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Antibody Engineering for Pursuing a Healthier Future


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Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.
Content may be subject to copyright.
published: 28 March 2017
doi: 10.3389/fmicb.2017.00495
Frontiers in Microbiology | 1March 2017 | Volume 8 | Article 495
Edited by:
Kuldeep Dhama,
Indian Veterinary Research Institute,
Reviewed by:
Maryam Dadar,
Razi Vaccine and Serum Research
Institute, Iran
Hafiz M. N. Iqbal,
Monterrey Institute of Technology and
Higher Education, Mexico
Ruchi Tiwari,
DUVASU Mathura UP, India
Shihua Wang
Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Microbiology
Received: 26 January 2017
Accepted: 09 March 2017
Published: 28 March 2017
Saeed AFUH, Wang R, Ling S and
Wang S (2017) Antibody Engineering
for Pursuing a Healthier Future.
Front. Microbiol. 8:495.
doi: 10.3389/fmicb.2017.00495
Antibody Engineering for Pursuing a
Healthier Future
Abdullah F. U. H. Saeed, Rongzhi Wang, Sume i Ling and Shihua Wang*
Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical
Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China
Since the development of antibody-production techniques, a number of
immunoglobulins have been developed on a large scale using conventional methods.
Hybridoma technology opened a new horizon in the production of antibodies against
target antigens of infectious pathogens, malignant diseases including autoimmune
disorders, and numerous potent toxins. However, these clinical humanized or chimeric
murine antibodies have several limitations and complexities. Therefore, to overcome
these difficulties, recent advances in genetic engineering techniques and phage display
technique have allowed the production of highly specific recombinant antibodies.
These engineered antibodies have been constructed in the hunt for novel therapeutic
drugs equipped with enhanced immunoprotective abilities, such as engaging immune
effector functions, effective development of fusion proteins, efficient tumor and
tissue penetration, and high-affinity antibodies directed against conserved targets.
Advanced antibody engineering techniques have extensive applications in the fields
of immunology, biotechnology, diagnostics, and therapeutic medicines. However,
there is limited knowledge regarding dynamic antibody development approaches.
Therefore, this review extends beyond our understanding of conventional polyclonal
and monoclonal antibodies. Furthermore, recent advances in antibody engineering
techniques together with antibody fragments, display technologies, immunomodulation,
and broad applications of antibodies are discussed to enhance innovative antibody
production in pursuit of a healthier future for humans.
Keywords: antibody engineering, hybridoma technology, antibody fragments, scFv, phage display technology,
immunomodulation, immunology
In recent years, the development of polyclonal and monoclonal antibody by means of laboratory
animals has become a vital approach to protect against a number of pathogenic contagions
(Marasco and Sui, 2007). These immunoprotective molecules provide defense against transmissible
diseases and can eliminate the infection. Their prophylactic and therapeutic protection ability
was first discovered in the late nineteenth century by the passive transmission of antibodies from
a diseased animal that provided immunity against diphtheria. Subsequently, immune sera from
various herbivores and humans were obtained, pooled, and used as therapeutics. Since then,
the management of infectious diseases such as diphtheria, tetanus, pneumococcal pneumonia,
meningococcal meningitis, and toxin-mediated diseases has considerably improved patient survival
(Casadevall, 1999).
Saeed et al. Antibody Engineering and Recent Advances in Immunology
Antibodies consist of two heavy chains [variable (VH), joining
(JH), diversity (D), and constant (C) region] and two light
chains [variable (VH), joining (JH), and constant (C) region], that
are linked by non-covalent bonding and disulfide (s-s) bridges
(Hamers-Casterman et al., 1993). Antibodies bind antigen with
the help of a VHH fragment that can identify specific and
unique conformational epitopes by the presence of its long
complementary determining regions (CDR3). Escherichia coli
expression systems are unique for the validation of the correct
functioning of antibody fragments in the periplasmic space or
cytoplasm. Conversely, periplasmic expression systems help VH
and VLpairing by providing optimal conditions to allow the
production of functional molecules (Sonoda et al., 2011).
Polyclonal antibodies contain large and diverse
concentrations of different antibodies with unknown specificities.
They are broadly used for the detection of different antigens
in research and diagnostics. However, non-human polyclonal
antibodies induce immune responses in humans that impede
their clinical use such as treating snake bites (Wilde et al., 1996).
Monoclonal antibodies have revolutionized scientific research.
Production of these molecules is based on the fusion of antibody
generating spleen cells from immunized mice, rats, or rabbits
with immortal myeloma cell lines. These monoclonal antibodies
are a highly specific class of biological reagents that facilitate
enhanced clinical diagnostics in the medical arena. Subsequently,
various antibodies are used clinically as prophylactic or
therapeutic agents. The first monoclonal antibody developed by
hybridoma technology was reported in 1975 and subsequently
licensed in 1986 (Köhler and Milstein, 1975; Nelson, 2010). This
development technique signifies a novel way to target specific
mutations in nucleic acids and provide extensive expression in
disease and other conditions (Nelson et al., 2010).
Antibody production was primarily dependent on animal
immunization until the late 1980s by using experimental mice,
rabbits and other related laboratory animals (Wang et al.,
2010). The main difficulty in the production and application of
monoclonal antibodies is the incompetent immune response to
highly toxic or conserved antigens. Furthermore, most clinical
antibodies are of human origin or are at least humanized
in some aspect to avoid immunogenicity (Reichert, 2013).
Therefore, transgenic mice and rabbits with human antibody
genes have been developed to solve this immunogenicity
problem but not the necessity of an effective immune response
after immunization. Finally, to overcome this problem, human
antibodies were generated in vitro by antibody engineering
technologies such as phage display, construction of antibody
fragments, immunomodulatory antibodies, and cell-free systems
(Edwards and He, 2012).
Expression of recombinant antibodies in vitro experienced
a boost with the advent of new molecular tools using various
model organism such as yeast, bacteria etc., and new techniques
for the selection of genetically engineered recombinant libraries
using phage display technology. The phage display technique
was first established by George P. Smith, when he validated
the display of exogenous proteins on filamentous phage by
fusing the peptide of interest to gene III of the phage. The first
recombinant antibody fragments were constructed in bacteria 17
years ago (Roque et al., 2004). The goal of antibody production
technology is to achieve high-titers of highly specific, and high-
affinity antisera. Antigen preparation and animal immunizations
are carried out following the guidelines of production techniques
via hybridoma technology and recombinant technology (Smith,
1985). Moreover, therapeutic antibodies have been developed
by modulation to the fragment crystallizable (Fc) receptor
function and contribution of Fc glycan to immunoglobins,
and the regulation of the antibody glycosylation in relation to
immunoglobins-based therapeutics (Shade and Anthony, 2013).
Human diseases have been known for ages. The comfort
of global travel and better interdependence have supplemented
layers of intricacy to comprehend infectious diseases. These
life threatening contagions effect human health in relation to
unpredicted illnesses, deaths, and interfere many other normal
life activities. Moreover, the diseases take a significant human
toll as well as cause public fear (Morens and Fauci, 2013).
To date, limited knowledge is available on extended aspects
of the production of antibodies by hybridoma technology,
antibody engineering techniques, construction of antibody
fragments, display technologies, and their extended applications
(Fauci and Morens, 2012). Therefore, to cope these health
threats and limitations, extraordinary advances in hybridoma
technology and antibody engineering techniques for the
development of countermeasures (diagnostics, and treatment
by therapeutic antibodies) have been discussed in the present
review. Additionally, widespread antibody applications have
been described in detail for pursuing a healthier future for
humans, and to live a happy life.
Antigen interactions are essential for the normal functions of
antibodies that are widely used in research or therapeutics. The
antigen-specific and membrane-associated receptor antibody
response is mediated by T and/or B cells. Consequently, upon
binding with a suitable antigen, B lymphocytes are induced
to proliferate, and divide by a number of activating signals,
thus increasing the numbers of B cells. These B cells are
then differentiated into specific antibody producing plasma cell
clones that recognize specific antigen epitopes via the antigen
receptor. B cells are activated after recognizing their specific
antigen (Figure 1A;Andersen et al., 2006). Some antigens are
highly multifarious and exhibit abundant epitopes recognized
by several lymphocytes. Consequently, lymphocytes multiply
and differentiate by activation of these multifarious antigens
into plasma cells that produce polyclonal antibody responses
(McCullough and Summerfield, 2005).
Polyclonal antibodies (pAbs) can be produced rapidly in
several months (compared to monoclonal antibodies), with low
cost, low technical skill, and these are stable over a broad range of
pH and salt concentrations. In addition, they have applications
as therapeutic immunoglobulins (Lipman et al., 2005). PAbs
recognize multiple linear epitopes with minimal conformational
changes and contain numerous antibodies of varying affinities,
which are useful for the immunoprecipitation of composite
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Saeed et al. Antibody Engineering and Recent Advances in Immunology
FIGURE 1 | Interaction of antibodies with numerous antigens present on the surface of target cell. (A) Interaction of polyclonal antibodies with specific
surface antigen activates B lymphocytes to divide and differentiate into plasma cell clones producing more antibodies that recognize antigens. (B) Interaction of
monoclonal antibodies with specific surface antigen activates B lymphocytes to divide and differentiate into plasma cell clones that further recruit homogeneous and
mono-specific antibodies.
antigens to form a large precipitating lattice. Mice are used
often to produce pAbs because of their small size and blood
volume. As an alternative, pAbs are produced as ascites in mice.
Moreover, these antibodies have superior specificity compared
with monoclonal antibodies because they are generated by a large
number of B-cell clones each producing antibodies to a specific
epitope (Hudson et al., 2012; Zhuang et al., 2014).
Monoclonal antibodies (mAbs) are clinically significant
homogeneous and mono-specific scientific biomolecules
produced from hybridoma cells by hybridoma technology
(Zhang, 2012). mAbs arise from single cell clone compared to
multiple cell clones for pAbs (Figure 1B;Andersen et al., 2006).
