Artificial Antibodies- A History,

A Comparison to Natural
Antibodies, and its Change
Throughout Time
By: Erika Raina, Sharda Raina, and Nikki Nguyen
University of Washington

I.

Introduction (History)

The history of the discovery of the antibody, and its subsequent creation to the artificial
antibody has been a giant step for man and for science. In the 1940s, continuous scientific
research including the discovery of deoxyribonucleic was made open to the public as physicians
and scientists worked together to find and make a discovery worthy of a Nobel peace prize and
to understand the mechanism that is the human body. German Physician Paul Ehrlich, a 1940s
medical scientist, proposed the side chain theory. The side chain theory stated that cell had a
series of receptors that bind molecules, and interact with disease-causing toxic molecules,
causing an irreversible interaction that prevents nutrient intake. The theory states that the body
made replacement side chains, or antibodies, that are unable to fit on the surface of cells and thus
are circulated throughout the body ass it neutralizes the toxin and then remains in the body to
prevent invasions from other pathogenic toxins.(5). It was an aid the immune system in curing
illness, and as a result, lower mortality rate. As research continued, those who studied
antibodies diverged into two groups of study- those who believed antibodies to be specifically
protein and in a Y shaped structure, and those who found smaller no protein compounds to also
be considered antibodies. For the purpose of this paper, an antibody will follow the definition of
the former, as that is how antibodies are described in modern day textbooks.
Over time, 3 types of synthetic antibodies were produced for other diagnostic and
therapeutic uses beyond the original capabilities of the natural antibody. The mechanisms for
creating the antibodies evolved from homologous recombination in transgenetic mice to using a
protein scaffolding. The chemical inertness of the artificial antibodies, its role in society, and its

overarching benefits are undeniable despite alternative antibodies being produced with lower
production costs and increased solubility.
II. Natural Antibodies
ooIn essence, there are two parts to understanding antibodies. One is the natural humanmade antibody and its contrast of the artificial antibody. As a result, there must be a clear
understanding of the natural antibody. According to Nesters microbiology textbook, an antibody
is an immunoglobulin protein that is manufactured with the purpose to identify and neutralize
pathogens. Within this protein, there are 2 functional regions- two arms and a stem. The arms,
also known as the Fab region or the variable region, is made of a light polypeptide chain with a
specific epitope at the end that binds to the antigen (1). This binds similar to an enzyme-substrate
in its lock and key selectivity. A hyper variability region is created from three loops on the
structural beta sheet being brought together from the two chains. An antigen binds to the hyper
variability (high-sensitivity region of an antibody with repeating nucleotides like a
complementary, binding ligands) in a covalent nonreversible reaction. In some cases, amino
acids create electrostatic interactions such as hydrogen bonding while hydrophobic interactions
contribute to binding in polar amino acids (12).

Figure 1: Y structure of Antibodies, in conjunction with the heavy and light chains and bonded by disulfide
bonds(1)

Within the human body, there are antibodies made from 5 classes of Immunoglobins (Ig),
and ultimately there are two that are important for this paper. The first type of antibody made is
Immunoglobulin M (IgM) and are the first to react to an antigen and initially react with the
antigen. However, as the immune response continues on, some B cells switch off a variable
region that determines the antibody to be an IgM.antibody and in turn changes it to a different
class of antibodies- Immunoglobulin G, or IgG antibodies. Millions of different antibodies are
produced in this same format- but with varying regions in the antibodies- the constant gene
region, the diversity gene region, the variable region, and the J region, as different genes are
turned off and on and as a result express different and similar combinations of amino acids.
(1,12)
As many amino acids are aromatic, or have 4n+2 pi electrons, the amino acids participate
in hydrophobic and Van der Waal interactions. In contrast, the stem, also known as the Fc region
or constant region, is made of 2 polypeptide chains, or heavy chains .The heavy polypeptide
chains acts as a flag to other parts of the immune system in adaptive immunity. The heavy and
light chains of an IgG antibody are connected by 12 intrachain disulfide bonds, thus following
the classical model of sulfur bonding. The heavy chains are connected at the hinge regions with
disulfide bonds, although the number varies between 2-11 disulfide bonds pending on the
subclass of the IgG (the variance however is irrelevant to the paper and will not be discussed).
The light and heavy chains are connected through the disulfide bonds of cysteine residues that
are located in the hinge region or in the 3rd cysteine of the heavy chain. (13).

