsions
and those infected with vaccine virus produce complement-fixing anti-
bodies which are partly organ specific. The specific antigen is more
abundant in the white matter and parallels the myelin content. Jorpcs
(13) has studied the fibrin-like "stromatin" in horse and sheep red
  cell stroma.
  Enzymes have again been shown to be antigenic by Ten Broeck
(14)' who found that trypsin, chymotrypsin, and chymotrypsinogen
actively sensitize guinea pigs with some degree of specificity. The last
two do not readily form precipitins in rabbits. Walton & Segura (15),
however, found no evidence of antipapain formation in dogs, the
enzyme precipitating both normal and 5nmune" dog serum.
   As for the vegetable proteins, Ishigami (16) has found rice and
soy bean glutelins to be antigenic, but the partial hydrolysis products
of the latter are immunologicalIy inactive. Additional evidence has
been presented that the allergens of ragweed pollen are proteins (17).
   The bacterial antigens have received much attention. In the
Sa&zonella group White (18) has continued his antigenic analysis,
finding that in B. protetcs X 19 the "0" or heat-stable somatic complex


               IMMUNOCHEMISTRY            571
contains an alkali-labile factor responsible for the "0" agglutination
in homologous antiserum and an alkali-stable facto? responsible for
the cross reaction with typhus serum. From members of the group,
in addition to the "Q" protein, White has removed with a higher acid-
alcohol concentration a "T" protein flocking at pH 7.2, common to the
group, and probably not on the bacillary surface. Antibodies to this
protein agglutinate "R" and "g" strains but not alcohol-washed "S"
or young living cultures. The special serological properties of the e
form depend on carbohydrate haptens (20). By extraction of B.
Aertrycke with 0.25 N trichloroacetic acid in the cold, Boivin and
collaborators (21) have isolated an antigenic complex which is split
by boiling acetic acid into a non-antigenic specific polysaccharide and
a phosphatide, the latter yielding a crystalline fatty acid3 on further
degradation. Other organisms gave analogous products. Similar re-
sults with B. Aertrycke have been reported by Raistrick & Topley
(22). Grasset (23) has detoxified extracts of the typhoid group of
organisms with formalin and reported success in the immunization of
humans.
Day has continued his study of pneumococcus antigens (24)) find-
ing a type-specific antigen which may be extracted with dilute hydro-
chloric acid, but which is dissociated by alkaIi or by pneumomoccus
autolytic enzymes to a species-specific antigen tid is then destroyed.
An acid-resistant, alkali-labile antigen, yielding protective antisera,
was also extracted from very virulent hemolytic streptococci (25).
Boor & Miller (26) have studied gonococcus and meningococcus
"nucleoproteins" [cf. also Rake & Scherp (27) J , finding them almost
as toxic as the organisms themselves and failing to obtain evidence of
separate endotoxins. Antisera to the proteins contained antibodies
reacting with the specific polysaccharides, but since the antigens were
not filtered to remove bacterial fragments their presence was not ex-
cluded. Similar observations have, however, frequently been made in
the reviewer's laboratory in the case of filtered streptococcus and
tubercle-bacillus proteins.4
Strains of the Brucella group studied by Huston, Huddleson &
Hershey (28) contained a small amount of a water-soluble "albumin"

2 Probably a specific polysaccharide according to Castaneda (19).
s Unpublished experiments by the reviewer and Menzel have shown that
magnesium palmitate is split from the high-rotating polysaccharide of tubercle
bacilli by treatment with alkali.
+ Unpublished experiments.


572       ANNUAL REVIEW OF BIOCHEMISTRY

which disappeared rapidly if enzyme action was not avoided, and 4 to
7 per cent of a water-soluble nucleo-protein which split off nucleic
acid when treated with alkali.

Tomcsik & Szongott (29) have given more study to the type-
specific "protein" isolated from the capsule of the anthrax bacillus,
which, however, fails to give most of the protein reactions. Its chemi-
cal behavior appears to the reviewer to be remarkably like that of the
specific polysaccharide of Type I pneumococcus.

Analyses of cholera-vibrio proteins are reported by Linton and
co-workers (30). Krueger (31 j has given a method of disintegrating
bacteria by extraction with buffer in a mechanical grinder. This was
adopted by Huston et aJ. (28), using ice to minimize enzyme action
and water instead of buffer.

