A Brief History of Antibodies

The acquisition of immunity to a disease that a patient has already encountered has been
documented for many centuries. Arguably some of the earliest work in the field that has now become
known as immunology was performed in the period around 1714-1717. Lady Mary Wortley Montagu,
Emanuel Timoni and James Pylarini pioneered a smallpox inoculation, a course of action
unparalleled in medical advance up to that point. Variolation, as it was known, used live smallpox
virus in the liquid taken from a smallpox blister in a mild case of the disease and carried in a nutshell
(1). In 1798 the first smallpox vaccination was more notably demonstrated by Edward Jenner. This
was performed by inoculating a boy with the fluid from a cowpox pustule giving him immunity to the
very similar but much more serious disease smallpox (2, 3).
The earliest reference to antibodies came from Emil von Behring and Shibasabura Kitasato in 1890.
In a landmark publication they showed that the transfer of serum from animals immunised against
diptheria to animals suffering from it could cure the infected animals (4). The potential for treatment
in humans was immediately apparent and Behring was later awarded the Nobel Prize for this work in
1901.
In 1900 Paul Ehrlich, who is regarded as one of the fathers of modern immunology, proposed the
side-chain theory, where he hypothesised that side chain receptors on cells bind to a given
pathogen. He was the first to propose a model for an antibody molecule in which the antibody was
branched and consisted of multiple sites for binding to foreign material, known as antigen, and for
the activation of the complement pathway (5). This model agreed with the lock and key hypothesis
for enzymes proposed by Emil Fischer (6, 7) and still in general terms holds true today.
Astrid Fagraeus in 1948 described that plasma B cells are specifically involved in antibody
generation and by 1957 Frank Burnet and David Talmage had developed the clonal selection theory
(8). This stated that a lymphocyte makes a single specific antibody molecule that is determined
before it encounters an antigen, which was in contrast to the instructive theory developed by Linus
Pauling in 1940 where the antigen acted as a template for the antibody (9).
By 1959 Gerald Edelman and Rodney Porter independently published the molecular structure of
antibodies (10, 11), for which they were later jointly awarded the Nobel Prize in 1972. The first
atomic resolution structure of an antibody fragment was published in 1973 (12) and this was quickly

followed by the invention of monoclonal antibodies in 1975 by Georges Khler and Csar Milstein
(13) signalling the start of the modern era of antibody research and discovery.
Antibody structure >>
<< Antibody overview

References
1.

Case, C.L., and Chung, K.T. (1997). Montagu and Jenner: The Campaign Against Smallpox.
SIM News 47, 5860.

2.

Jenner, E. An Inquiry Into the Causes and Effects of the Variol Vaccin, Or Cow-Pox.

3.

Riedel, S. (2005). Edward Jenner and the history of smallpox and vaccination. Proc (Bayl
Univ Med Cent) 18, 2125.

4.

Behring, E., and Kitasato, S. (1890). Uber das Zustandekommen Der Diphtherie- Immunitat
Und der Tetanus-Immunitat Bei Thieren. Dtsch Med Wochenschr 49, 11131114.

5.

Davies, D.R., and Chacko, S. (1993). Antibody structure. Acc. Chem. Res. 26, 421427.

6.

Fischer, E. (1894). Einfluss der Configuration auf die Wirkung der Enzyme. Berichte Der
Deutschen Chemischen Gesellschaft 27, 29852993.

7.

Lemieux, R.U., and Spohr, U. (1994). How Emil Fischer was led to the lock and key concept
for enzyme specificity. Adv Carbohydr Chem Biochem 50, 120.

8.

Edelman, G.M. (1959). Dissociation of -globulin. Am. Chem. Soc. 81, 31553156.

9.

Burnet, F.M. (1957). A modification of Jernes theory of antibody production using the
concept of clonal selection. The Australian Journal of Science 20, 6769.

10.

Pauling, L. (1940). A Theory of the Structure and Process of Formation of Antibodies. J. Am.
Chem. Soc. 62, 26432657.

11.

Porter, R.R. (1959). The hydrolysis of rabbit y-globulin and antibodies with crystalline papain.
Biochem. J. 73, 119126.

12.

Inbar, D., Hochman, J., and Givol, D. (1972). Localization of antibody-combining sites within
the variable portions of heavy and light chains. Proc. Natl. Acad. Sci. U.S.A. 69, 26592662.

