precipitate formed by an antibody and its precipitin, as well ae in that

formed by an antibody and the precipitin to normal globulin, In agreement with

this, Smith and Marrack © found that a precipitate formed by a precipitin and

WO

 

F, C, Smith and J, Marrack, Brit, J, Bx, Path. 12, 494 (1930).

 

& serum containing diphtheria antitoxin has the power of combining with
diphtheria toxin; and similar reqults have also been obtained recently by

uy

Heidelberger and Treffers ~ in the case of specific predipitates formed by

pneumoceeous antibody with homologous antiserum,

4h Personal commnication of unpublished material by Profeesor XM, Reidelberger
and Dr. H., P. Treffera, ,

e. Factors Affecting the Rate of Antibody Production apd the Specificity of

Antibodies.-—In order that an antibody be effective, the surface region of the

 

 

antisen covered by an antibody end must be large enough so that the integrated
attractive forces constitute an antibody-antigen bond of significant strength.
With a dond of average strength the antibody molecule will be dissociated fron
its anticen template, perhaps with the aid of the auxiliary mechanien mentioned
in Section VI, until an equilibrium or steady-state concentration is built un
in the serum. Wow if the antigen surface contains a large number of strong
groups, capable of interacting strongly with complementary structures in the
antibody, the antibody-antigen bond may be so strong that the antibody ie not
able to separate itself from the antigen, and only a small antibody concentration
can be built up in the serum. We hence conclude that an antigen containing weak
sgroups will in general be a cod antizen, whereas one containing many strong
groups will be a poor antigen, with respect to antibody production.

Thie prediction, which at firat thought seens paradoxical, is in fact borne

 

out by experiment. Thus a bland protein such 2s egg albumin is a good antigen,
as are also conjugated proteins with weak groups attached. An axoprotein with
many strong groups attached (arsenic acid, sulfonic acid, nitro, etc; the azo

group itself 18 a rather strong group, capable of forming hydrogen bonds) is
eT

a poor antigen; in order to obtain serum to such haptens an azoprotein containing

a limited mumber of the groups muat be used. I am told by Dr, Landsteiner that

this observation was made in the saarly days of the study of exovroteine’@,

 

12 see K, landsteiner and H, Lampl, loc.cit.

 

Pertinent data have been reported in recent years by Haurowitz and his
collaborators (loc.cit.), whe found the optimum arsenic content for the production
of antihapten by azoprotein made from arsanilic acid to be between 0.5 and 1.0%;
very little antibody is produced by antigens with over 2% arsenic, although strong
precipi¢in reaction 1s shown by azoprotein with an arsenic content as great as 10%.
A second deduotion, relating to specificity, can also be made. To achieve
a sufficiently strong antibody-antigen bend with an antigen containing only weak
groups a large surface region of the antigen must come into play, whereas with
an antigen containing strong groups only a small region (in the limit one group)
ia needed. Hence antibodies to antigens containing atrong groups show low
specificity, and those to antigens containing weak groups show high specificity.
Thies vredioction is substantiated by many observations. Bez albumin, hemoglobin,
and similar proteine give highly specific sera, whereas azoproteins produce sera
which are less specific, etrong cross-reactions being observed among various
proteins with the same hapten attached. This shows, indeed, that a single
hapten group gives a sufficiently strong bond to hold antibody and antigen
together. In such a case the approximation of the antibody to a strong hapten
4s very close, and great specificity 1e shown with regard to the hapten iteelf,
this specificity being the greater the stronger the hapten. Many examples of
these effects are to be found in Landateiner's work.
f. The Sffect of Denaturing Agents.—-We made the fundamental postulate that the
end parts of the polypeptide chains of the globulin molecule are characterized

by having a very large number of accessible confieurotions with nearly the sane
energy, whereas there is only one stable configuretion for the central part.

It is accordingly probable that the end configurations, giving characteristic
proverties to the antibodies, would be destroyed before the central part of

the molecule is affect+d and, moreover, that the sensitivity to denaturing agente
or conditions of antibodies to different antigens would be different. The
available meager experimental information seems to be compatible with these

tea},

 

1
‘7 See Marrack, loc.clt., op. 48-53,

 

Some remarks may ve made regarding the difference in behavior of anti-
bodies and antigens in the presence of denaturing cerents. An antigen molecule
may undergo a considerable change in configuration without losing comletely its
power of reveting with the homologous serum; if some of the surface regions
remain essentially unchanged after partial denaturation of the pretein, the
antibody molecules complementary to these regions will retain the power of
combining with them, whereas the antibody molecules comlementary to the regions
vhich have been greatly changed by denaturation will no longer be effective.

In particular some native proteins may be built of cuperimposed layers, as
described in Section IV, the antigenic regions on top of the top layer and on
the bottom of the bottom layer would still be effective after the partial
denaturation of the molecule uy the umleafing of the layers, whereas the
anticenic regions at the sides of the original molecule would in large part

loge their effectiveness by this unleafing. The observation by Rothen and
Landsteiner’ that ecg albumin spread into surface films 10 i thick retaing

ay

 

A, Rothen and K, Landsteiner, Science 99, 65 (1939).

 

the ability to combine with anti-ezg-albumin rabbit seraa is most simply
explained by the assumptions that the native egg albumin molecule hag the layer

structure suggested above and that the process of surface denaturation of this
29

molecule involves the unleafing of the layers without the loss of their structure.
As mentioned above, it is probable that for nost antibodies the end rezions
are affected by denaturing agents more eseily than the centr] rerion, and that
the firet step in denaturation of an antibody involves these end regions and
leads to loss of their specific properties. It has been shown by Danielli,
Dandelli, and Marrack!® that the reactivity of antibodies ie destroyed by

 

we J. F. Danielli, M, Danielli, and J. R, Marrack, British J. Exp, Path.
29. 393 (1938).