Since their discovery, these molecules have been used as research
tools and have revolutionized the fields of biotechnology,
immunology, diagnostics, and medicine. The technology was
described for the first time by Köhler and Milstein (1975) in the
mid-1970s in the journal Nature, and they were later awarded
the Nobel Prize (Saeed and Awan, 2016).
Currently, mAb products approved by the US Food and Drug
Administration (FDA) are increasing worldwide i.e., about four
new products per year. Currently, 47 mAb products in the US,
Europe and global markets have been approved for the treatment
of a variety of diseases (Table 1;Ecker et al., 2015). At the current
rate, about 70 mAb products will be on the market by 2020,
and collective global trade will be approximately $125 billion
(Ecker et al., 2015). Improvements in hybridoma technology are
based on research demand, cost effectiveness, human labor, and
reduced development time. Similarly, the production of mAbs
requires multiple phases, long duration, and high cost. Currently,
mAbs have been produced against a number of mycotoxins such
as fumonisin B1 (Yuan et al., 2012; Ling et al., 2014, 2015b),
citreoviridin (Jin et al., 2014), marine toxins (Saeed and Wang,
2016), and other exo- and endo-antigens. Similarly, mAbs against
transmembrane enzymes have been produced (Yuan et al., 2012).
Method of Antibody Production
Hybridoma technology has been a significant and essential
platform for producing high-quality mAbs (Zhang, 2012). It
permits generation of therapeutic antibodies in a native form.
However, technical difficulties in hybridoma production have
updated the mainstream antibody production into new ways like
display and transgenic mice techniques. Nevertheless, hybridoma
technology is a classical and established route of generating
specific antibodies all around the globe (Glukhova et al., 2016).
The technology begins with immunization of test animals with
an antigen of interest and serum antibody titer is determined
by enzyme linked immunosorbent assay (ELISA). Subsequently,
the spleen is aseptically removed and splenocytes are fused
with myeloma cells to produce hybridoma cells. Hybridoma
cells are then cultured in 96-well plates in the presence of
hypoxanthine-aminopterin-thymidine (HAT) selection medium
for high throughput screening. Later, hybridoma cells producing
desired antibodies are screened by conventional ELISA and
novel nanoparticle-probed immunoassay (colloidal gold or silver
nanoparticles; Figure 2). Cell culture systems in vitro with
specific mAb cell lines were then subjected to mass generation by
media selection, shaker flasks, and bench-scale bioreactors (Ling
et al., 2014).
The optimal conditions such as temperature, percent carbon
dioxide, and humidity for cell cultures should be determined
(Sen and Roychoudhury, 2013), and then transferred to a
pilot scale for scalability and toxicology studies. In addition,
clinical materials should be produced on a large scale under
the existing good manufacturing practice (cGMP) regulations.
After production on a small scale, products that are already
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TABLE 1 | Monoclonal antibody products in the US, Europe, and global markets approved for diseases.
Brand name Company reporting US
Company reporting EU
Year of
AlprolIX (Factor IX Fc fusion
Biogen Idec Sobi and Biogen Idec 2014 Hemophilia B
Cyramza (ramucirumab) Eli Lilly and Co. Eli Lilly and Co. 2014 Gastric cancer and non-small cell lung cancer
Eloctate (Factor VIII Fc
fusion protein)
Biogen Idec Sobi and Biogen Idec 2014 Anti-hemophilic Factor
Entyvio (vedolizumab) Takeda Pharmaceutical
Takeda Pharmaceutical
2014 Ulcerative colitis (UC)/Crohn’s disease (CD)
Keytruda (pembrolizumab) Merck & Co. Merck & Co. 2014 Melanoma
Sylvant (siltuximab) Johnson & Johnson Johnson & Johnson 2014 Multicentric Castleman’s Disease (MCD)
Inflectra (infliximab
N/A Hospira 2013 Tumor necrosis
Kadcyla (ado-trastuzumab
Roche Roche 2013 Metastatic breast cancer
Lemtrada (alemtuzumab) N/A Sanofi 2013 Relapsing form of multiple sclerosis (MS)
Gazyva (obinutuzumab) Roche Roche 2013 Chronic lymphocytic leukemia (CLL)
Remsima (infliximab
N/A Celltrion 2013 Rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, Crohn’s
disease, ankylosing spondylitis, and plaque psoriasis
Perjeta (pertuzumab) Roche Roche 2012 Human epidermal growth factor receptor 2 (HER2)/neu-positive)
metastatic breast cancer
Abthrax (raxibacumab) GlaxoSmithKline GlaxoSmithKline 2012 Inhalational anthrax
Prolia (denosumab) Amgen GlaxoSmithKline 2011 Osteoporosis
Adcetris (brentuximab
Seattle Genetics Takeda Pharmaceutical
2011 Hodgkin lymphoma
Benlysta (belimumab) GlaxoSmithKline GlaxoSmithKline 2011 Systemic lupus erythematosus (SLE/lupus)
Eylea (aflibercept) Regeneron
Bayer Healthcare
2011 Macular degeneration
Nulojix (belatacept) Bristol-Myers Squibb Bristol-Myers Squibb 2011 Prevention of transplant rejection
Xgeva (denosumab) Amgen Amgen 2010 Prevention of bone fractures and other skeletal bone tumor
Arzerra (ofatumumab) GlaxoSmithKline GlaxoSmithKline 2009 Chronic lymphocytic leukemia (CLL)
Ilaris (canakinumab) Novartis Pharmaceuticals Novartis Pharmaceuticals 2009 Systemic Juvenile Idiopathic Arthritis (SJIA)
Actemra (tocilizumab) Roche Roche 2009 Moderate to severe rheumatoid arthritis
Simponi Aria (golimumab) Johnson & Johnson Merck & Co. 2009 Rheumatoid arthritis
Stelara (ustekinumab) Johnson & Johnson Johnson & Johnson 2009 Plaque psoriasis, psoriatic arthritis and Crohn’s disease
Removab (catumaxomab) N/A NeoPharm Group 2009 Malignant ascites
Arcalyst (rilonacept) Regeneron
2008 Familial Cold Auto-inflammatory Syndrome/Muckle-Wells
Cimzia (certolizumab pegol) UCB UCB 2008 Rheumatoid Arthritis
Nplate (romiplostim) Amgen Amgen 2008 Low blood platelet counts
Soliris (eculizumab) Alexion Pharmaceuticals Alexion Pharmaceuticals 2007 Paroxysmal nocturnal hemoglobinuria (PNH), and Atypical
hemolytic uremic syndrome (aHUS)
Lucentis (ranibizumab) Roche Novartis Pharmaceuticals 2006 Macular degeneration
Vectibix (panitumumab) Amgen Amgen 2006 Metastatic colorectal cancer
Orencia (abatacept) Bristol-Myers Squibb Bristol-Myers Squibb 2005 Rheumatoid arthritis
Avastin (bevacizumab) Roche Roche 2004 Various cancers and eye disease
Tysabri (natalizumab) Biogen Idec Biogen Idec 2004 MS and Crohn’s disease
Erbitux (cetuximab) Bristol-Myers Squibb Merck KGaA 2004 Metastatic colorectal cancer, metastatic non-small cell lung
cancer, head and neck cancer
Humira (adalimumab) AbbVie AbbVie 2002 Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis,
ankylosing spondylitis, plaque psoriasis, and hidradenitis
Enbrel (etanercept) Amgen Pfizer 1998 Rheumatoid arthritis
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TABLE 1 | Continued
Brand name Company reporting US
Company reporting EU
Year of
Herceptin (trastuzumab) Roche Roche 1998 Breast cancer/stomach cancer
Remicade (infliximab) Johnson & Johnson Merck & Co. 1998 Crohn’s Disease
Simulect (basiliximab) Novartis Pharmaceuticals Novartis Pharmaceuticals 1998 Prevention of organ rejection
Synagis (palivizumab) AstraZeneca Abbvie 1998 Lung disease caused by respiratory syncytial virus (RSV)
Rituxan (rituximab) Roche Roche 1997 Non-Hodgkin’s lymphoma or chronic lymphocytic leukemia
ReoPro (abciximab) Lilly Lilly 1994 Prevention of blood clots
FIGURE 2 | Illustration showing the production route of hybridoma technology. Monoclonal antibodies are generated by immunizing laboratory animals with a
target antigen. B cells and myeloma cells are fused and then selected in HAT medium. Finally, hybridoma cells producing the desired antibodies are screened.
cultured in a laboratory are then transferred to pilot scale
commercialization. The process then performs characterization,
scaling, technology transfer, and validation. Commercial cell
culture for the production of a biological product is completed
by pilot scale laboratory methods (Li et al., 2010). Recently,
commercialization is initiated by process characterization, scale-
up, technology transfer, and validation of the manufacturing
process (Li et al., 2006).
ELISA is enzyme-based colorimetric assay, requires
large sample volumes, several incubation steps and has low
detection sensitivity (Tang and Hewlett, 2010). Conversely,
nanotechnology and nanoparticles (NPs) use nanomaterials with
length scale of 1–100 nanometers (nm). Nanomaterials have
unique biological properties such as small size, large surface-to-
volume ratio, sharp melting temperature, magnetic properties,
unusual target binding properties, and size based multi-coloring
(Qi and Wang, 2004). NPs such as gold particles have been used
to improve assay sensitivity and specificity of antibodies, low
limit of detection (LOD), dose response over 10,000-fold and the
detection sensitivity by 1,000-fold compared to ELISA (Tang and
Hewlett, 2010).
Similarly, an essential factor of NPs selection is that
nanometer-sized (colloidal gold or silver) particles can be
conjugated with targeting ligands, including mAbs, peptides,
or small molecules with functional and structural properties
that are not available from either discrete molecules or bulk
materials. Nanoparticles are probed with mAbs that can be
used to target any antigen of interest (Ling et al., 2015a).
The use of colloidal gold is a rapid, less time consuming, and
cost effective technique. This technology has been used in the
development of immunochromatic strip assays based on mAb.