13.

Khler, G., and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of
predefined specificity. Nature 256, 495497.

Akuisisi kekebalan terhadap penyakit yang pasien telah mengalami telah didokumentasikan selama
berabad-abad. Boleh dibilang beberapa pekerjaan awal di bidang yang sekarang telah menjadi
dikenal sebagai imunologi dilakukan pada periode 1714-1717 sekitar. Lady Mary Wortley Montagu,
Emanuel Timoni dan James Pylarini memelopori inokulasi cacar, suatu tindakan yang tak tertandingi
di muka medis sampai saat itu. Variolation, seperti yang diketahui, digunakan virus cacar hidup
dalam cairan yang diambil dari blister cacar dalam kasus ringan penyakit dan dibawa dalam
Singkatnya (1). Pada tahun 1798 vaksinasi cacar pertama lebih terutama ditunjukkan oleh Edward
Jenner. Hal ini dilakukan dengan menginokulasikan anak dengan cairan dari bintil cacar sapi
memberinya kekebalan terhadap sangat mirip tapi jauh lebih serius penyakit cacar (2, 3).
Referensi paling awal untuk antibodi berasal dari Emil von Behring dan Kitasato Shibasabura pada
tahun 1890. Dalam publikasi tengara mereka menunjukkan bahwa transfer serum dari hewan
diimunisasi diptheria untuk hewan yang menderita itu bisa menyembuhkan hewan yang terinfeksi
(4). Potensi untuk pengobatan pada manusia adalah segera jelas dan Behring kemudian
dianugerahi Hadiah Nobel untuk pekerjaan ini pada tahun 1901.
Pada tahun 1900 Paul Ehrlich, yang dianggap sebagai salah satu bapak imunologi modern,
mengajukan teori sisi-rantai, di mana ia hipotesis bahwa rantai samping reseptor pada sel mengikat
ke patogen tertentu. Dia adalah orang pertama yang mengusulkan model untuk sebuah molekul
antibodi yang antibodi itu bercabang dan terdiri dari beberapa situs untuk mengikat bahan asing,
yang dikenal sebagai antigen, dan untuk aktivasi jalur komplemen (5). Model ini setuju dengan 'kunci
dan kunci' hipotesis untuk enzim yang diusulkan oleh Emil Fischer (6, 7) dan masih secara umum
berlaku saat ini.
Astrid Fagraeus pada tahun 1948 dijelaskan bahwa sel plasma B secara khusus terlibat dalam
generasi antibodi dan oleh 1957 Frank Burnet dan David Talmage telah mengembangkan teori
seleksi klonal (8). Hal ini menyatakan bahwa limfosit yang membuat molekul antibodi spesifik
tunggal yang ditentukan sebelum bertemu antigen, yang berbeda dengan teori instruktif yang
dikembangkan oleh Linus Pauling pada tahun 1940 di mana antigen bertindak sebagai template
untuk antibodi (9).
Pada tahun 1959 Gerald Edelman dan Rodney Porter independen menerbitkan struktur molekul
antibodi (10, 11), yang mereka kemudian bersama-sama dianugerahi Hadiah Nobel pada tahun
1972. Struktur resolusi pertama atom fragmen antibodi diterbitkan pada tahun 1973 (12) dan ini
segera diikuti oleh penemuan antibodi monoklonal pada tahun 1975 oleh Georges Khler dan Csar
Milstein (13) menandakan dimulainya era modern penelitian antibodi dan penemuan.

Antibody Structure
In simplistic terms antibodies perform two main functions in different regions of their structure. While
one part of the antibody, the antigen binding fragment (Fab), recognises the antigen, the other part
of the antibody, known as the crystallisable fragment (Fc), interacts with other elements of the
immune system, such as phagocytes or components of the complement pathway, to promote
removal of the antigen.

Figure. Schematic representation of an IgG.