  Proteins of a human strain of tubercle bacillus have been frac-
tionated by Heidelberger & Menzel (32) by the method originally
used for Streptococcw. Fractions of differing optical rotation and
phosphorus content were obtained, and of these the neutral extract-
able protein was antigenically distinct from the portion extracted by
strongly alkaline media. All fractions showed a strong tuberculin
activity, differing in this respect from the relatively inactive "albumin"
and "globulin" isolated from the bacillus by Gough (33) without pre-
cautions to avoid enzyme action. Pedersen-Bjergaard (34) has found
that tubercle-bacillus phosphatides prepared according to Anderson
stimulate the production of complement-fixing antibodies. Since most
of the nitrogen in the phosphatides could be liberated as ammonia it
was considered that the antigenic properties could not be due to pro-
tein. Gough (35) has purified the skin-active material in tubercle-
bacillus filtrates by precipitating benzoic acid in the liquid, drying the
precipitate, and removing the benzoic acid with acetone. No analyses
are given. Spiegel-Adolf & Seibert (36) found that nucleic acid
could be removed from purified tuberculin by ammonium sulfate f rac-
tionation without impairing its activity. Seibert & Munday (37)
found no marked differences in the composition of ammonium sulfate-
purified tuberculins from human, bovine, avjan, or timothy strains.
The presence of polysaccharide influenced the nitrogen partition.
Cystine nitrogen was lowest in the human tuberculin. Schaefer &
Sandor (38) found that the proteins in tubercle-bacillus culture fluid
gave rise to complement-fixing antibodies distinct from those formed
in response to the bacillary lipoids. Boquet & Sandor (39) found a
specificity difference in the proteins of unheated tubercle-bacillus cul-


              IMMUNOCHEMISTRY           573
tures and those of heated tuberculin. Kall6s & Hoffmann (40) report
that the activity of a polypeptide, "@-tuberculin," isolated by ultrafil-
tration from bouillon cultures or the blood, urine or skin of tubercu-
losis patients is proportional to the tryptophane content.
Pyl (41) has pointed out physicochemical analogies between the
virus of foot and mouth disease and proteins and enzymes. Galloway
(42) has shown that the fixed virus of rabies is still antigenic after
inactivation by the combined action of dyes and light.
Much work has been done with bacteriophage, Schlesinger (43)
has effected a far-reaching purification of an anti-B. coli phage, get-
ting twenty to fifty times the usual concentration on a synthetic
medium. By ultrafiltration, removal of the phage from the membrane,
and fractional centrifugation at high speed, the phage was obtained
free from agglutinogen as a sulfur-like solution with properties much
like those of nucleoprotein. Fifteen mg. failed to show respiratory
or fermentative activity. The particles were usually agglutinated by
antiserum, but could be neutralized without agglutination. Andrewes
& Elford (44) found that the presence of phage-antiphage complex
failed to retard neutralization of fresh phage by antiphage, which was
taken as evidence against a mass-law explanation of the reaction.
There was also no Danysz effect. It was considered that different
phage particles varied in their resistance to antibody, since a given
amount of serum neutralized 95 per cent of the phage in four hours
regardless of the phage dilution. In confirmation of Asheshov & Ser-
tic's observation, the inhibition of phage-antiphage interaction (4.5)
by bacterial extracts has been traced by Burnet & Gough (46) and
Levine & Frisch (47) to a specific polysaccharide which Burnet &
Gough have found to be alkali-labile with respect to its phage-inhibit-
ing power but not as to its antiserum-precipitating property. Burnet
(48) has also studied the chemical inactivation of phage, as have Wells
& Sherwood (48~) ; and Krueger and co-workers (49) have shown
that the inactivation by mercuric or cyanide ions may be completely
reversed by suitabIe removal of the inactivating ion, so that the inacti-
vation appears to be Iike that of an enzyme. Meyer & Taslakowa (SO)
conclude, however, from the antigenic invariance of phage on sub-
strates of different antigenicity that assimilative activity and forma-
tion of new substance are shown.
Bacterial bemolysins have also been studied extensively. In the
action of staphylolysin on the red cells of different animals Forssman
(51) reported that it behaved much as an enzyme, and coutd some-


574       ANNUAL REVIEW OF BIOCHEMISTRY
times be found in undiminished amount when lysis was complete. The
great difference from lysis by antibody, or immune hemolysin, is
stressed. Birch-Hirschfeld (52) has shown that hemolysin and pro-
tease run parallel in extracts of S~u~k.ylococci grown on cellophane
agar, but may be separated. Schwachman, Hellerman & Cohen (53)
have found that Paewnococcats hemolysin behaves like sulfhydryl com-
pounds, being reversibly inactivated by cuprous oxide and organic
mercury compounds capable of forming stable mercaptides. Its oxi-
dation-reduction behavior is much like that of urease and papain.
Again there has been much work on the neutralization of toxins
by various substances, and many methods, mainly modifications of
older methods, have been used for the purification of toxins, but since
these have not yet led to the actual isolation and study of a chemically
well-defined, pure toxin, they cannot be taken up in a brief review.
Only Krestownikowa & Rjachina (54), who described purified ery-
sipelas and meningococcus toxins, have reported for comparison
parallel experiments on the broth used. Wheeler (54a) did not find
any correlation between protein synthesized and toxicity in diphtheria
cultures on synthetic media.
Schmidt (55) has made a detailed study of the mechanism of
diphtheria-anatoxin formation by formaldehyde, finding that it differs
from "toxoid" in its non-reversibility, stability, and lower amino nitro-
gen content. Contrary to Burney, it could be prepared from purified
toxin with very low concentrations of formaldehyde. Hooker & Fol-
lensby (56) have given evidence that scarlatinal toxin may contain
at least two separate toxins characterized by different chemical and
physical behavior.