Figure 2: Cysteine is seen as a polar uncharged amino acid (3)

Figure 3: Classical Structure of Disulfide bonding from cysteine structure (13)

The function of antibodies in the human body is to eliminate foreign pathogens or microbes,
through various processes as listed below: (1)
1. opsonization- an antibody is marked for removal by a phagocyte. When a phagocyte is
opsonized, it is then marked for destruction in a process called cell-mediated cytotoxicity. The
antibodies are part of the adaptive immune system and are supported by a part of the passive
immune system known as the complement pathway of the immune system.

2. Immobilization/Prevention (Crosslinking): By binding to the flagellum of bacterium or by to

different bacteria and one another, the movement of the bacterium is compromised.
3. Neutralization: In the presence of an antigen, the antibody may attach to multiple receptors on
a virus or toxin, rendering it unable to bind to other molecules/other cell receptors.
The classical complement pathway in the immune system is important for its activation of
antibodies. Rejection may occur when an individual receives an organ transplant or an allogenic
skin graft because the immune system sees the antibody as foreign antigen that must be removed.
A dysfunction in the graft used for transplant, creatinine is elevated and associated with
endothelial cell swelling and exposure of an antigen. The antibody binds to the antigen and
activates the classical complement pathway of the immune system, attacking the graft/organ and
as a result, its new self. (15)
Antibodies have also been used for a variety of other diagnostic testing, which includes and is
not limited to (3, 12)
1. Immunosignaturing- the use of peptide sequences to test the presence of antibodies in
blood using fluorescent secondary antibodies. This is used for diagnosis of valley fever,
cancer, and vaccine efficacy.
2. Western blotting- a detection of a protein or antibody by separating macromolecules
through electrophoresis, or an electric current that separates molecules based on size and
charge, transferring the macromolecules onto a nitrocellulose or polyvinylidiene
difluoride membrane and complexed with an antibody, and creating a product that can be
detected by chemiluminescent detection.
3. ELISA-enzyme linked immunosorbent assay - a technique that this technique uses the
antibodies in a well that captures an antigen as well as enzyme labeled antibodies that

bind to the antigen. With the result of an enzyme, a color change occurs as covalent
bonds are created and electrons decrease in energy states, releasing energy in the process
4. Immunoaffinity chromatography - the separation of antibodies or antibody fragment with
immobilized antigens on a solid matrix for identifying and qualifying antigens
5. Immunosensors - the use of analytical devices that detects an antigen-antibody complex
and converts to an electrical signal for the presence of an antigen when tests like ELISA
are not an option.
In contrast, synthetic antibodies will be defined as any immunoglobulin protein not
biologically manufactured in the human body that is made for specific molecular recognition of a
target molecule. A synthetic antibody often times has similar chemical structuring of amino
acids, but varies in source, origin, and specificity. Similar to natural antibodies, synthetic
antibodies rely on stereoisomerism and intermolecular forces and in many cases, relies on the
structure of the protein on the surface of the pathogen (2). Within synthetic antibodies, there are
3 sets, each presently used but with different dates of discovery: animal antibodies, plastic
antibodies, and synbodies.
II. Animal Created Antibodies
Animal Antibodies were the most traditional way of making an antibody until the 2000s.
This genome can be found in any living organism as long as there is the presence of specific IgG
human genome meaning that while the immunoglobulin protein is manufactured by a eukaryotic
organism and using the immunoglobulin from the human genome, it is not necessary the
mechanisms of the human body producing the antibody .
From the 1900s to the early 2000s, transgenic animals were the main form of obtaining
antibodies through means of animal-created antibodies. In 1989, M. Bruggelmann discovered the

similarity of Ig loci in mammals and the ability to use integrated loci in the animals immune
system. The transgenic animal, often times mice, would be injected with germline genetic
material containing IgG and a foreign antigen. (11)The gene for the antibody is rearranged to
highly express the variable region (V) region of the gene, and in many cases the diversity(D)
region and the J region. (1) The immunoglobulin rearranges in the transgenic mice(although the
specifics are unknown) to create chimeric immunoglobulin or which can then be adopted into the
human genome, which is called chimeric human transloci as the constant region is the genes
sequence of the host(mouse).In very rare cases (1%), the constant region of the gene is also of
the human sequence, creating a fully human transloci. As a result IgG antibodies are made, and,
found in the human body by creating covalent hydrophobic bonds with the antigens.(13) One of
the limitations on this, however, is the need to find an eukaryote that expresses similar Ig loci, so
that the human Ig gene may be incorporated by homologous recombination. In homologous
recombination, the chemical structure of the DNA is similar, however the new gene has amino
acids with a higher binding affinity than the original gene. The new gene is incorporated into the
DNA sequence and is replicated by the organism in the presence of an antigen. The antibodies
are extracted as serum from the animal and injected into the human, creating a mouse
monoclonal antibody (mAbs). (14)
One of the major differences in structure of transgenic animal IgG antibodies (mAbs) and
human antibodies is that there is an increase of free sulfhydryls. While in most cases of IgG, the
cysteines in IgG make a disulfide bonded state, higher levels of free sulfhydryls from unbounded
cysteines are found in the denatured state as the disulfide bonds are degraded. Within the heavy
chain variable domain of the antibody, hydrophobic interaction shows incomplete disulfide
bonding that can be fixed with the presence of copper sulfate. The presence of a trisulfide bond