An antibody consists of two heavy chains (blue) and two light chains (green) folded into
constant and variable domains. The enlargement of the variable domain shows a ribbon
representation of the -sheet framework and CDR loops.
Antibodies all have the same basic structure consisting of two heavy and two light chains forming
two Fab arms containing identical domains at either end attached by a flexible hinge region to the
stem of the antibody, the Fc domain, giving the classical Y shape. The chains fold into repeated
immunoglobulin folds consisting of anti-parallel -sheets (1), which form either constant or variable
domains. The Fab domains consist of two variable and two constant domains, with the two variable
domains making up the variable fragment (Fv), which provides the antigen specificity of the antibody
(2) with the constant domains acting as a structural framework. Each variable domain contains three

hypervariable loops, known as complementarity determining regions (CDRs), evenly distributed

between four less variable framework (FR) regions. It is the CDRs that provide a specific antigen
recognition site on the surface of the antibody and the hypervariability of these regions enables
antibodies to recognise an almost unlimited number of antigens (3).

Figure. Structural representations of an IgG.

The heavy chain is shown in blue, light chain in green and glycosylation in orange. On the
left is a ribbon representation showing the secondary structure elements and on the right
hand side is a space-filled model of the same molecule. PDB accession number of the
mouse IgG1 is 1IGY.
Antibodies are glycosylated proteins, with the position and extent of glycosylation varying between
isotypes. As displayed in the image above the Fc region of an IgG consists of two paired CH3
domains and, in contrast, two CH2 domains that are separated and do not interact but have two
oligosaccharide chains interposed between them. These chains cover the hydrophobic faces that
would normally lead to domain pairing. The N-glycans contain a common core region of two Nacetyl-glucosamine residues (GlcNAc) linked to an asparagine (N297 in human IgG1) via an amide
bond and three mannose residues. This core structure may contain additional terminal sugars, such
as mannose, GlcNac, galactose, fucose and sialic acid, generating a large amount of heterogeneity
(4).

Antibody Isotypes & Subtypes

In mammals, antibodies are classified into five main classes or isotypes IgA, IgD, IgE, IgG and
IgM. They are classed according to the heavy chain they contain alpha, delta, epsilon, gamma or
mu respectively. These differ in the sequence and number of constant domains, hinge structure and
the valency of the antibody. Antibody light chains fall into two classes in mammals, kappa and
lambda, with kappa light chains being the more common of the two. Although these are relatively
dissimilar in protein sequence they share a similar structure and function.

Figure. Immunoglobulin isotypes.

Schematic representation of the five immunoglobulin classes or isotypes in mammals.
IgG is the most abundant antibody in normal human serum, accounting for 70-85% of the total
immunoglobulin pool (1). It is monomeric with a molecular weight of approximately 150 kDa, is the
major antibody of the secondary immune response and has the longest half-life (20-24 days) of the
five immunoglobulin classes. IgG consists of four human subclasses (IgG1, IgG2, IgG3 and IgG4)
each containing a different heavy chain. They are highly homologous and differ mainly in the hinge
region and the extent to which they activate the host immune system. IgG1 and IgG4 contain two
inter-chain disulphide bonds in the hinge region, IgG2 has 4 and IgG3 has 11. Details of the
functional differences between the IgG sub-classes in human will be described in the section on Fc

effector function. In mice the IgG class is divided into five sub-classes (IgG1, IgG2A, IgG2B, IgG2C
and IgG3) and in rat there are four (IgG1, IgG2A, IgG2B, IgG2C). Sub-class nomenclature has
arisen independently for each species and so there is no general relationship between the subclasses from each species.
IgM accounts for 5-10% of the immunoglobulin pool and is the predominant antibody in the primary
immune response (1). Unlike IgG, IgM does not contain a hinge region but does contain an
additional constant domain and an 18 amino acid tailpiece at the carboxy terminus, which contains a
cysteine and is involved in multimerisation of the molecule. It is classically represented as a
pentamer of the basic four chain structure held together by a J chain but can also exist in a
hexameric form without the J chain (2) and as a monomer on the surface of B-cells. Soluble IgM is in
excess of 1 MDa and thus due to its size is largely confined to the intravascular pool.
IgA represents approximately 5-15% of the antibody pool and either exists as a monomer or a dimer
(1). IgA is the predominant antibody in mucous secretions such as saliva, tears, milk and intestinal
juice. Like IgM, the multimeric form of IgA contains a tail piece at the carboxy terminus and is held
together by the J chain. The secretory component is a 75 kDa polypeptide chain synthesised by
epithelial cells of the gut for linkage to the dimeric form of IgA. As the name implies the secretory
component facilitates IgA transport across epithelial cells but is also involved in protecting IgA
secreted into the lumen of the gut from proteolytic digestion.
IgD accounts for less than 1% of the total plasma immunoglobulin but is present in large quantities
on the membrane of B-cells (1). IgD has the same basic structure as IgG but with an extended hinge
region which is very susceptible to proteolytic digestion. The precise function of this class of antibody
is still unknown.
IgE is very scarce in the serum but is found on the basophils and mast-cells of all individuals (1). In a
similar manner to IgM, IgE has two additional constant domains in place of the hinge region. This
class may play a role in immunity to parasites but is more commonly associated with type I
immediate hypersensitivity, where an IgE immune response occurs to innocuous environmental
antigens such as pollen and peanuts.