 

46

surface denaturation ~.

nae Rothen and Landeteiner (los.cit.) have pointed out that from these facts
regarding surface denaturation the conclusion can be drawn that "the specifie
reactivity of antibodies is to a large extent dependent upon structures
aifferent from those which mainly determine the specificity of antigens".

 

 

An interesting possible method of producing antibodies from serum or
flobulin solution outside of the animal is suggented by the theory. The globulin
would be treated with a denaturing agent or condition aufficiently strong to cause
the chain ends to uncoil; after which this agent or condition would be ranoved
slowly while antigen or hapten is present in the solution in considerable
concentration. The chain ends would then coil up to assume the configurations
stable under these conditions, which would be configurations complementary to
thoee of the antigen or hapten.

Many of the experiments suggested above are being undertaken in our
Laboratories, with the collaboration of Dr. Dar Campbell.

VI. Processes Auxiliary to Antibody Formation

It seems not unlikely that certain processes auxiliary to antibody formation
oceur, ‘The reported increase in globulin (aside from the antibody fraction) after
immunization suggests the operation of a mechanisem whereby the presence of
antigen molecules accelerates the synthesis of the globulin polypeptide chains.

There is little basis for suggesting possible mechaniems for this process at present.
The occurrence of the anamestic reaction—-the renewed production of
antibodies to an antigen caused by injection of a second antigen--may be explained
by the asaumtion that following the synthesis of an antibody a nechani sm cones
into operation in the cell to facilitate the removal of the antibody from the
antigen, perhaps by changing the hydrogen-ion or salt concentration or
dielectric constant. This would assiet in removing antibodies not only from the
second antigen but aleo from those molecules of the first antigen which had
remained, covered with homologous antibody attached too firmly for spontaneous
removal, in the cell. The evidence indicates that the anamnestic reaction is
not in general strong. At a tice after inoculation with typhoid bacillus or
erythrocytes long enough that the corresponding agglutinins are no longer
detectable in the serum injection of ancther antigen gives rise to the presence |
of there agglutinins in amounts detectable by the very sensitive ag:lutination

test; but Kabat and Heidelberger’? found that the amount of additional antibody

 

WT 8, A, Kabat and M. Heidelberger, J. Exp. Med. G8, 229 (3957).

 

to serum albumin produced by injection of ege albumin or typhoid toxin was too
amall to be detected by their method of analysis.

aus The mechanian for catching the antibody molecule and holding it in the
region of clobulin aynthesia may be closely related to that of antibody
production--possibly a partially liberated clobulin chain which forms a bond
or two bonds with an antigen molecule directly above it fie rrevented from
freeing ite central part from the cell wall, and eo serves as an anchor,

fhe renewed production of antibody in the serum after bleoting ie to be

attributed to the presence of trapned antigen molecules in the celis. The greater
duration of active than of passive irmmmnigation may be attributed to this or to

the presence of complexes of antigen and surrounding antibodies, the outer ends

of which could combine with additional antigen.
Acknov. ent

My interest in immunology was awakened by conversations with Dr. Karl
landeteiner; I am glad to express ny gratitude to him, and to acknowledge ay
indebtedness to him for ideas as well ae for facts, I wish also to thank
Professors Michael Heidelberger and Dan Campbell for advice and assistance.

Summary

It 18 assumed that antibodies differ from normal seran globulin only in the
way in which the two end parte of the globulin polypeptide chain are coiled, these
parte, as a result of their amino-acid composition and order, having accessible
a very grest many configurations with nearly the same stability; under the
influence of an antigen molecule they assume configurations complementary to
surface regions of the antigen, thus forming two active ends. After the frasing
of one end and the liberation of the central part of the chain this part of the
chain folds up to form the central part of the antibody molecule, with two
oppositely-directed ends able to attach themselves to two antizen molecules.

Among the points of comparison of the theory and experiment are the
followin:: the heterogeneity of sera, the bivalence of antibodies and multi-~
valence of antigens, the framework structure and molecular ratio of antibody-
antigen precipitates, the use of a single antizen molecule as template for an
antibody molecule, criteria for antigenic activity, the behavior of antigens
containing two differant haptens, the antigenic activity of antibodies,
factors affecting the rate of antibody production and the specificity of

antibodies, and the effect of denaturing agents. It is shown that most of the

reported experimental results are compatible with the theory. Some new

experiments suggested by the theory are mentioned,

Pasadena, California Recel ved

 
Fig.

Fig.

Fir,

Fir.

Fig.
Fig.
Fig.

Fig.

1.

2.

4,

5.

32
Legende for figures

Diagrams representing four stages in the process of formation of a
molecule of normal serum globulin (left side of figure) and six stages
in the process of formation of an antibody molecule as the result of
interaction of the globulin polypeptide chain with an antigen
molecule, There is also shown (lower right) an antigen molecule
surrounded by attached antibody molecules or parts of molecules and
thus inhibited from further antibody formation.
(A) Diagram representing agclutinated cells. (B) Diagram of the
region of contact of two cells, showing the postulated structure
and mode of action of agglutinin molecules,
A portion of an ideal antibody-antigen framework, One plane of the
atructure corresponding to the value twelve for the valence of the
antigen molecules is shown.
4 portion of an antibody-antigen network formed in the region of
antibody excess,
A portion of the network formed in the region of antigen excess.
Repwesentative soluble complexes formed with excess antigen,
A soluble complex formed with excess antibody.
The folding of polypeptide chains into a layer held together by

imino-carbonyl hydrogen bonds.