       CHEMICALLY ALTERED PROTEINS AS ANTIGENS
Wormall's views, as against those of Bruynoghe and Adant, on
the serological relationships of bromo and iodo proteins, have been
confirmed by Finkelstein (57). Hopkins & Wormall (58) have made
a study of phenylureido- and p-bromophenylureido proteins formed
by the action of the aryl isocyanates at pH's not exceeding 9.5. The
precipitin and inhibition tests support the view that the free amino
groups in intact protein (also the reactive groups with isocyanates)
are the s-amino kqoups of lysine. The specificity changes were much
like those produced by the introduction of aryl azo groups into pro-
teins. The ureido gelatin derivatives were not antigenic but precipi-
tated antisera to the ureido horse-serum globulins and ureido caseino-


              IMMUNOCHEMISTRY           575
gens. With the ureido amino acids complete inhibition of specific
precipitation was obtained only with those from lysine and e-amino-
n-hexoic acid. Somewhat at variance with the above is Lewis' finding
(5&z) that deaminized casein seemed immunologically identical with
casein. Doerr & Girard (59) have found that atoxylazo-racemized
egg albumin is not antigenic, but is precipitated by antisera to atoxyl-
azo egg albumin. Reiner (59u) has reported greatly impaired immu-
nizing power for toxin or toxoid put through the coupling process
with diazotized atoxyl.

RAPTENS

General.-As will be seen below, the distinction between haptens
and antigens is no longer absolute, but "haptim" is so convenient a
term for the portion of an antigen determining a particular specificity
that it will undoubtedly continue to be used. Medveczky (60) has
proposed a logical but unnecessary and somewhat cumbersome system
of nomenclature for the haptens.

Erlenmeyer, Berger & Leo (61) have continued their work on
the immunological relationships of substances with equal force fields,
improving their technique to meet objections previously raised. They
found crossing between CaH, * CO . NH s CaHl * NT and C4HsS *
CO - NH. C,H, * N2-, also a relation between -SOsH and -SeO,H,
as predicted by the theory. CaH, * CO * C,H, . NT and C1,H, . CO *
NH - CoH, * N,L were not related to the first group nor was -SO,H to
the second. Berger & Erlenmeyer's views (61~) on the relation be-
tween molecuIar size and affinity for antibodies require more and
better supporting evidence.

Boyd & Hooker (62) have found that more diazotized atoxyl can
be combined with protein than can be accounted for by the tyrosine
and histidine content. Other amino acids failed to yield colored
compounds under the conditions used, so that the question of how
the excess is combined in the protein is left open. The possibility of
diazoamino compound formation does not seem to have been con-
sidered. Hooker & Boyd (62a) found that atoxylazo gelatin (A) did
not precipitate in homologous antiserum which, however, gave pre-
cipitates with atoxylazo casein or atoxylazo egg albumin (B). On
the basis of inhibition tests it is concluded that A gives rise only to
azohistidine antibody, while I3 forms anti-azotyrosine as well. This
hypothesis is supported further by inhibition tests with atoxylazo-
phenol (C) and atoxylazoimidazole, instead of the corresponding tyro-


ANNUAL REVIEW OF BIOCHEMISTRY

sine and histidine compounds. Since the latter differ by containing
two azo groups instead of one, and C has a j-hydroxy group instead of
the o-hydroxy group in the tyrosine compound, the evidence of the
tests loses greatly in force. Finally, the differences between crystalline
hen- and duck-egg albumins were studied and explained on the above
basis of the existence of antigenic determinants of diverse specificity
in a single protein. It is concluded that one of the two antibodies pro-
duced by hen-egg albumin is different from that produced by duck-egg
albumin, while the other is similar, but not necessarily identical.

That multiple reactive groups determine the outcome of specific
precipitation has also received strong support from Heidelberger &
Kendall (63), who found that the cross reaction between crystalline
egg albumin and antiserum to R-salt-azo-benzidineazo-crystalline egg
albumin differed remarkably in its quantitative aspects from the
homologous egg albumin-anti egg albumin and dye-antidye reactions.
It was concluded that the egg albumin formed a highly dissociated
complex with the antidye, and this was shown to be in agreement with
Landsteiner's work on azohaptens and specificity. It was also shown
that in this particular instance it was not necessary to assume the
presence of more than a single antibody in the antidye sera.