The technique has widespread applications for the detection of a
number of antigens (exogenous antigens, endogenous antigens,
autoantigens, neoantigens, viral antigens, and tumor antigens)
with high specificity and binding affinity (Schumacher and
Schreiber, 2015).
Antigen Preparation
Antigen preparation including quality and quantity is essential
for the production of antibodies. The purification of antigens
is difficult and time consuming but antigen purity is vital for
adequate immune responses (Leenaars and Hendriksen, 2005).
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Insignificant impurities (<1%) can be immunodominant in
the case of various microbial antigens. They show non-specific
activity against the antigens of interest and specific activity
against impurities. Higher concentrations of specific antibodies
are obtained after purification and the exclusion of impurities
by extensive absorption procedures such as antibody affinity
chromatography (Leenaars et al., 1997; Mazzer et al., 2015).
Before scheduling immunization, antigen contamination
should be considered by the researcher. The antigen and diluents
should be free of endotoxins such as lipopolysaccharide, or
chemical residues that have been utilized to neutralize the
microorganism. Additionally, the pH must be adjusted to
prevent undesirable effects in the animal to be immunized
(Hendriksen and Hau, 2003). Moreover, sterile working
conditions, antigen concentration, animal preparation, and
injection quality must be confirmed. These conditions are
necessary to avoid suppression, sensitization, tolerance, or other
superfluous immunomodulation and to induce effective immune
responses. The required antigen concentration (µg to mg) is
based on the intrinsic properties of the antigen. Usual doses of
antigen conjugated with Freund’s adjuvant for rabbits are in
the range of 50–1,000 µg; for mice, 10–200 µg; and for goats
and sheep, 250–5,000 µg (Leenaars et al., 1997). The inherent
properties of antigen quantity include purification, animals to be
immunized, type, and quality of the adjuvant (to elicit high-titer
serum responses), route and the immunization (injection)
frequency (Hanly et al., 1995).
Antibody production involves the immunization or injection
of laboratory animals with an immunogenic protein and test
sampling of antiserum. The immunization is performed in
specific animal species and the adjuvant is processed to form an
immunogenic emulsion that is insoluble in water (Leenaars and
Hendriksen, 2005). Conjugation of smaller or less immunogenic
antigens i.e., haptens (numerous myco- and marine toxins)
to carrier proteins is essential before animal injection. After
immunization, the animal is monitored daily or three times a
week for side effects (clinical and pathological examination).
Severe pathological changes such as tissue reactivity, infection
and anaphylactic reactions in case of booster injections have been
reported in the absence of visual clinical or behavioral changes
(Leenaars et al., 1997).
The immunization route is based on the choice of animal
species, adjuvant, concentration and quality parameters of the
antigen (Apostolico Jde et al., 2016). Suitable immunization
routes include subcutaneous (s.c.), intradermal (i.d.),
intramuscular (i.m.), intraperitoneal (i.p.), and intravenous
(i.v.). Blood sample handling should not interfere with the
immunization site to avoid pain and distress to the animal while
taking blood sample (Leenaars et al., 1997). The i.v. route for
water-in-oil emulsions such as Freund’s complete and incomplete
adjuvants (FCA and FIA) may be fatal as embolisms caused
by large particulates or viscous gel adjuvants (e.g., aluminum
salts) can occur (Hanly et al., 1995). The s.c. administration
of FCA is immunologically effective (0.1 mL in mice and 0.25
mL in rabbits) with some reports of pathological changes.
The i.p. administration of FCA is not suggested for antibody
development because it induces infection, peritonitis, and social
variations. There is high risk of anaphylactic shock for booster
injections by the i.v. or i.p. route (Leenaars et al., 1997).
The volume of the immunogenic mixture depends on the
quality of the antigen and the degree of the lesions formed.
Therefore, smaller volumes of mixtures are injected to induce
antibody responses except when increased concentrations of
antigen are required (Leenaars and Hendriksen, 2005). The
immunization volume also depends on the animal species, route,
and chemistry of the injection mixture. The volume of FCA
has lethal effects on animal physiology and the extent of lesions
produced is associated with high volumes of FCA (Ramos-Vara
and Miller, 2014).
Booster injections have a significant effect on the outcomes of
immunization and induction of B memory cells. Class switching
of B cells depends on the time interval between two consecutive
injections. A small volume of antigen can be used for a booster
injection without adjuvant (Leenaars et al., 1997). A booster
injection is used when the antibody titer has reached a plateau
or begins to decline. Antibody titers typically peak at 2–3 weeks
after immunization in the absence of the depot-forming adjuvant
used in the first immunization. A booster injection is used after 4
weeks in the presence of depot-forming adjuvant. FCA should be
used in the first s.c. injection to avoid subsequent (Mycobacteria
proteins) severe tissue reactions (Hendriksen and Hau, 2003).
Hybridoma Screening
Hybridoma technology is essential for the production of high-
quality mAbs as well as research and diagnostic reagents, and
it is currently the most rapidly growing class of therapeutics
(Hnasko and Stanker, 2015). MAbs can be effectively screened
by a number of techniques such as ELISA, phage display,
and other related technologies. Screening identifies and picks
only specific antibody producing hybridomas. In hybridoma
technology, laboratory animals are immunized with an antigen
of interest and specific antibody-producing B cells are isolated
from the spleen and then fused with immortal myeloma cells
(such as sp2/0; Bradbury et al., 2011). Subsequently, expansions
of clonal populations are produced from serially diluted sub-
cloned individual hybridoma cells in a microtiter plate. Next,
the hybridoma supernatant is checked by ELISA or other related
immunoassays for desired antibody activity (El Debs et al., 2012).
After screening, hybridoma cells are expanded in bigger wells
or in culture flasks to maintain the hybridoma and provide
sufficient cells for cryopreservation and supernatants for later
investigations (Nelson et al., 2010; El Debs et al., 2012). However,
this technique restricts the number of clones that can be screened
to no more than a few thousand. Consequently, insufficient
numbers of cells remain available for clonal expansion and
cell immortalization. Therefore, improved techniques such as
antigen-based microarrays have been used for the screening of
>105clones in <12 h, or lithographically fabricated microwells
for individual cell compartmentalization. Nevertheless, these
techniques only screen for binding activity and do not allow
functional assays (El Debs et al., 2012).
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Characterization of Monoclonal Antibodies
MAb characterization is based on a determination of its
physicochemical and immunochemical character, heterogeneity,
purity, impurities, biological activity, potency, and concentration
(Berkowitz et al., 2012). For physicochemical properties, the
isotype (class, subclass, light chain composition) and primary
structure of the mAb are determined. Immunological properties
include binding assay, affinity, avidity, immunoreactivity (cross-
reactivity with other structurally homologous proteins), analysis
of CDR, epitope characterization, and determination of effector
functions (CHMP, 2008).
Heterogeneity (including chromatographic and
electrophoretic methods) and quantity are determined for
the characterization of mAb purity, impurity, contaminants, and
concentration. Specificity of mAbs is also checked. Peptide map,
anti-idiotype immunoassay, potency, and other appropriate
methods are used to determine its identity and biological activity
(CHMP, 2008).
Applications of Monoclonal Antibodies
MAb-based products exhibit superior specificity for a particular
antigen. This characteristic feature of the immunoglobins makes
them an ideal tool for many applications including disease
diagnosis and therapy (Table 2;Modjtahedi et al., 2012; Redman
et al., 2015; Steplewski et al., 2015). Diagnostic applications
include biochemical analysis and imaging. It involves a number
of immunoassays for the detection of hormonal, tissue, and
cell products. Imaging is carried out using radiolabeled mAbs
for diagnostics of infectious diseases. Therapeutic mAbs have a
wide range of applications. They are used in the treatment of
cancer, transplantation of bone marrow and organs, autoimmune
diseases, cardiovascular diseases, and various infectious diseases.
TABLE 2 | Summary of various applications of monoclonal antibodies.
Application Features Diagnosis, treatment, and gains
Biochemical analysis Diagnostic tests are regularly used in radioimmunoassay (RIA)
and ELISA in the laboratory to quantify the circulating
concentrations of hormones and several other tissue and cell
Pregnancy: human chorionic gonadotropin; cancers: colorectal cancer,
prostate cancer, tumor markers; hormonal disorders: thyroxine,
triiodothyronine, thyroid; infectious diseases, sexually transmitted
diseases (STDs) include Neisseria gonorrhoeae, herpes simplex virus.
Diagnostic imaging The technique is also called immunoscintigraphy.
Radiolabeled—mAbs are used in the diagnostic imaging of
the diseases. The radioisotopes commonly used for labeling
mAbs are iodine—131 and technetium—99. Imaging tool
include single photon emission computed tomography
(SPECT) cameras.
Myocardial infarction: myocardial necrosis; deep vein thrombosis:
thrombus in thigh, pelvis, calf, knee; atherosclerosis: coronary and
peripheral arteries; immunohistopathology of cancers: colon, stomach,
pancreas, liver, germ cells of testes, choriocarcinoma, prostate cancer,
melanoma; hematopoietic malignancies: hematopoietic stem cells
malignancy; bacterial infections.
Direct therapeutic agents Monoclonal antibodies can be directly used for augmenting
the immune function of the host triggering minimal toxicity to
the target tissues or the host.
Opsonization and phagocytosis of pathogenic organisms: hepatitis
B-virus, E. coli, haemophilus influenza, streptococcus sp.
pseudomonas sp; cancer treatment: ADCC, CDC, phagocytosis of
cancer cells, colorectal cancer, lymphoma, melanoma;
immunosuppression of organ transplantation; treatment of AIDS;
treatment of autoimmune diseases: rheumatoid arthritis, MS.
Targeting agents in therapy Toxins, drugs, radioisotopes etc., can be attached or
conjugated to the tissue-specific mAbs and carried to target
tissues for efficient action.