formation, stable at pH of 6.5 is also created through a nucleophilic attack of hydrogen sulfide
with a disulfide bond. (13). While the changes in structure and stability are not well understood,
the introduction of a trisulfide bond and lower stability may trigger an immune response when
introduced to the human body and provide unknown effects on the stability and biological
functions of the molecule. (11)

Caption : The change from Mouse Monoclonal Antibodies to a Transgenic Human Antibody
through chimeric recombination.(11)

Animal antibodies, as they are relatively similar to full human monoclonal antibodies, are
used for the same diagnostic techniques including blotting, immunostaining, western blotting,
and ELISAs, although as they lack the same constant region (heavy chain region), they may be
less accurate. Because of its natural setting, the antibodies have a high affinity from affinity
maturation inside the laboratory setting/through testing. Fully human IgMs of monoclonal
antibodies, or mAbs, when used, can have a high affinity to HIV by detecting the antigen p24
(14). The antibody can also act in neutralizing tumors by acting with circulating antigens for

neutralization or removal as it binds to the cell surface receptors of tumors and with natural killer
cells, monocytes, and macrophages (passive immunity) by the Fc region as in the case of chronic
lymphocytic leukemia. (11).
. However, it raises ethical questions of the use of animals in creation of antibodies, and
the harmful effects that it may have on both the animal and the human. An exclusive drawback to
monoclonal antibodies (mAbs) made from transgenic animals is its support of available lines. In
many cases, once the antibody is made, the scientist often times dismisses the line of mice. The
antibody is also made by the expression of a partly human genome of IgG and may lead to the
need of humanize the antibody. However, the mouse cellular system, similar to the human
cellular system has cytotoxic T cells that can create a cellular response against the antibody,
thinking the antibody is pathogen. For the human, if the body does not accept the antibody and
there is the existence of a partial immune system, the human partial immune response may be
activated against the self and cause more damage to the humans already compromised immune
system, causing sepsis and even death. (11)
III. Plastic Antibodies
In the late 1900s, molecular imprinting was introduced and as a result, plastic antibodies
plastic antibodies were made through molecular imprinting of a template and the formation of a
rigid monomer that binds to the antigen. Molecular imprinting, synonymous with genetic
imprinting, is used for molecular recognition and based on the lock and key model of an enzyme
in which the enzyme has a unique geometric structure from specific amino acid affinities to fold
that create specific binding sites that specific substrates can react with. A template molecule of
the antigen is used to create a polymer matrices as functional groups cause interaction of

monomers to create a cavity. The antibodies act similar to normal microbiota, neutralizing the
antigen by binding to the receptor sites of an antigen. (10)
Molecular imprinting involves the creation of a print molecule or template that creates
reversible covalent interactions with polymerizable monomers that then turn into a macroporous
monomer with recognition sites and functional groups complementary to the crosslinked
monomer it once attached to. The technique for molecular imprinting can vary, but below is an
example of preparation for molecular imprinting. 1 mmol of the antigen is dissolved with the
imprinting porogen in a 3:2 ratio. This solution is mixed with methacrylic acid, in a 1:10 mmol
ratio. A crosslinking polymer with high rigidity such as ethylene glycol dimethylacrylate is used
in a 70-90% increase by volume. An initiating reagent is added and the solution is degassed of
nitrogen and polymerized at thermolytic (45-60) or photolytic (366 nm) conditions for 10 hours.
The small particles tend to have a poor particle separation from the template (assay samples), so
the particles must be washed extensively with an 8:7:5 ratio of ethanol:water:acetic acid with 1
M ammonium acetate to extract some particles, ethanol to more effectively extract the imprint
antigen from the polymer, and water to access the highest recognition sites. (2)
Due to the hydrophobic nature of the polymer from weakened noncovalent hydrogen
bonds ionic bonds that require polymerization to be performed in dry solvent, the polymers are
unable to be used in techniques such as immunodiffusion, immunoelectrophoresis,
immunoblotting, and tissue immunofluorescence, but instead used for solid support. (2)
Despite its limitations, the use of artificial antibodies has expanded over the years. While
plastic antibodies have been used primarily as a diagnostic tool in areas including bio sensing,
affinity separation, and medical treatment, one of the newest applications of plastic antibodies is
in cosmetics, specifically in deodorant to fight body odors. This is done by creating a cavity in