Landsteiner & van der Scheer (64) sensitized guinea pigs with
azoproteins prepared f r0rn.paminosuccinanilic and -suberaniIic acids
and were able to shock them fatally with the homologous bisazo com-
pound prepared by coupling the hapten with resorcinol.

Goebel, Avery & Babers have continued their fundamental study
of carbohydrate haptens linked to protein through the diazo reaction,
Introduction of an acetyl group into a l?l-glucoside was found to abol-
ish the cross reactions otherwise observed with unacetylated a-gluco-
side (65). The paminophenol glucosides of the disaccharides cello-
biose, maltose, gentiobiose, and lactose were diazotized and coupled
with protein (66). The immune reactions of these products appeared
in the main to be determined by the glucoside molecule as a whole, the
configuration of the terminal hexose molecule, and the position of
linkage of the two hexose units in the carbohydrate radical.

Specific poE_ysaccharides.-The nature of the discrepancy between
the type-specific polysaccharide (SI) of Pneumococcus I, as originally
isolated, and the antigenic products first reported by Perlzweig &
Steffen and later by Schiemann & Casper, Enders, Felton, and Wads-
worth & Brown (67) has been cleared up by Avery & Goebel (6g),
who showed that preparations made without the use of alkali con-


             IMMUNOCHEMISTRY           577
tained 5.9 to 6.9 per cent of acetyl, failed to precipitate at the iso-
electric point as did the original SI, protected mice in very small doses
against infection by the homologous organism, produced purpura
when given in large doses, and removed all protective antibodies from
Type I antiserum. Treatment with alkali spIit off the acetyl groups
and gave SI as originally isolated, which was non-antigenic in mice
and failed to remove all protective antibody, precipitin, or agglutinin
from antiserum. Both products, however, reacted with antiserum at
dilutions of one to several millions, and it was this circumstance of
equal "titers" which originally led the reviewer to believe that alkali
could safely be used in the preparation. The quantitative, absolute
method of precipitin determination later developed by the reviewer
and Kendall would, however, have made the difference in the alkali-
treated material evident at once.

Pappenheimer & Enders (69) also found SI to be a degradation
product of an "A" substance which they isolated and which was then
found (70) to be identical with the acetyl SI. A similar product
appears to have been obtained by Sevag (71) after repeatedly freezing
aqueous Pneumococcus-I suspensions in liquid air and removing pro-
tein by shaking with chloroform and amyl alcohol.6 The same method
was used to isolate a polys-accharide from egg white to which immuno-
logical activity is rather incautiously ascribed, especially in view of
Ferry & Levy's negative findings (72).

Francis (73) has found both the acetyl and deacetylated SI to be
antigenic when given intradermally in man. Oram (74) has shown
differences between SI and pneumococcus "leucocidin."

Lancefield (75) has found that hemolytic streptococci of animal
origin fall into a number of groups characterized by group-specific
polysaccharides which differ from that of group A, the human patho-
gens. In the B, or bovine group, type-specific carbohydrates were also
encountered. Julianelle & Wieghard (76) have similarly found that
human pathogenic strains (Type A) of Stafihylococcus possess a
specific polysaccharide chemically distinct from that of the Type B,
avirulent, non-pathogenic strains. Linton & Shrivastava (77) have
isolated, among the hydrolysis products of the specific polysaccharides
of agar-grown cholera vibrios, a non-reducing fraction, glucurono-
galactose, and galactose from some strains and arabinose from others.

Sordelli, Deulofeu & Ferrari (78) have found that the antigen in

8 [aID of the product is erroneously given as 21.9' instead of 217".


578       ANNUAL REVIEW OF BIOCHEMISTRY
agar-grown B. anthraci, which produces antibodies which precipitate
agar, may be easily dissociated by washing the organisms. Zozaya &
Medina (79) have shown their previously reported cross reactions
of specific polysaccharides to be partially due to agar. Pneumococcus
`V substance absorbed agar antibodies from serum, indicating a
chemical relationship between the polysaccharides.
The partial hydrolytic products of the specific polysaccharide of
Type III pneumococcus, with the exception of the ultimate aldobionic
acid unit, were found by Heidelberger & Kendall (80) to precipitate
homologous horse antiserum, but not rabbit antisera.. Reaction never-
theless occurred in the latter case, as higher concentrations inhibited
specific precipitation of S III. A somewhat similar weakening in
precipitating power toward rabbit sera, but not horse sera, was found
by Levine & Frisch (81) in phage-inhibiting extracts of B. Aertrycke
heated with dilute acid at 80". At the same time the intensity of the
phage-inhibiting action was greatly increased, suggesting the un-
masking of specifically reactive groups, as had been found by Heidel-
berger, Avery 1L Goebel (82) in the partial hydrolysis of gum arabic.
Munday & Seibert (83) have called attention to the higher values for
reducing sugars obtained in tubercle-bacillus-polysaccharide hydroly-
sates with the Hagedorn-Jensen method than with the Shaffer-Hart-
mann method, ascribing the dieerence largely to the pentose present.
The errors introduced by the presence of products of protein hydroly-
sis were also evaluated. Hydrolytic enzymes for various specific poly-
saccharides have again been encountered (84).
Lipoids and ~misceltctneous suhstun.ccs. - Evidence is given by
Jorpes & Norlin (85) that of the blood-group specific substances A,
originally thought to be lipoids, then carbohydrates, the actual hemag-
glutinin contains relatively intact proteins, occurring in the "alloxy-
protein" fraction of urine and separable from the polysaccharide
sheep-hemolysin-inhibiting factor by precipitation with tannin. Their
activity was destroyed by proteolytic enzymes. The polysaccharide
factor was founcl by Freudenberg (L Eichel (85a) to contain galactose,
amino sugar, and acetyl. Misawa (86) and Landsteiner & Jacobs (87)
have shown that adsorption of purified Forssman hapten on various
substances does not convert the hapten into an antigen, as it does the
crude extract used by Gonzales and ArmanguC.
Rudy (88) has found that the so-called lipoid hapten of the brain
becomes more and more water soluble as it is purified, giving reactions
for acid, nitrogen, and sugar. He has aIso reviewed the subject of