Immunotoxins: diphtheria toxin, pseudomonas exotoxin, toxins used
for cancer treatment; drug delivery: antibody-directed enzyme pro-drug
therapy (ADEPT), liposomes coupled mAb drug delivery; dissolution of
blood clots: thrombus in coronary or cerebral artery; immunotherapy
(RAIT): yttrium-90, indium-111.
Protein Purification MAbs are produced against protein of interest and
conveniently used for the purification of that particular protein.
In a single step, it is likely to reach more than 5,000-fold purification of
Catalytic mAbs (ABZYMES) The antibody enzymes, appropriately regarded as abzymes,
are the catalytic antibodies. Hapten-carrier complex is formed
that resembles the transition state of an ester undergoing
hydrolysis. This hapten conjugate is used to produce
anti-hapten mAbs.
Widespread applications include splicing of peptides and
deoxyribonucleic acids, dissolution of blood clots, and killing of viruses.
Autoantibody fingerprinting Recently, a new class of individual specific (IS) autoantibodies
have been documented in recent years. These
IS-autoantibodies are developed after birth and extend
maximum in number by 2 years, and then stay persistent
Autoantibodies collected from blood, saliva, semen and tears are used
for detection, and identification of individuals especially for forensic
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Therapy can be carried out by direct use of mAbs as therapeutic
agents and as targeting agents respectively (Modjtahedi et al.,
Computational and Bioinformatics Tools
for Antibody Selection
Computational and bioinformatics approaches play an essential
role for antibody selection and epitope prediction. It is
an interdisciplinary science and the term can be defined
as “the application of computer tools to handle biological
information.” Several computational and bioinformatics tools for
prediction of antibody binders include RANKPEP, nHLAPred,
NetMHC, kernel-based inter-allele peptide binding prediction
system (KISS) with support vector machine (SVM; Bhasin and
Raghava, 2007; Lundegaard et al., 2008). Moreover, these tools
use databases that contain known epitopes from SYFPEITHI,
MHCBN, LANL, and IEDB for protein epitope prediction (Soria-
Guerra et al., 2015).
Furthermore, other tools include position specific scoring
matrices (PSSM) used for sequence alignment, IEDB analysis
resource database uses NetMHCpan for peptide affinity,
quantitative matrices (QM), whole antigen processing pathway
(WAPP), Matthews correlation coefficient (MCC), and spatial
epitope prediction of protein antigens (SEPPA) (Soria-Guerra
et al., 2015). Recently, a simulation tool C-ImmSim was
developed for the study of a number of different immunological
processes. The processes include simulations of immune
response by representing pathogens, as well as lymphocytes
receptors, amino acid sequences and T and B cell epitope
prediction (Rapin et al., 2010).
The first fully human mAb was developed over 25 years ago
by phage display and a selection of antigen-specific binders
from blood lymphocyte libraries (Gavilondo and Larrick, 2000).
This technique employed transgenic animals such as mice
and rabbits with integrated human immunoglobulin (Ig) loci.
Germline-configured chimeric constructs confirmed that human,
mouse, and all mammalian Ig loci function in very similar ways
(Neuberger and Bruggemann, 1997). Antibody development
has progressed from hybridoma technology to a recombinant
deoxyribonucleic acid (DNA) approach. In the last few years, a
number of engineered antibody drugs have been approved or
investigated in phase II or III clinical trials (Table 3;Nelson et al.,
2010; Dantas-Barbosa et al., 2012; Nixon et al., 2014).
MAb immunoglobulins (IgG) are the starting material for
the generation of smaller antibody fragments in lymphoid or
non-lymphoid cells (Klimka et al., 2000). Traditional hybridoma
technology has several limitations such as being exclusively
murine based, time consuming, and exhibiting low-affinity in
conventional assays. Therefore, antibody engineering, display
system, and immunomodulation methods are now used to
produce efficient therapeutic antibodies (Klimka et al., 2000).
The first study of recombinant antibodies in bacteria was
difficult because of interference from disoriented proteins in
the bacterial cytoplasm. A new antibody expression technique
was developed to produce smaller antibody molecules (Fab or
Fv fragments; Okamoto et al., 2004) where numerous types
of vectors (phagemid) are used with competent E. coli. This
technique involves the expression of antibody fragments for
recombinant antibody construction. In this way, a number of
genetically engineered antibodies have been constructed, such
as Fab fragments, Fv fragments, single-chain variable fragments,
bivalent antibodies, and bispecific antibodies (Little et al., 2000).
Antibody Fragment Display
Different antibody fragments are used in phage display
technology: scFv (single chain fragment variable), Fv (fragment
variable), Fab (antigen binding fragment) and their derivatives,
V-gene domain, bispecific or bivalent antibodies, and other
oligomers (Figure 3;Nelson, 2010). Fab fragments are the linkage
of VH–CHand VL–CLby disulfide bridges, and radiolabeled
Fabs are used in tumor imaging. Fv is used for the construction
of VLand VHdomains or their modifications such as scFv,
which is the most popular fragment. A (Gly4Ser)3 linker is
used for the stabilization of VL–VHand proper antigen binding
site formation without the loss of antibody affinity. Chelating
recombinant antibodies (CRAbs) are scFv segments with a high
binding affinity. These constructs consist of two scFv fragments
specific to the identical antigen and adjacent epitopes. These
fragments are connected by a short linker (up to 10 amino acids)
for the dimerization and formation of diabodies (Wright and
Deonarain, 2007; Nelson, 2010).
Single-Chain Variable Fragment (scFv)
ScFv has high affinity, highly solubility, multi-domains, and
high binding specificity with their target antigen and they
are used for antibody engineering, biotechnology, cancer
research, and biomedical applications. ScFv have been engineered
to improve the effector functions of full-length antibodies,
carrying toxins to kill cells, or cytokines to activate the
immune system. Furthermore, bispecific antibodies have been
constructed to target multiple receptors (AlDeghaither et al.,
2015). Recombinant antibody engineering and recombinant
DNA technology has facilitated successful expression and cloning
of widespread antibody fragments in bacteria (E. coli), as well
as mammalian (Chinese hamster ovary (CHO), or myeloma
cell lines e.g., Sp2/0), yeast (Pichia pastoris), plant (Arabidopsis),
and insect cells (Drosophila melanogaster) (Frenzel et al., 2013).
These smaller fragments have several advantages over full-
length antibodies such as tumor and tissue penetration, blood
clearance, short retention times, and reduced immunogenicity.
Likewise, they have better fusion in bacteria, and display on a
filamentous phage. These fragments permit the production of
homogenous proteins for diagnostic and therapeutic purposes
as well as structural studies (Frenzel et al., 2013; Wu et al.,
ScFv fusion proteins are constructed by the association of
heavy (VH) and light chains (VL) of immunoglobulins via
a short peptide linker. Antigen specific scFv can be easily
generated by phage display. These fusion proteins have extensive
applications in cancer therapeutics such as in lymphatic invasion
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TABLE 3 | Engineered antibody drugs in phase II, phase III clinical trials or approved.
drug code
Company/sponsor Type/source Target antigen Progress status Disorder(s) Administration
Human IgG4 monoclonal
Programmed cell death
protein 1 (PD-1)
Approved (2017) Locally advanced or
metastatic urothelial
Eli Lilly and
Humanized IgG1
monoclonal antibody
Epidermal Growth Factor
Receptor (EGFR)
Approved (2015) Cancer (NSCLC) IV
Eli Lilly and
Human IgG1 monoclonal
Vascular endothelial growth
factor receptor 2 (VEGFR2)
Approved (2015) Cancer (NSCLC, breast,
metastatic gastric
CAT technology and
Human Genome
Recombinant Human IgG1λ
Monoclonal Antibody
Protective antigen (PA)
component of anthrax
(Bacillus anthracis)
Approved (2012) Prophylaxis, anthrax Oral or IV
Human IgG1κmonoclonal
Human cytotoxic
T-lymphocyte antigen 4
Approved (2011) Melanoma, Metastatic IV
GlaxoSmithKline Recombinant human IgG1λ
monoclonal antibody
B-lymphocyte stimulator
Approved (2011) Autoantibody-positive,
systemic lupus
Dyax Corp. human IgG1 monoclonal
Plasma kallikrein Approved (2009) Hereditary angioedema SI
Amgen Inc. Peptide-Fc fusions or
Thrombopoietin receptor
Approved (2008) Immune thrombocytopenic
Genentech Recombinant humanized
IgG1κmonoclonal antibody
Vascular endothelial growth
factor A (VEGF-A)
Approved (2006) Neovascular (wet) age-related
macular degeneration
Abbott Laboratories Recombinant human IgG1
monoclonal antibody
Tumor necrosis factor-α
Approved (2002) Rheumatoid arthritis, juvenile
idiopathic arthritis, psoriatic
arthritis, ankylosing
spondylitis, crohn’s disease,
ulcerative colitis, plaque
Recombinant factor VIII
Factor VIII Approved (2008) Hemophilia A IV
(CNTO 1959)
Human IgG1 monoclonal
Interleukin 23 (IL-23p19) Phase III Psoriasis IV or SI
Gantenerumab Roche Human IgG1 monoclonal
Beta-amyloid Phase III Alzheimer IV or SI
(AMG 386)
Amgen Inc. Angiopoietin
1/2-neutralizing peptibody
Angiopoietin 1 and 2
neutralizing peptibody
Phase III Cancer (ovarian, peritoneal,
fallopian tube)
Sanofi/Crucell Human IgG1κmonoclonal
Rabies virus glycoprotein Phase III Prophylaxis, inhaled anthrax IV
Human IgG1λmonoclonal
Activin receptor IIB (ActRIIB) Phase IIb/III Pathological muscle loss and
ZMapp Mapp
Chimeric monoclonal
antibody cocktail of
MB-003, ZMab, c2G4 and
Ebola virus protein sGP Phase II Ebola virus disease (EVD) IV
GRN1005 Angiochem Inc. Peptide-drug conjugate Lipoprotein receptor
Phase II Non-small Cell Lung Cancer
(NSCLC) With Brain
(AMG 479)
Amgen Inc. Human IgG1 monoclonal
Insulin-like growth factor
receptor (IGF-1R)
Phase II Cancer (pancreatic, colorectal
breast, NSCLC)
Eli Lilly and Co. Human IgG1 monoclonal
Insulin-like growth factor-1
receptor (IGF-1R)
Phase II Cancer (NSCLC, metastatic
melanoma of the eye, liver)
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TABLE 3 | Continued
drug code
Company/sponsor Type/source Target antigen Progress status Disorder(s) Administration
MM-121 Merrimack
partner with Sanofi
Human IgG2 monoclonal
ErbB3 gene Phase II Cancer (advanced ovarian,
hormone sensitive breast
cancer, NSCLC, and HER2
negative neoadjuvant breast
BIIB 033 Biogen Idec Human aglycosyl IgG1
monoclonal antibody
Leucine-rich repeat and
immunoglobulin-like domain
containing, Nogo
receptor-interacting protein
Phase II Acute optic neuritis, MS IV
Mapatumumab GSK company Human IgG1 monoclonal
apoptosis-inducing ligand
receptor 4 (TRAIL-4)
Phase II Cancer (NSCLC,
non-Hodgkin lymphoma, liver,
AstraZeneca Human IgG4 monoclonal
Interleukin-13 Phase IIb Ulcerative colitis, pulmonary
fibrosis, asthma
MedImmune Human IgG4 monoclonal
stimulating factor receptor
Phase IIb Rheumatoid arthritis IV
Bertilimumab Immune
Human IgG4 monoclonal
Eotaxin-1 (CCL-11 gene) Phase II Ulcerative colitis IV
MorphoSys/ GSK
Recombinant Human IgG1
monoclonal antibody
Granulocyte macrophage
colony-stimulating factor
Phase II Inflammatory diseases
(rheumatoid arthritis)
BHQ880 Novartis
Human IgG1 monoclonal
Dickkopf-1 (DKK1 gene) Phase II Multiple myeloma IV
(CNTO 888)
Centocor Research
& Development, Inc.