Antibody Effector Functions

Antibodies act by a number of mechanisms, most of which engage other arms of the immune
system. Antibodies can simply block interactions of molecules or they can activate the classical

complement pathway (known as complement dependent cytotoxicity or CDC) by interaction of C1q

on the C1 complex with clustered antibodies. Critically antibodies also act as a link between the
antibody-mediated and cell-mediated immune responses through engagement of Fc receptors.

Figure. Antibody modes of action.

Antibodies have several modes of action: i) they can block ligand-receptor interactions; ii)
cause cell lysis through activation of complement dependant cytotoxicity (CDC); iii) interact
with Fc receptors on effector cells to engage antibody dependent cellular cytotoxicity; iv)
signal for ingestion of a pathogen by a phagocyte.

Fc receptors
Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated
(humoral) immune response to cellular effector functions. Receptors for all classes of
immunoglobulins have been identified, including FcR (IgG), FcRI (IgE), FcRI (IgA), FcR (IgM)
and FcR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64
(FcRI), CD32 (FcRIIa, FcRIIb and FcRIIc) and CD16 (FcRIIIa and FcRIIIb). FcRI is classed
as a high affinity receptor (nanomolar range KD) while FcRII and FcRIII are low to intermediate
affinity (micromolar range KD) (1).
In antibody dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (natural
killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is
bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of
various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which
mediate in the destruction of the target cell. The level of ADCC effector function various for human

IgG subtypes. Although this is dependent on the allotype and specific FcvR in simple terms ADCC
effector function is high for human IgG1 and IgG3, and low for IgG2 and IgG4. As shown in the
model below FcRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge
of the binding site has resulted in engineering efforts to modulate IgG effector functions see Fc
engineering section for more detail.

Figure. Human IgG1-FcRIII complex.

A model of human IgG1 in complex with Fc receptor III, which binds asymmetrically across
the hinge and upper CH2 region of the antibody. The left hand image shows a ribbon
representation and the right hand side a space-filled model. The antibody heavy and light
chains are shown in blue and green respectively, glycosylation in orange and FcRIII in red.
Model produced from PDB accession numbers 1IGY and 1E4K.

Other Antibody Interactions

FcRn
It was recognised in the 1960s that the two processes of IgG transport from mother to her young and
the protection of IgG from catabolism were mediated by receptors that share many features (1).
Originally these were referred to as the neonatal transport receptor (FcRn) and the IgG protection
receptor (FcRp) respectively. It wasnt until 1996 that it was conclusively shown that these were the
same receptor (2-4). These have since been unified under the term Brambell receptor (FcRB) in
honour of their discoverer, although the receptor is still more commonly referred to as FcRn.

Figure. Human IgG1-FcRn complex.

A model of human IgG1 in complex with the FcRn-B2M heterodimer, which binds in the CH2
and CH3 regions of the antibody. The upper images show space-filled models and the lower
images are ribbon representations. The antibody heavy and light chains are shown in blue
and green respectively, glycosylation in orange, FcRn heavy chain in red and 2microglobulin (B2M) in yellow. Model produced from PDB accession numbers 1IGY and
1I1A.
FcRn is a heterodimer of a 2-microglobulin (B2M) light chain and a major histocompatibility
complex (MHC) class I-like heavy chain. As shown in the image above, FcRn binds IgG at the
interface between the CH2 and CH3. Critically the binding site contains a number of histidine
residues which results in a pH dependent binding that is crucial to its function (5, 6). Endocytosis of
IgG is followed by binding of IgG to FcRn in the acidic environment (pH 6.0) of the endosome. The
IgG-FcRn complex is then trafficked through cellular conduits to bypass lysosomal degradation and
finally IgG is released back into the serum at physiological pH. This cycle is depicted in the image

below. FcRn-mediated recycling results in IgG1, 2 and 4 having the longest serum half-life of all
proteins, at approximately 21 days. Although IgG3 binds FcRn recycling of this sub-class is less
efficient due to a single amino acid change at the binding site (7), resulting in a half-life of only 7
days.