              IMMUNOCHEMISTRY            579
lipoid haptens (89). Wadsworth, Maltaner & Maltaner (90) have
found that highly purified cephalin or lecithin react neither as anti-
gens nor haptens when the authors' quantitative complement-fixation
method is used, involving adequate controls and proper consideration
of the anticomplementary action, especially of cephalin when exposed
to air.. Maltaner & Maltaner have also found that union of cephalin
with serum proteins to form water-soluble complexes did not change
the protein specificity or convert cephalin to a hapten. Cholesterol-
swine-serum mixtures yielded antisera which showed only non-specific
fixation of complement, as did normal serum-cholesterol mixtures,

On the other hand, tubercle-bacillus lipoids, particularly the un-
usually constituted phosphatides, appear to be definitely antigenic (34,
91), and Tropp & Baserga (92) have isolated a spleen polydiamino-
phosphatide, which, in contradistinction to cerebron and IignoceryI-
sphingosin, gave rise to complement-fixing antibodies when injected
with pig serum. Seibert, Long & Morley (92a) found S-tubercle-
bacillus strains to contain more lipoid than R strains. Hettche (92b)
has found the bactericidal and hemolytic action of B. pyocyaneum
lipoids to be due to the liquid fatty acids. The bactericidal action of
fatty acids of known constitution was also studied.

Wedum (93) was unable to observe any evidence of immunologi-,
cal activity on injection into guinea pigs of a number of synthetic
glucosides ranging in molecular weight from 222 to 1147.

ANTIBODIES AND COMPLEMENT

Buttle (94) has reported ingenious experiments which failed to
locate the source of production of diphtheria antitoxin in rabbits.
McMaster & Hudack (95) obtained evidence of the formation of
agglutinins in the lymph nodes of mice. References to the influence
of d:et and various substances, or treatments, on antibody formation
are grouped under reference 96.

The nature of antibodies and the mode of their formation were
discussed by Eastwood (97) with the aid of a stereochemical analogy
but with gloomy distrust of the chemical approach to an understanding
of immunity. Mudd (98) has put forward a theory of antibody
formation much like that of Breinl & Haurowitz,B but more specific,
and based on the analogy to the temporary union of an enzyme with
active groups in a polypeptide. Baldassi (99) observed that develop-

6 Cf. Heidelberger, M., Ann. Rev. Biochem., 1, 664 (1932).


580       ANNUAL REVIEW OF BIOCHEMISTRY

ment of diphtheria antitoxin in horses was marked by an increase in
the optical rotation of the serum, in some cases even before the ap-
pearance of antitoxin.
Studies on the purification of antibodies again point to their pro-
-tein nature. Silber 8z Demidowa (100) were unable to reduce the
protein content of typhoid agglutinins below 0.16 per cent ; on heat-
ing, agglutinin disappearance and protein denaturation ran parallel.
The resemblance of the heat destruction of tetanus antitoxin to pro-
tein denaturation was pointed out by Gerlough & White (101). Fel-
ton & Kauffmann (102) have given additional data on highly purified
pneumococcus antibody ; dissociated f rotn combination with specific
polysaccharide it showed minimum sohrbility at pH 6 -8, differing
from the pseudoglobulin associated with it in serum only by its rela-
tive insolubility in water and its basicity. The antibody protein was
digested by pepsin and trypsin with loss of its immunological proper-
ties. The dissociated precipitin gave positive tests for agglutinin,
precipitin, bacteriolysin, opsonin, complement fixation, and protection,
supporting the unitarian theory. Girard & Lourau (103) have also
observed that agglutinins and hemolysins differ from other serum
proteins in their higher isoelectric points as shown by cataphoresis.
Kirk & Sumner (104) have effected a high degree of purification of
antiurease by specific precipitation with urease, dissociation of the
enzymatically active precipitate with 0.05 N HCI, which aIso destroys
the urease, neutralization to p1-I 5, and removal of the denatured en-
zyme. Pepsin and papain'digest the purified antibody. Its combining
relationships with urease were studied, but the data, on careful analy-
sis, lead to opposite conclusions than those drawn. Ramon (lOS),
having separated antitoxin specificaI!y from the accompanying non-
specific pseudoglobulin, has ignored Marrack & Smith's careful work
in concluding that antitoxin is possibly non-protein.
References on the distribution of antibodies and antitoxins in the
protein fractions of the sera of various animals, and the failure of
serum lipoids to affect either distribution or reactivity are grouped
under 106; the last reference deals with the separation of serum lipase
from antitoxin.
An absolute, quantitative method for the determination of agglu-
tinins has been proposed by Heidelberger 8z Kabat (107). As in the
precipitin method from the same laboratory, the amount of antibody
nitrogen precipitated by an excess of antigen (in this case a measured
amount of bacterial suspension) is measured, deducting the nitrogen