the antibody that is specific for amidinium functionalities by hydrolyzing esters that fits the
precursors of the malodorous compounds in sweat before they are acidified and create an odor.
(16)
IV. Synbodies
In the early 2000s, the most recent type of antibody was discovered to be used as an antibody: A
class of affinity reagents (small molecules that bind/are attracted to larger targets), the molecules
were called synbodies and are often used as anti-infective agents. Synbodies are produced by
exposing an antigen, or target of interest with a peptide microarray to find low affinity
peptides. This is then scaffolded to create high infinity high binding agents with an orthogonal
functional group. While very little is known about the mechanisms involved in synbodies, its use
as primarily an antibacterial agent will be discussed.
The recently discovered synbodies have been in use for antibacterial activity. As
antibiotics were discovered and overused throughout the 1920s, an increasing need has risen in
the medical world for how to fight antibiotic-resistant bacteria. A group led by Valeriy
Domenyuk has searched and found a way to create synbodies with antibacterial activity. For
antibacterial activity, monoclonal antibody based therapeutics had been found to be anti-infection
agents despite high costs from development and production. Antimicrobial peptides have been
discovered in use through amino acid substitutions, but it leads to a high toxicity. Synbodies are
screened on a peptide array to show affinity to an antigen, then acquired onto a scaffold to induce
high affinity. In this case, bacteria is screened on a 10,000 random sequence peptide microarray
and pathogen specific peptides that have binding or lytic action that causes infection is
isolated. .A peptide is a chain of amino acids bonded by an amide bond from the carboxyl group
of the amino acid reacting with the amine group of another. In the microarray, A collection of

varying peptides are linked onto a glass or plastic surface which is then incubated with the
desired sample of binding. In this scenario, the antigen would be used. A secondary antibody is
then added with a fluorescent label. The bacteria is then grown in conditions with and without
activated peptides and a synbody is then designed to adapt the character of the peptide. (7)

II.

Drawbacks to Artificial Antibodies

In natural-human made antibodies, the highest drawback is external environment for the
antibody. With all the benefits to artificial antibodies, one might ask, what are the risks? The
biggest issue in the creation of the artificial antibody is time. The immune system itself can make
as many as 108 different antibodies by assembling a different combination of variable, joining,
and diversity regions in the gene that makes up the epitope on the variable region. As millions of
epitopes exist, each with a differing chemical structure, it is a long process to determine the
specific arrangement of polypeptides that bind to create the shape of the epitope. In many cases,
a pathogen must be injected into an animal and have the animal create natural antibodies that
must then be recreated or made harmless for humans. Another process, although very recently
found, is a technique of creating synbodies by constructing and linking amino acid sequences
and exposing it to various types of human proteins and other pathogens. The chains are bonded
by disulfide bonding, which are easily broken by the presence of high concentrations of salt or
hydrogen concentration (acidic pH) as a hydrogen sulfide complex is created.(3)
Another disadvantage to a synthetic antibody, or at least one made by means of
transgenic animals, is the size of the antibody. Often times, due to the size of the antibody, a
eukaryotic expression system is required which means that the animals must be grown in lab, and
their optimization and fermentation of antibodies may be not only costly, but only prevalent

towards specific antigens. By isolating the IgG domain, or shortening the reading frame of the
IgG genes, the use of the antibody can be increased with a heightened binding affinity.
Ultimately, the cost of research and to determine an antibody is both costly and ineffective,
although widely accepted. (11)
III.