Figure. FcRn mediated recycling.

In the early endosome IgG interacts with FcRn at pH 6.0. The FcRn-IgG complex is then
recycled back to the cell surface and IgG released at neutral pH thus rescuing IgG from
lysosomal degradation.

Protein A and G
Although not a function of antibodies per se, two bacterial proteins, Protein A and Protein G, bind to
the Fc region of some antibodies. Protein A is a 56 kDa surface protein originally found in the cell
wall of Staphylococcus aureus and Protein G is a 65 kDa protein from Streptococcal bacteria. In a
similar manner to FcRn, protein A and G bind at the interface between the CH2 and CH3 domains,

as depicted in the image below. Although overlapping the three epitopes are distinct and this results
in differential binding of Protein A and G to different isotypes, subtypes and species of
immunoglobulin. A summary table of the binding affinities of these proteins to different antibodies can
be found here. Due to their unique ability to bind pH dependently with high affinity to IgG in
particular, these proteins have become widely used for the purpose of purifying antibodies.

Figure. A model of human IgG1 in complex with both Protein A and Protein A.
The upper images show space-filled models and the lower images are ribbon
representations. The antibody heavy and light chains are shown in blue and green
respectively, glycosylation in orange, a Protein A fragment in purple and a Protein G
fragment in yellow. Model produced from PDB accession numbers 1IGY, 1L6X and 1FCC.

Antibodies as Tools
Due to their high specificity and selectivity antibodies have always had the potential to be of great
use as biochemical tools for a range of applications including selection, identification, purification
and as therapeutics. Broadly speaking antibodies are categorised into two groups (polyclonal or
monoclonal) and are utilised in three main areas (research, diagnostics and therapeutics).

Figure. Categories of antibodies.

Comparison of polyclonal antibodies, which bind to the same antigen but different
epitopes, with monoclonal antibodies which all bind to the same epitope on a target
antigen.
Polyclonal antibodies (pAbs) are a heterogeneous mixture of antibodies directed against various
epitopes on the same antigen. The antibodies are generated by different B-cell clones of the animal
and as a consequence are immunochemically dissimilar, with different specificities and affinities.
The true potential of antibodies as specific targeting agents was not realised until ground breaking
work by Khler and Milstein in 1975 resulted in the production of monoclonal antibodies or mAbs (1).
These were produced by the fusing of antibody producing mouse spleen cells with an immortal
mouse myeloma cell line, resulting in the formation of an immortal cell line, known as a hybridoma,
expressing a single antibody with specificity for one particular epitope on an antigen, i.e. a
monoclonal antibody. Once produced hybridomas can be cultured in vitro indefinitely allowing the
relatively easy purification of large quantities of monoclonal antibody.

Research
Antibodies are vital tools in many of the laboratory techniques that are used to answer basic
research questions. Due to their outstanding specificity they make exquisite tools that allow

researchers to identify molecules that cannot be seen by the naked eye and thus enable conclusions
to be drawn about the target molecule and pathway of interest. Routine procedures such as western
blot, flow cytometry, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and
many others all rely antibodies.

Diagnostics
Antibodies have become a critical component of many diagnostic assays. Uses included but are not
limited to the detection of infections, recognition of allergies and the measurement of hormones and
other biological markers in blood.

Therapeutics
The ability of antibodies to bind an almost unlimited number of target proteins with high specificity
always meant they were destined to be used as therapeutics. As early as 1900 Paul Ehrlich coined
the term magic bullets in reference to antibodies. Following the ground breaking publication on the
production of monoclonal antibodies (1) the early success of the first therapeutic antibody OKT3
(muromonab) as a treatment for transplant rejection was not immediately followed by the wave of
approvals that many anticipated. However, since the mid to late 1990s therapeutic antibodies have
become one of the fastest growing classes of therapeutics in the biological drugs market (2). Some
of the work that led to this breakthrough will be discussed in more detail in the antibody engineering
section.