IMMUNOCHEMISTRY           581

in the bacteria used. In this way it is possible to determine agglutinin
in mg. per cc. with a high degree of accuracy instead of recording a
vague "titer." The method has as yet been perfected only for pneumo-
coccus S [Dawson "M" (lOS)] and R (Dawson "S") strains. With
the method it has been possible to show an exact quantitative corre-
spondence between anticarbohydrate as precipitin and aggiutinin.
Heidelberger, Kendall & Soo Hoo (109) have studied antibody for-
mation to the red dye, R-salt-azobenzidineazo-crystalline egg albumin
with the aid of the absolute quantitative method, finding that as little
as 0.55 mg. of the antigen, given in a series of minute doses, may
stimulate the production of more than 200 times its weight of precipi-
tin. With larger amounts of antigen relatively less antibody was
formed, but the sera were of higher antibody content. The variation
of antibody content in different rabbits was followed through a num-
ber of courses of injections, as was the variation in individual sera
over long periods of storage. The same red hapten has been used
by Marrack (110) as an aid in tracing the behavior of aggiutinins.
Vincent ( 111)) in elaborating his ideas on toxin formation, has quoted
Bourdin as calculating that a horse may produce enough antitoxin to
neutralize more than 1,000 times as many L, doses of toxin as were
used to stimulate the antitoxin production-another blow to the theory
that antigen fragments are contained in antibody. Brown (112) has
found excellent correspondence, in most cases, between precipitation
of homologous antisera and antibody solutions with "cellular," or
better, acetyl polysaccharide of Type I pneumococcus and the mouse-
protection values, confirming earlier work on antisera in which the
deacetylated SI had been used.7

Griitzner ( 113) has shown that-old anti&in sera could be restored
by treatment with 0.05 N to 0.01 N NaOH, and has purified antiricin
by extracting dried sera in this way. In some instances the activity
of fresh sera was increased, leading to belief in a pre-stage of antibody
formation requiring activation. Mudd et al. (114) have found that
frozen sera, evaporated in a high vacuum, form fluffy masses easily
soluble in water and retaining their activity well.

Hooker & Boyd (I 15) have discussed the relation of antibody to
antigen and have shown, as did Berger & Erlenmeyer, that antisera to
atoxylazoprotein contain no more arsenic than normal sera. It was
also shown that it was necessary for atoxylazocasein to contain at least

r Cf. Heidelberger, M., Al&n. Rev. Blochem., 2, 503 (1933).


58.2       ANNUAL REVIEW OF BIOCHEMISTRY
thirteen arsenic azo groups in order to precipitafe with anti-atoxylazo-
egg white, but calculations of the numbe.r of anti-hapten groups per
molecule of antibody are less convincing.
The specificity changes undergone by antibodies when iodinated,
formalinized, racemized with sodium hydroxide, and coupled with
diazotized aromatic amines have been studied by Breinl & Haurowitz
(116). With the last reagent agglutinins and species specificity were
destroyed to about the same extent, while iodination affected the
former more strongly. The findings were considered consistent with
the hypothesis that antibodies are proteins, and that, just as in the
case of antigens, aromatic groupings are important in determining
their specificity. Mudd & Joffe (117) found that formalin treatment
shifted the isoelectric point of agglutinins toward the acid side and
reduced the end titer.
Lumsden & ;Macrae (117a) have reported antibodies to cancer
cells.
Silber & Schafran (118) have called attention to a number of
properties of complement which vary in the same direction as the
protein denaturation taking place. Gordon & Thompson (119) studied
the effect of salts on complement activity, and concluded that this
activity is associated with a particular state of aggregation of serum
proteins. Bancroft, Quick & Stanley-Brown (120) have given evi-
dence that complement and prothrombin are not identical, while
Maltaner & Maltaner (121) have shown that the inhibition of com-
plement by cephalin parallels the activity of cephalin in the process of
coagulation and may be reversed by calcium chloride.