Human IgG1κmonoclonal
Monocyte Chemoattractant
Protein-1 (MCP-1), CCL-2
Phase II Prostate cancer IV
Recombinant Human IgG1
Oxidized low-density
lipoprotein (LDL)
Phase II Stable atherosclerotic
vascular disease
IV or SC
Amgen Inc. Recombinant Human IgG1
monoclonal antibody
Epithelial cell adhesion
molecule (EpCAM)
Phase II Colorectal cancer (CRC), liver
and breast metastases
Sanofi/Genzyme Human IgG4 monoclonal
Transforming growth
factor-beta (TGF-β) 1, 2,
and 3
Phase II Primary brain tumors, primary
focal segmental
glomerulosclerosis, diopathic
pulmonary fibrosis, cancer
BI-505 BioInvent Human IgG1 monoclonal
Intercellular Adhesion
Molecule 1 (ICAM-1) or
Cluster of Differentiation 54
Phase II Cancer (multiple myeloma) IV
NSCLC, non-small cell lung cancer; IV, intravenous; SI, subcutaneous injection; IM-intramuscular injection.
vessels, colon cancer, hepatocarcinoma, and diagnostics of
human disease (Tonelli et al., 2012). Moreover, scFv have been
widely used with phage display panning i.e., an affinity selection
technique, to construct ligands to detect toxins produced by
various pathogenic entities in vitro or in vivo. Similarly, co-
expression vector systems have been used to prevent and cure
diseases by scFv. Currently, various scFv fragments have been
constructed against toxins and virulence factors of pathogens
(Tonelli et al., 2012; Wang et al., 2013, 2016). Additionally,
a method for rapid and effective high-affinity GFP-based
antibody production corresponding to scFv was developed. It
was demonstrated by inserting CDR3 into green fluorescence
protein (GFP) loops for improved disease diagnosis and therapy
(Wang et al., 2014b). Skp co-expressing scFv has high solubility
and binding activity to antigen thermolabile hemolysin (TLH)
(a pathogenic factor of Vibrio parahaemolyticus) and was
developed by using pACYC-Duet-skp co-expression vector. This
scFv was constructed to detect TLH directly in real samples.
Furthermore, an antibody was developed against TLH that
showed strong neutralizing effects on TLH-induced cell toxicity
(Wang et al., 2006, 2007, 2012, 2013, 2014a; Chen et al.,
Library Construction of scFv
Phage-displayed antibody libraries have been widely used
for the construction of high-affinity target-specific antibodies
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FIGURE 3 | Complete antibody and various types of antibody fragments. These fragments are constructed by antibody engineering techniques for enhanced
therapeutic applications.
(Chen et al., 2008). ScFv is a non-covalent heterodimer that
consists of VHand VLdomains. Moreover, antibody repertoires
from phage-displayed libraries are constructed by harvesting
messenger ribonucleic acids (mRNAs) from peripheral blood
lymphocytes, hybridoma, spleen, bone marrow, tonsil, and
similar other sources (Chen et al., 2008). Large libraries with
a diverse range of antibodies and genes are created using
reverse transcribed (RT) process into cDNA to function as a
template for antibody gene amplification (PCR) (Lim et al.,
Libraries are also created by PCR assembly, phagemid,
and sequential cloning or combinatorial infection. The VH
and VLchains are combined (linker orientation dependent)
and cloned to construct a combinatorial scFv library for
antigen selection (Ahmad et al., 2012). Another technique
for recombinant antibody production is the utilization of
phage recombinants displaying antibodies at their tips, and
which undergo biopanning for the in vitro selection of scFv
from large libraries of variable domains circumventing the
traditional hybridoma method. Numerous scFv fragments have
been constructed against haptens (Wang et al., 2006, 2014a),
proteins, carbohydrates, receptors, and tumor antigens for
medical therapies and diagnostic applications (Wang et al., 2012,
Screening by Phage Display
Phage display is a powerful biological technique for screening
specific peptides or proteins. Screening of antibody libraries by
phage display permits the rapid selection of scFvs to isolate
VHand VLchains for mAb transformation. Thus, therapeutic
antibodies against noxious or highly conserved antigens, plasma
membrane proteins, and receptors can be obtained in their native
conformation while avoiding animal immunization (Rader and
Barbas, 1997). The peptide libraries are incubated on a plate
coated with the antigen of interest. Next, unbound phages are
washed away. Then, the phages are amplified using host bacteria
for high-affinity elution and binding/amplification cycles to
expand the pool in favor of binding sequences. Finally, individual
clones are characterized after 3–5 rounds by DNA sequencing
and ELISA to achieve the resultant structural and functional
details (Catanese et al., 2007).
Expression of scFv
Numerous expression systems such as E. coli, yeast, mammalian,
insect cell, wheat germ cell-free expression system, and plant-
based expression system have been used for the successful
isolation of scFv and display as fragments (Wang et al.,
2013). The expression and activation of scFv is performed by
appropriate folding and in vitro refolding for aggregation. The
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expression systems for the production of active scFv antibody are
selected, designed, and constructed based on hosts. The bacterial
expression system is the most suitable and widely used method
for the production of scFv antibody fragments compared to other
available expression strategies (Frenzel et al., 2013).
E. coli is a valuable tool for expression systems in the fields of
genetics and biochemistry. The system has numerous advantages
including enhanced folding, low cost, high throughput, well-
studied physiology and genetics, rapid growth, high yields
up to 10–30% of total cellular protein, and simple handling.
Additionally, it can be used for multi-plexed cloning, expression,
and purification of proteins for structural genomics (Rosano and
Ceccarelli, 2014).
Characterization of scFv
Antibody characterization involves peptide mapping, glycan
characterization analysis, purification, fragmentation of antibody
pharmacokinetics, and quality assurance for many applications
in basic research (Roth et al., 2012). The scFv antibody is
usually characterized by affinity, isotype, cross binding, phage-
ELISA, and immunoblot. Biochemical characterization includes
the expression of scFv antibody in a soluble form in infected
E. coli cells. In addition, further characterization is carried out by
immunofluorescence antibody test (IFAT), mass spectrometry,
sequencing and indirect immunofluorescence (IFI) assays,
cytotoxicity analysis, surface plasmon resonance, and NMR
spectroscopy (Wang et al., 2012; Yuasa et al., 2014; Levenhagen
et al., 2015).
Other Antibody Fragments
Antibody engineering has become an aesthete discipline covering
a wide range of production technologies. Moreover, techniques
include the modification of clinically significant therapeutic
drugs by antibody fragments especially for clinical imaging and
to target multiple disease associated antigens (Spiess et al., 2015).
Fab antibody fragments are smaller with better tissue and tumor
penetration than intact mAbs. Fab lack a constant region and
therefore, antibody effector functions (Nelson, 2010). Moreover,
Fab bind to specific antigens and are used in non-clinical studies
(e.g., staining experiments) and clinical therapeutics such as anti-
TNFαPEGylated fab fragment. This fragment has a 14 day serum
half-life and is used to improve anti-tumor activity and to reduce
immunogenicity (Chames et al., 2009).
Bispecific Antibody
The tumor necrosis factor (TNF) and interleukins 1 and 6
(IL-1 and IL-6) proinflammatory cytokines cause multifactorial
disease such as cancer and systemic inflammations. Moreover,
these factors are involved in redundancy of disease-mediation
and crosstalk between signal cascades (Arango Duque and
Descoteaux, 2014). Similarly, upregulation of alternative
receptors and pathway switching is often related to resistance to
therapy (Dong et al., 2011). The obstruction of several targets
or multiple sites on one target is associated with improved
therapeutic efficacy. Over the past decade, dual targeting with
bispecific antibodies has gradually switched to combinatorial
or cocktail therapy. This targeting technique is based on the
targeting of multiple disease-modifying reagents with one
drug. Several bispecific molecules such as diabodies, IgG-like
tetravalent Di-diabodies, IgG-scFv fusion proteins, and bispecific
AdnectinsTM have been developed to target tumor mediated
receptors such as members of the epidermal growth factor (EGF)
receptor family, i.e., EGFR, HER2, HER3, and HER4,45 and the
insulin-like growth factor 1 receptor (IGF-1R; Tao et al., 2007;
Kontermann and Brinkmann, 2015). The application of a single,
bispecific molecule is advantageous because it is less complicated
to administer to patients, requires reduced preclinical and clinical
testing, and has cost effective manufacturing (Kontermann and
Brinkmann, 2015).