Alternatives

As antibodies has been seen as a high priority to the scientific world and widely accepted,
few alternatives have been discovered, although they do exist. In essence, the biggest difference
in the alternative is the lack of a Y shaped structure that is essential to the definition of the
antibody. It is the Y shaped structure that allows for a binding to the antigen through the Fab
(variable) region but still be indicative to the rest of the immune system through the Fc (constant)
region.( As a result, an antibody mimetic is usually listed as an alternative, including affinities,
monobodies and darpins. While used for similar functions, differing structures exist that allows
for higher solubility and stability towards heat as well as substantial costs. Some of the benefits
of the reagent is the ability to target proteins in multiple ways as opposed to a natural antibody
with a specific antigenic determinant. Without the use the stereotypical antibody structure, the
use of the aptamer includes intracellular live cell imaging According to estimates, researchers
spend up to 800 million dollars a year on the research and development of antibodies that may
not even function. (4)
Mathias Uhlen of the Royal Institute of Technology in Stockholm, Sweden found that out
of the 5346 antibodies received, about half could not detect their antigen on a Western blot or
standard immunohistochemistry assay. To this the group uses aptamers- short molecules of single
or double stranded DNA or RNA that are 1/10 the size of a natural antibody.

An aptamer is ss-RNA or ss-DNA oligonucleotides (small number of nucleotides) that recognize

a specific 3D structure. As a result of being oligonucleotides, the reagents lack a disulfide bond
and lack cysteine residue characteristic of the Y shape in an antibody. It switches often times
from an aptamer sequence to a functional aptamer that binds to an antigen. Due to its small size,
its affinity is higher than natural antibodies-pieces of the aptamer can be used to activate B and T
cells. , and because of the intrinsic property of the nucleotides as the basis of formation, aptamers
can be heated up to 90 degrees Celsius without denaturing. In the case of RNA and DNA, a
phosphodiester bond is created from the electronegative phosphate group being hydrolyzed by 2
hydroxyl groups to create an ester bond linkage on the 3 OH. However, scientists believe that
the affinity of aptamer folding stems from the presence of a 2 OH group and the phosphodiester
linkages are sites of nuclease hydrolysis and the substitution of the 2 OH group with a 2 fluoro,
2amino or borano phosphosphate increases nucleus resistance by increasing electronegativity.
(9)
The creation of an aptamer, similar to molecular imprinting, is a form of trial and error.
The process, called SELEX, involves the synthesis of 100000 unique randomly binded sequences
with a phosphodiester bond with a fluorochrome reporter on a primer to monitor the progress of
reaction. RNA polymerase is added to the primer for transcription in the 5 to 3 direction. The
library is then heated and cooled for the formation of 3D structures, then mixed with the target
for binding enrichment. By the use of capillary electrophoresis, a current is run through the
mixture and those that bind are separated from those unbind based on electro reactivity. This
process further amplifies the sequence through polymerase chain reaction, or the heating of the
DNA to split the DNA, cooling for the binding of the primer, then reheating for synthesis of
DNA. However, often times the sequences are highly glycosylated and are unable to react. (6)

Engineered protein scaffolding has been used as an alternative. While little is known
about protein scaffolding, it is known that scaffold proteins primarily regulate signaling
pathways in the body like signal reduction and as a result enhance signaling efficiency. They are
not immunoglobulins from the lack of a Y shaped structure, and are in essence are nonimmunoglobulin scaffolds that can be adapted for specific binding functions at a specific target
site. In essence, in thinking of the structure of a protein, only 1 active site has been adapted for
specific binding. Scaffolds have been shown to have a higher increase of being applied towards
the development of drug candidates for in vivo diagnostics. The scaffold is created by a small
monomeric protein like Kunitz inhibitors, or from the folded extra membrane of a cell surface
protein that is then modified to existing or create a new binding site for a target. It has been
found to have a heightened production yield in comparison to traditional techniques. Some of the
more known protein scaffolds include affibodies (a three helix bundle of 58 residues with an
interface on 2 alpha helixes), Kunitz domain, anticalins, and DARPins. The protein scaffolds are
all similar in its reliance on beta sandwich with randomized loops at the end that often expose a
concave surface for enzyme substrates or ligands. (4)
VI. Conclusion
Antibodies have been a central role in research since the realization of their molecular
properties in the 1940s. A significant part of this research focuses on translating the adaptability
and specificity of natural antibodies into artificial counterparts such as transgenic syntheses,
molecular imprints and synbodies. The development of artificial antibodies paves the way for
improved diagnosis treatment of immunological conditions without the complications that come
from conventional immunotherapy, such as graft-versus host reactions and chemical instability.
Even so, while there are varying types of antibodies, one of the main benefits to the artificial

antibody in its essence is its stability, or chemical inertness. Chemical inertness is the ability of a
molecule or atom to remain bonded to another in the presence of a different molecule. The
antibody is used as a rigid crosslinked polymer, while also used to treat medical diseases
including cancer, and addiction. While new techniques appear as a replacement to antibodies, the
reliability of these trusted methods cannot be denied.

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