      THE CHEMISTRY OF IMMUNE REACTIONS

  General .-Moriyama (122) has attempted a colloidal interpreta-
tion of the mechanism of immune reactions. According to Fuchs
(123) the "residual nitrogen" diminishes in serum when antigen and
antibody combine, the decrease being represented by a change from,
for example, 0.38396 mg. per cc. to 0.36254 mg. ! Since the effect is
very slight at best, and purified antigen and rintibody are known to
combine in the absence of `(residual nitrogen," the reviewer is skep-
tical of this and subsequent work from the same laboratory on tumor
antibodies.
Agglutination.-Iv~novics ( 124) has used a "quantitative" method,
accurate to -+lO to 15 per cent, an.d concludes that the binding of
agglutinin to dysentery bacteria is a physical process, not chemical.


              IMMUNOCHEMISTRY            583
Duncan (125) and Miles (126) have discussed the factors influencing
agglutination in optimal proportions and have studied and expIained
the great difference in the optimum depending on whether antigen or
antibody is diluted. In the ,former case, corresponding to the Dean-
Webb procedure, true antigen-antibody equivalence is more nearly
represented by the optimum. An absolute method for agglutinin de-
termination, conforming to the criteria of quantitative chemical analy-
sis, has been referred to already (107).
Jones & Little (127) have studied the increase in volume occurring
when bacteria are agglutinated by immune serum and find it to be
greater than can be accounted for by the amount of protein absorbed.
Olitzki (128) has discussed the electric charge of bacteria sensitized
with purified agglutinins, but has failed to realize that precisely these
effects would be expected if antibodies were proteins. SCdallZn &
CIavel (129) have recovered up to 50 per cent of agglutinin and pro-
tective antibodies by treating agglutinated streptococci with dilute
hydrochloric acid.
The precipitin reuction.-Frequent reference to this reaction has
already been made. An absolute, quantitative method for the measure-
ment of precipitins is given by Heidelberger et ol. (109). Culbertson
(130) has given the details of his modification (accurate to about
10 per cent) of this method and has applied it to the determination
of blood volume and the measurement of circulating antibody be-
fore and after the injection of egg albumin.. It is concluded that
circulating antibody accounts for the egg albumin which disappears,
and that cellular antibodies are not immediately available ( 131). The
influence of pH on the egg albumin-anti egg albumin reaction was also
taken up (132). Duncan has studied the optimal proportions method
in the precipitin reaction much as in the case of agglutination (i33).
Taylor (134) has also applied the method to the egg albumin-anti egg
albumin system, but the cumbersome technique and discussion of the
difficulties do not inspire confidence in its use for precise work. With
Adair & Adair (135) he has checked the method by nitrogen analyses,
finding that the Dean-Webb optimum corresponds closely to the
equivalence point. Maximum precipitation (total nitrogen) occurred
when 1.6 to 2.4 times the optimal amount of egg albumin was present.
There was no inhibition with very high concentrations of antibody.
The weights of the precipitates agreed closely with those calculated
from the nitrogen content, confirming the protein nature of the pre-
cipitate. Jones (136) has proposed measurement of the volume of


584       ANNUAL REVIEW OF BIOCHEMISTRY

the precipitate as an accurate titration of precipitin, but its accuracy
is doubtful since it indicates a linear relation between the amount of
antigen added and the amount of precipitate, up to and somewhat
beyond the equivalence point. In inhibition tests with atoxylazoprotein-
antibody, Haurowitz & Breinl (137) obtained positive results with
arsanilic acid but not with the corresponding stibonic acid as should
have followed from Berger & Erlenmeyer's theory of similar force
fields. Lumierc & Meyer (138) have studied the albumin and globulin
content, viscosity, surface tension, colloid osmotic pressure, and vol-
ume of the proteins in the supernatant fluid, concluding that the reac-
tion is purely physical and that the molecules in the supernatant fluid
have a greater volume than in normal sera owing to increased hydra-
tion. The results, however, are easily explained on a chemical basis,
particularly as the proportions of antigen and antibody used were
such as to throw the reaction partly into the inhibition zone, where
large molecules of soluble antigen-antibody compound would be pres-
ent. Following the demonstration by Heidelberger & Kendall that the
high antibody-hapten ratios observed in the precipitin reaction between
Type-III-pneumococcus specific polysaccharide and antibody are due
to the low molecular weight of the polysaccharide, Hooker & Boyd
(139) have made an interesting comparative study of the equivalence
point ratios of other systems as well, including egg albumin, hemo-
globin, pseudoglobulin, and hemocyanin, with molecular weights rang-
ing from 4,ooO to 2,000,OOO. With certain assumptions a mathematical
expression was derived from which equivalence point ratios corre-
sponding to given molecular weights were calculated. In some in-
stances remarkably close agreement was obtained with the observed
values. While there is undoubtedly a relation between the antigen-
antibody ratios observed in the precipitin reaction and the relative
molecular weights of the reactants, there is still uncertainty regarding
almost every factor involved in the quantitative formulation of such
a relation. Hooker & Boyd point out that a typical specific polysac-
charide may contain 500 times as many molecules per unit weight as,
for example, hemocyanin, thus accounting for the high limiting dilu-
tions observed in precipitin reactions involving the carbohydrates and
the relatively low limiting titers of protein-antiprotein systems., The
same workers (140) have also used the precipitin reaction to bring
forward very definite evidence that serum albumin to which heparin
had been added was still albumin, and had not been converted to
globulin as claimed by A. Fischer. The precipitin reaction has been