Other Gene Engineered Antibody
Smaller antibody fragments permit in depth tissue penetration
associated with the affinity of the antibody fragments. Moreover,
a high concentration of complete antibody restricts its ability
to infiltrate tumors (Weiner and Adams, 2000). Additional
engineered antibody fragments include CDRs, Fab, F(ab’)2,
monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent
IgG, bispecific diabody, trispecific triabody, scFv-Fc, minibody,
new antigen receptor (IgNAR), variable new antigen receptor (V-
NAR) domains in sharks, camelid heavy chain IgG (hcIgG), and
VhH (Nelson, 2010; Rodrigo et al., 2015).
Phage display is a selection technique for fusion proteins and
phage coat proteins that are expressed on the phage surface. The
library is developed by careful genetic manipulation (Figure 4;
Chan et al., 2014). Peptide or protein coding genes are inserted
into a vector fused to the 5-terminal of pIII or pVIII that
are phage coat proteins. The bacteria are transformed with
phagemid libraries, and then infected with a helper phage to
assemble phage particles that express fusion proteins on their
surface. Subsequently, the displayed proteins/antibody fragments
are rooted to the surface of the coat protein, and permit affinity
purification with its analogous genes (Barbas et al., 1991; Chan
et al., 2014).
Principles of Phage Display
Filamentous bacteriophages used in phage display techniques are
viruses that belong to the Inoviridae family. There are fewer
of these filamentous phages in this genus compared with tailed
phages. Inovirus virions are 7 mm in diameter, contain circular
DNA enclosed in a protein capsid, and infect both Gram negative
and positive bacteria. They do not lyse host cells, instead, they are
packed and extrude at the surface (Marvin et al., 2014).
The genomes of these viruses consist of double strand DNA
(dsDNA), single stranded DNA (ssDNA), double strand RNA
(dsRNA), and single strand RNA (ssRNA). The viruses enter
host cells via pilli and are involved in genome replication,
and virion structure, assembly and regulation (Stassen et al.,
1994). These viruses undergo extensive recombination, act as
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FIGURE 4 | Various phage display systems. Gene pIII is represented as an
orange box, the foreign DNA insert as a blue box, and the fusion products as a
green circle.
vectors (phagemid) for gene transfer and are closely related
phenotypically and genotypically. They can integrate into the
host genome by phage-encoded transposases and host-encoded
XerC/D (Hassan et al., 2010). Filamentous phages have three
distinct classes, including M13, f1, fd, and M13 that infects E. coli.
Most proteins are displayed at phage proteins pIII (fusion) and
pVIII (preserving functional coupling with 6–7 residues; Hess
et al., 2012).
This phage can be easily manipulated due to its small genome.
Therefore, these are the best-studied viruses and are extensively
used in phage display technology. Large phage particles can be
produced by the insertion of DNA into non-essential regions,
are stable in extreme conditions, and can be produced in high
amounts (Jo´
nczyk et al., 2011).
Phage Display Technology (PDT)
A number of in vitro techniques have been established for the
development of antibodies in comparison with in vivo methods
that involve animal immunization. PDT is a powerful tool for
screening of specific recombinant protein binders against a large
number of target antigens, including peptides, glycoproteins,
glycolipids, saccharides, nucleic acids, and haptens. PDT is the
most commonly used in vitro technology (Bradbury et al., 2003a).
A feature of this technology is a direct physical link between
genotype and phenotype of the displayed protein/variable
antibody fragments. Screening of the displayed protein by
antigen in vitro is analogous to the selection of protein fragments
in natural immunity (Petrenko and Vodyanoy, 2003).
Production Method
Phage display technology has facilitated the production of protein
libraries, which are formed with large numbers of phage particles
displaying different molecules (106–1011 different ligands in a
population of >1012 phage molecules). Specific binder screening
with biopanning allows the enrichment of the desired molecule
(Bazan et al., 2012). The first step is the incubation of the
display library with an immobilized surface (for example,
microplate, magnetic beads, column matrix, PVDF membrane,
or immunotubes) of the entire cell. The non-binding phages are
then removed by extensive washing and the binders are eluted
by acid or salt buffer. Then, binders are amplified using an
appropriate bacterial host cell such as E. coli. To obtain high-
affinity targets, up to five rounds of biopanning are performed
(Figure 5). Finally, DNA sequencing of the primary structure is
carried out to produce individual clones of the target protein
(Bazan et al., 2012; Wu et al., 2016).
Many factors including proper biopanning design, type
of immobilized surface, binding time, washing, and antigen
concentration affect the level of selection and the screening
of antibodies to unique epitopes (Bazan et al., 2012).
Numerous screening cycles are essential in biopanning to
attain the preferred binding activity of the acquired monoclonal
phage antibodies. Several tests including ELISA, fluorometric
microvolume assay technology (FMAT), and chromophore-
assisted laser inactivation (CALI) are used to analyze this activity
(Tonelli et al., 2012).
Various types of phage libraries are used for the screening of
specific recombinant proteins/antibodies in relation to antigen
specificity: (1) construction of specific high-affinity antibody
library for specific antigens by the use of immunized animals
with a dissociation constant in the nanomolar range, (2) a single-
pot (general) non-specific library produced against antigens, and
(3) construction of secondary mutant antibody phage libraries
for the screening of antibodies with high specificity (Hust
and Dubel, 2004). The development of an antibody library
from immunized animals has become obsolete because of the
construction of diverse universal single-pot libraries for the
isolation of numerous antigens. PDT has many applications
in biotechnology, production of recombinant multifunctional
antibodies, cancer, immunotherapeutics, and the enhanced
validity of protein fragments (Yau et al., 2003).
Applications of PDT
The study of epitopes and mimotopes in the interactions of
antigen-antibody binding was the earliest application of PDT
(Wu et al., 2016). Mimotopes are miniscule peptides that mimic
linear, intermittent, or non-peptide epitopes. It was noted that
scFv, Fab fragment, and VHH domains could be displayed on the
phage successfully (Tonelli et al., 2012). This laid the foundation
of new molecular recognition techniques to determine protein
folding, stability, structure-to-function relationships, and other
related protein-protein interactions. The fusion of many ligands
with phage particles has enrich phage displayed cDNA libraries
significantly (Vithayathil et al., 2011).
Several novel molecular techniques have been established
for screening functional molecules. These techniques include
the identification of peptide agonists, receptor antagonists, the
determination of binding specificity of domains, mapping of
simple carbohydrates and functional epitopes, the identification
of tumor inhibitor targets, and molecular imaging by
fluorescently labeled phages (Fukuda, 2012). Recently, PDT
has been widely used in medical sciences for the production of
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FIGURE 5 | Schematic illustration of the biopanning technique. The target is attached to a phage library that is immobilized on a solid surface. Unbound phages
are washed out, and specific phages are eluted and amplified. After several rounds of biopanning, the phages are analyzed to obtain diagnostic and therapeutic
a large number of humanized antibodies and the production of
new therapeutics. These antibodies have preclinical and clinical
applications (Rothe et al., 2006).
Transfusion Medicine
A large number of antibody reagents are being developed
for hematological applications such as cell subpopulation
identification, directed therapeutics, and in vivo imaging. Anti-
ABO, anti-Rh, and anti-Kell hemagglutination antibodies have
been developed against red blood antigens (Marks et al., 1993).
Anti-Rh (D) and anti-HPA-Ia bispecific diabodies developed by
PDT that are useful for hemagglutination assays. These diabodies
are being used for the treatment of neonatal alloimmune
thrombocytopenia (Watkins et al., 1999).
Moreover, various antibody reagents have been raised against
fetal red blood cells (Huie et al., 2001). Additionally, this
technique has helped the production of antibodies against
dendritic cells, white blood cells (WBC) (Fitting et al., 2011),
hairy cell leukemia (Kreitman et al., 2012), myeloma protein
(paraproteins) (O’Nuallain et al., 2007), B and T cells (Maeda
et al., 2009), clotting factors, AITP, GPIa, and GPIIIa antigens,
CD antigens (Chu et al., 2006), and 11-dehydro-thromboxane B2
(11D-TX) antigens (Siegel et al., 2003).
Autoimmune Diseases and Neurological Therapeutics
Human immune libraries developed by PDT facilitate the
study of autoimmune and neurological disorder physiology,
clinical diagnostics, and the treatment of AITP (platelet
disorder caused by anti-platelet autoantibodies), MS, myasthenia
gravis (MG) [antibodies against nicotinic acetylcholine receptor
(AcChoR)], thrombotic thrombocytopenic purpura (TTP),
Cogan’s syndrome (CS) caused by systemic vasculitis, acute
anterior uveitis (AAU), ocular inflammation, insulin dependent
diabetes mellitus (IDDM) caused by the destruction of pancreatic
beta cells, Wegener’s granulomatosis (Finnern et al., 1997),
autoimmune thyroid disease (Latrofa et al., 2003), primary biliary
cirrhosis (PBC), and Sjögren’s syndrome (SS). Additionally,
the technique has therapeutic uses in blistering skin diseases,
pemphigus vulgaris (PV) (Payne et al., 2005), pemphigus
foliaceus (PF) (Ishii et al., 2008), autoimmune hepatitis
(AIH), primary biliary cirrhosis (PBC), mixed cryoglobulinemia
(CryoII), systemic sclerosis (SSc), autoimmune cholangitis
(AIC), antiphospholipid syndrome (APS), vitiligo rheumatoid
arthritis, Crohn’s disease, Graves’ disease (GD), and celiac disease
(genetic inflammatory disorder) (Zohreh and Hossein, 2008).