              IMMUNOCHEMISTRY           585

followed in pneumonia by Viktorow & Masel (141), who emphasize
the necessity of controlling pH and salt content.in urines set up with
antibody. Beale (142) has used the precipitin reaction for the meas-
urement of plant viruses. An interesting development in virus dis-
eases was Hughes' discovery (143) that the serum of monkeys acutely
ill with yellow fever contained a precipitinogen in the albumin fraction
which was entirely independent of the virus, and was possibly formed
by the action of the virus on the body proteins. This substance induced
precipitin formation in the sera of recovered cases in man and mon-
keys. An apparently similar observation in trypanosomiasis has been
made by Poindexter (144).
Anaphylaxis and allergy.-Jones & Fleischer (145) have found
the pseudoglobulin of normal horse serum or diphtheria antitoxin to
be more active in causing serum sickness in rabbits than the euglobulin
or albumin, the activity possibly being due to some other substance
precipitated with the pseudoglobulin, or to variations in the configu-
ration of this fraction in individual sera. Davidsohn (146) believed
the Forssman antigen in the injected horse serum to be the responsible
factor. Rentz & Kaktin (147) reported that daily peroraI administra-
tion of acid to guinea pigs increased the severity of anaphylactic
shock about 33 per cent, while alkali administration diminished it
about 50 per cent. Paul & Popper (148) have shown that in dogs
and guinea pigs, but not in rabbits, complement is diminished in
histamine shock as well as in anaphylactic shock. Landsteiner &
Jacobs (149) were able to induce a specific skin sensitivity in a num-
ber of guinea pigs by repeated injections of salvarsan, p-C,H, (NH,) *,
p-ONC,H,N( CH,),, 1,2,4-CaH,C1 (NO,) *, and suberanilic acid azo-
resorcinol.
Nemolysis and compEeme*td jixation.-Diacono (150) has con-
tributed a review and reported the recovery of about 80 per cent of
the anti-sheep-cell hemolysin in guinea pig antiserum ; it was present
in the globulin fraction precipitable by carbon dioxide. Serum titers
were influenced by the animals' diet.  Cholesterol in tivo did not
influence the reaction; in vitro, it inhibited the alexin only. Gyiirffy
(151) h  d It
   as ea with inhibiting effects in beef-serum systems. Siera-
kowski & Zablocki (152) found that the pH-velocity curve of hemoly-
sis greatly resembIed that of tryptic action, with an optimum from
pH 7.6 to 8.0, in line with the finding of Maltaner & Maltaner (153)
that the reversible inhibition of hemolysis or clotting after adsorption
with magnesium hydroxide is due to the increased alkalinity. Randall


586       ANNUAL REVIEW OF BIOCHEMISTRY
(154) has traced the inhibiting effect of sodium cyanide to its action
on complement, while Klopstock & Neter (155) have studied the
inhibiting action of tannin.
Hambleton (156) has discussed complement fixation as a sec-
ondary effect due to the acquisition of appropriate surface properties
by suitably modified antigens.
MiscelheoPcs reactions .-Ward & Enders (157) found that anti-
body to pneumococcus specific polysaccharide appeared to be the only
phagocytosis-promoting factor in these immune sera. If sufficient
time was allowed, the same endpoint, was reached, with. or without
complement.
Alloway (158) has studied the substance responsible for the
change of R pneumococci (Dawson "S") into S forms (Dawson
"M").
Studies on isohemagglutination have been reported by Ottensooser
& Lenzinger (159), and the water-soluble group-specific substances
have been further purified by Hallauer [(X0), cf. also (SS)].


IMMUNOCHEMISTRY

587

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588      ANNUAL REVIEW OF BIOCHEMISTRY
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              IMMUNOCHEMISTRY           589

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590       ANNUAL REVIEW OF EIOCHEMISTRY

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              IMMUNOCHEMISTRY            591

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592       ANNUAL REVIEW OF BIOCHEMISTRY

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