Neurological disorders are treated by intracellular antibody
fragments (intrabodies), which are potentially therapeutic.
Intrabodies select abnormal intracellular proteins. However,
there are several limitations in the extracellular binding and
internalization of DNA transfected by viral based vectors,
lipofection or electroporation (Jazi et al., 2012). These are
not efficient in vivo techniques and can alter cell viability.
This problem can be overcome by fusing protein transduction
domains (PTD) to antibodies (Langedijk et al., 2004). Phage
display libraries have been utilized for novel immunotherapeutic
strategies for the treatment of neurotoxins, Creutzfeldt–Jakob
disease (CJD), and Gerstmann-Sträussler-Scheinker syndrome
(GSS). They are also used for kuru disease, familial fatal
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insomnia by the accumulation of abnormal prion protein (PrPSc)
(Thanongsaksrikul and Chaicumpa, 2011), Huntington’s disease,
and Parkinson’s disease. Moreover, they have been employed in
inhibitory studies of β-amyloid formation, and enzyme therapy
of the brain vasculature and brain parenchyma (Chen et al.,
Peptide Homing in Organs and Molecular Imaging
Biopanning in vivo with phage display libraries has facilitated
the isolation of peptides homing to all types of organs in the
human body. Phage display is applied to stem cells for cell
based regenerative medicine (Gothard et al., 2011). Moreover,
this technique has assisted in the guided delivery of various
peptides/drugs such as proapoptotic peptides, cytotoxic drugs,
metalloprotease inhibitors, and cytokines to specific targets
(Nixon et al., 2014). The binding of peptides with the extracellular
domain of the LOX-1 receptor is associated with hypertension
and atherogenesis (Nixon et al., 2014). Other studies have
reported that the homing of an RGD-motif-containing peptide
to angiogenic vasculature was linked to a proapoptotic peptide
and was successfully used for the treatment of collagen-induced
arthritis in mice. Phage libraries have also been used for anti-
obesity, microparticle (MP), avb3 integrin angiogenesis therapy,
and in targeting vascular endothelial growth factor (VEGF)
(Cooke et al., 2001).
Similarly, phage displays are used for tumor targeting agents
e.g., the scFv (MFE-23) molecule is specific for carcino embryonic
antigen (CEA) (Edwards et al., 2008). This technique has
replaced radiolabeled antibodies that have multiple disadvantages
including reduced natural immunity (Adachi et al., 2011).
Furthermore, PDT has been used to isolate a number of peptides
for molecular imaging. Its advantages are small size, rapid
blood clearance, lack of immunogenicity, tissue penetration, and
increased diffusion. Numerous peptides for tumor targeting were
isolated using human B cell lymphoma (McGuire et al., 2006),
cervical (Robinson et al., 2005), colon (Rasmussen et al., 2002),
gastric (Liang et al., 2006), breast (Askoxylakis et al., 2005), lung
(Chang et al., 2009), glioblastoma (Wu et al., 2008), hepatic (Du
et al., 2006), prostate (Zitzmann et al., 2005), neuroblastoma
(Askoxylakis et al., 2006), and thyroid (Zitzmann et al., 2007)
carcinoma cell cultures. However, about 80% of these peptides
have not been reported to function in vivo. This inactivity was
observed in peptides that recognized mouse double minute 2
homolog-p53 protein (MDM2/p53) (Pazgier et al., 2009), IL-
11 receptor (Zurita et al., 2004), prostate specific antigen (PSA)
(Pakkala et al., 2004), heat shock protein 90 (Kim et al., 2006),
and growth factors (Hetian et al., 2002).
Hybridoma technology is a well-established method for the
generation of murine mAb cell lines by the fusion of splenocytes
(harvested from immunized mice) with myeloma cells. The
technology remains a feasible method for laboratories that
implement basic cell biological research. Hybridoma technology
is a comparatively simple procedure with minimal cost for
the steady production of native whole immunoglobulins
(Tomita and Tsumoto, 2011). Nevertheless, this technology
has various limitations such as antibodies produced by the
hybridoma technique are strictly murine proteins that limits their
therapeutic use in humans. In addition, they also trigger human
anti-mouse antibody (HAMA) responses (Tjandra et al., 1990).
Moreover, indefinite production costs, low fusion efficiency,
limited number of mAbs, difficulty in developing mAbs against
strictly conserved and toxin antigens and time consumption are
other disadvantages (Hnasko and Stanker, 2015).
The production and amplification of antibodies in vitro using
bacteria by PDT has a low turnaround time compared with
other methods. Additionally, the library comprises of diverse
variants up to 1013, which can be selected against a varied
range of biological and inorganic targets (Sblattero and Bradbury,
2000). The experimental conditions can be controlled, and the
required equipment and libraries are available commercially.
Disadvantages include a difficult procedure and lack of antibodies
displayed on the surface of each bacteriophage yielding a small
number of mAbs (Willats, 2002).
Antibody engineering is a remarkable modification technique
for production of highly specific and efficient antibody products.
However, antibodies are bulky macromolecules that encompass
challenges in construction, optimal pharmacokinetics,
manufacturing, stability, and process development. Nonetheless,
progress in antibody engineering technologies such as phage
display, yeast display, bacterial display, and ribosomal or cell-free
display continue to advance our capacity to rapidly screen
and refine stable binding immunoglobins. These engineering
techniques further improve biological properties significantly in
the effector domains of the mAbs (Filpula, 2007).
Engineered Immunomodulatory Antibodies
Immunomodulatory techniques are persistently progressing to
expand the clinical efficacy of therapeutic antibodies. Cell surface
antigens exhibit a wide array of targets that are overexpressed,
mutated or selectively expressed, and selected for modulated
antibody-based therapeutics. The technology functions through
engineering alterations in antigen or receptor function, the
immune system i.e., altering Fc function and T cell activation
and antibody conjugated drug delivery system (DDS) targeting
a specific antigen. Immunomodulatory antibodies have gained
significant clinical success (Scott et al., 2012).
The Fc region is modulated by engineering the effector
function, for example to increase or lessen binding to Fc gamma
receptors (FcγRs) or complement factors and the half-life of IgG.
The half-life can be extended by improving affinity of Fc for
Fc neonatal receptor (FcRn). Moreover, it can be prolonged by
engineering pH-dependent antigen binding to enhance recycling
of IgG via FcRn, and effective binding to the target molecule.
Engineering the Fc region permits the development of molecules
that are better suited to the pharmacology activity required of
them (Vincent and Zurini, 2012; Rath et al., 2015). Recently,
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a study investigates engineering the pH-dependent interaction
between IgG and FcRn. It involves modulation of constant Fc
part of monoclonal human IgG1 (hIgG1) antibodies to improve
effector functions and clinical efficacy of next-generation IgG1-
based therapeutics (Grevys et al., 2015).
Similarly, new opportunities have been created by the
development of antibody-drug conjugates (ADCs) to treat the
infectious diseases or target cancer cells. ADCs are being
developed by progressing in antibody generation, selection of
exceedingly cytotoxic molecules, and construction of stable
linkers that can be investigated in clinical trials (Vincent
and Zurini, 2012). Cytotoxic therapeutic mAbs often help
target cell-killing by eliciting immune effector functions.
These include antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP)
mediated by innate immune effector cells, and complement-
dependent cytotoxicity (CDC) mediated by humoral components
(Figure 6). in vitro studies, Fc engineering methods have been
specifically designed to modulate ADCC, ADCP, and CDC
envisioned for therapeutic mediation (Kinder et al., 2015).
Natural killer (NK) cells exhibit essential role in immunity
in the context of mAb treatment by exerting direct cytotoxicity
toward infected or tumor cells and contributing in modeling the
adaptive response (Cheng et al., 2013). Several T- or NK-cell
modulators such as ipilimumab and nivolumab were approved
for the treatment of metastatic melanoma (Berman et al., 2015).
Fc-engineered antibodies improve the ADCC/ADCP
potential and target CD19, CD20, CD40, and Her2.
Consequently, they enhance the therapeutic potential of
mAbs. NK cells are exclusive in exhibiting low-affinity activating
FcγRIIIa (CD16), and no inhibitory antibody receptors, featuring
a substantial role in ADCC. Several studies have established a
link between activating Fc receptors and the efficacy of mAb
therapy using mouse tumor models (Romain et al., 2014).
Recently, glyco-engineering technique has been used to
produce recombinant therapeutic proteins with optimized
efficacy, half-life, specificity, and antigenicity. Glyco-engineering
FIGURE 6 | The Fc region of an antibody mediates effector functions
such as CDC and ADCC.
of expression platforms is progressively documented as an
essential approach to advance biopharmaceuticals (Ferrer-
Miralles et al., 2009; Dicker and Strasser, 2015). The technique
has been applied to in vivo expression systems that include
mammalian cells, insect cells, yeast, and plants for the production
of recombinant proteins. The underlined approaches aim at
developing glycoproteins with homogeneous N- and O-linked
glycans of defined composition (Dicker and Strasser, 2015).
Moreover, multi-level glyco-engineering techniques have
been investigated to generate IgG with defined Fc-glycans in
eukaryotic cells (Dekkers et al., 2016). Additionally, E. coli
expression have been successfully employed to produce
recombinant human interleukin-2 (IL-2) (Kamionka, 2011).
Yeast, Bacterial, and Ribosomal Display
Antibodies are engineered with superior properties such as
binding affinity, stability, and catalytic activity by several other
display tools (for example, yeast and bacterial display) for
broad spectrum of biotechnology, medicine, and biomedical
applications. Yeast surface display exhibit development of
recombinant antibodies by displaying on the surface of
Saccharomyces cerevisiae via genetic fusion to an abundant cell
wall protein (Cherf and Cochran, 2015).
Yeast display technique has been used for engineering
protein affinity, stability, and enzymatic activity. Moreover, it is
extensively applied in protein epitope mapping, identification
of protein-protein interactions, and uses of displayed proteins
in industry and medicine (Cherf and Cochran, 2015). Several
recombinant antibodies have been generated by yeast display for
lethal infections such as highly pathogenic H5N1 avian influenza
virus (Lei et al., 2016), cell tumor (Li et al., 2017) and human
tumor endothelial marker 1 (TEM1) (Yuan et al., 2017).
Similarly, several bacterial display systems have been
established for Gram-negative bacteria and Gram-positive
bacteria (Lee et al., 2003). The display systems comprised of a
carrier protein as an anchoring motif, a target protein, and a host
strain. Proteins developed for use as anchoring motifs include
outer membrane proteins, lipoproteins, autotransporters,
subunits of surface appendages, and S-layer proteins (Han
and Lee, 2015). Bacterial display has widespread applications
including live vaccine development, screening-displayed peptide
libraries, biosorbents, whole-cell biocatalysts, and biosensor
development. Moreover, the promising technology is helping
in the remediation of pollutants, biofuel production, and
production of enantiomerically pure compounds (Han and Lee,
2015; Ramanan et al., 2016).
Ribosome display is a cell-free display system, and a technique
to perform entirely in vitro selection of proteins or peptides
to bind desired ligand. Ribosome display consists of both
prokaryotic and eukaryotic display systems (Zahnd et al., 2007).
It forms stable protein-ribosome-mRNA (PRM) complexes and
links individual nascent proteins (phenotypes) to their analogous
messenger RNA (mRNA) (genotypes). Ribosome display allows
synchronized isolation of a functional nascent protein, through
affinity for a ligand together with the encoding mRNA. The
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encoding mRNA is then transformed and amplified as DNA
for further manipulation, including repeated cycles or protein
expression. The advantages of ribosome display over other
cell based methods include displaying very large libraries,
generating toxic, proteolytically sensitive and unstable proteins,
and incorporation of modified amino acids or mutations at
distinct positions (He and Taussig, 2002; Zahnd et al., 2007).
Ribosome display systems have been investigated to identify
potential antigens of Clonorchis sinensis (Kasi et al., 2017), and
human tumor necrosis factor α(hTNFα) for diagnosis and
treatment (Zhao et al., 2016).
Advantages and Disadvantages
The large size of mAbs limits tumor penetration, and
their long serum half-life is not suitable for therapy and
imaging applications. Therefore, antibody fragments have been
constructed in various formats as they are small, monovalent,
penetrate tumor tissues efficiently, and are rapidly eliminated by
renal clearance (Chames et al., 2009).
Similarly, recombinant antibodies have several advantages:
(i) bacteria, yeast, plants, or animals can be used to produce
antibodies, (ii) no need for immunization, and (iii) intrinsic
properties (immunogenicity, binding affinity, pharmacokinetics,
specificity, and stability of antibodies) can be modified easily
using mutagenesis techniques. Genetically engineered antibodies
have integral characteristics that suit various downstream
applications or can be converted into functional whole
immunoglobulins (Bradbury et al., 2003b). Antibodies exhibit
strong immunity to defend against foreign antigens and non-self-
agents. However, a variety of recombinant antibodies is needed
to interact these hostile antigens. Over the last decade, the use
of antibody engineering or recombinant antibody technology has
shaped the genetic manipulation of a diverse range of antibody
fragments for research, diagnosis, and therapy (Kontermann and
Muller, 1999). This technology has resulted in better affinity and
specificity of manipulated antibody fragments and has facilitated
the replacement of hybridoma technology with various display
systems for unlimited antibody production against any known
antigen (Gram et al., 1992).
Conversely, engineered antibodies have various disadvantages
such as they exhibit greater expense and complexity in
manufacturing compared to antibodies developed by hybridoma
technology (Spiess et al., 2015). Due to their foreign nature,
engineered therapeutic antibodies lead to allotypic immune
responses that results in rapid clearance from body by kidney,
elicit on T-cell help, and have reduced antibody avidity.
Moreover, engineered antibodies exhibit reduced half-life due to
lack of an Fc domain and prevention of FcRn-mediated recycling.
Likewise, antibody based therapies have more limitations based
on the fact that many targets (sometimes in low level) have not
yet been determined for various diseases (Chames et al., 2009;
Attarwala, 2010).
In the 1890s, von Behring and Kitasato worked on tetanus
antitoxin that lead to the development of a new discipline,
immunology. They described antibodies for the first time and
discovered that inactive toxins can elicit a protective immune
response against active toxins in animals (Kantha, 1991). The
transfusion of serum from these protected animals elicited
an immune response in other animals. Therefore, antibodies
were originally called “antitoxins.” Since then, antibodies have
been shown to have a wider repertoire of antigen recognition.
Antibodies are widely used in diagnostic tests referred as
“immunoassays.” These are used to confirm diagnoses and for
rapidly growing antibody based technologies (Mukherjee et al.,
Similarly, antibodies have remarkable applications in the field
of diagnostics, therapeutics and targeted DDS. They have been
used to study various diseases such as cancer, metabolic and
hormonal disorders, and infections caused by bacteria, viruses,
fungi, algae, protozoa and other agents (Sundar and Prajapati,
2012). Moreover, these biomolecules have numerous application
in the diagnosis of lymphoid and myeloid malignancies.
Likewise, immunoglobins are used in tissue typing, ELISA, radio
immunoassay, serotyping of microorganisms, immunological
intervention with passive antibody, analyzing a patient’s antibody
profile, and antiidiotype inhibition (Siddiqui, 2010).
Detection and Immunodiagnostic Test Kits
The detection of antibodies against infectious agents and
the construction of rapid and sensitive antibody-based
immunodiagnostic test kits is an important part of basic
and clinical research. Assays have been developed against
pathogens, toxins, and infectious proteins/peptides (Ahmad
et al., 2012). The production of antibodies depends on specific
protein titers, extent of immunity, and identification of B
cell responses (Burbelo et al., 2010). Antibodies are routinely
detected by ELISA and other immunoassays. Accessibility of
full length DNA sequences is useful for the rapid and robust
systematic identification of antigens by recombinant proteins
using protein arrays for complete proteome analysis (Kierny
et al., 2012). Furthermore, numerous novel high-throughput
dominant immunoassays are used to detect antibody responses
against antigens (Burbelo et al., 2010).
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is the most common method for the quantitation of
pathological antigens. In some cases, modified ELISA have been
used in combination with various specific proteins/peptides. It
is rapid, consistent, relatively easy to analyze, and adaptable to
high-throughput screening (Alonso et al., 2015).
Principally, specific antibody is immobilized on high binding
ELISA plates by incubation overnight at 4C or for 1–2 h at 37C,
and then followed by 3–5 washes with PBST (137 mM NaCl,
2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, and 0.5%
Tween 20; Murayama et al., 1996). Then, plates are blocked with
irrelevant protein e.g., albumin (for example, PBS containing 4–
5% skimmed milk), and incubate overnight at 4C or for 1–2 h
at 37C. After washes, samples and standard dilutions are added
to the wells to be captured by bound protein, incubate for 1–2 h
at 37C and wash properly. Next, specific peroxidase-conjugated
enzyme labeled detection antibody is added to the wells to
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enable detection of the captured protein and incubate for 1 h
at 37C. After appropriate wash, colorimetric substrate is added
to the wells and incubated for 15–20 min at 37C for the color
development as catalyzed by the enzyme. For instance, addition
of 3,3,5,5-tetramethylbenzidine (TMB) substrate develops blue
color that can be read by microplate plate reader at the
wavelength of 562 nm. Additionally, addition of 0.16–2 M
sulfuric acid (H2SO4) as a stop reaction solution develops yellow
color that can be read at 405–450 nm correspondingly. In
addition, the incubation conditions and reagent formulations
should be preferably optimized based on the type of ELISA
(Grange et al., 2014; Thiha and Ibrahim, 2015).
There are four ELISA methods. Direct ELISA (dELISA) uses
competition between unwanted proteins for plastic binding
spots. Antigen is attached to the solid phase followed by an
enzyme-labeled antibody. This assay is used to detect various
pathogenic antigens (Brasino et al., 2015).
Indirect ELISA (iELISA) uses an antigen attached to a solid
phase followed by the addition of unlabeled primary antibody.
Instead, a peroxidase enzyme-conjugated secondary antibody is
added onto the first antibody. The iELISA is used to detect
specific antibodies in sera (Peng et al., 2016).
Competitive ELISA (cELISA) is used for the detection of
small molecules lacking multiple epitopes. Specific antibodies to
the analyte of interest are immobilized on a micro-titer plate.
Then, enzyme-conjugated antigen is incubated with capture
antibody and the same antigen in its unconjugated form. This
step yields a color following the addition of substrate. The signals
produced are directly proportional to the quantity of conjugated
enzyme bound and inversely proportional to the quantity of
unconjugated antigen present (Dupont-Deshorgue et al., 2015).
Sandwich ELISA (sELISA) is used for larger proteins with
multiple epitopes and two antibodies can be used consecutively.
The capture antibody is immobilized on a microtiter plate.
Then, unknown or known samples are added into the matrix
to minimize attachment to the solid phase. Peroxidase-enzyme
labeled antibody is then added for coloration, which is directly
proportional to the amount of antigen present (Qu et al., 2016).
ELISA has been extensively used in the detection of various
pathological antigens from viral, bacterial, fungal, protozoa, algal,
and numerous other sources (Thavaselvam and Vijayaraghavan,
2010). An improved ELISA has been used for detecting anti-
melanoma differentiation-associated gene 5 (MDA5) antibodies
that are expressed in patients with dermatomyositis. Clinical
study of this newly developed ELISA exhibited efficient detection
of anti-MDA5 antibodies and showed promising potential to
assist the routine clinical check of anti-MDA5 antibodies in
patients who supposed to have DM (Sato et al., 2016).
Western Blot
Western blot assay (WBA) is also called immunoblotting.
This technique is used for the determination of molecular
weight and amount of relevant proteins present in a sample.
Proteins in a sample are first separated by electrophoresis and
then transferred to a nitrocellulose or polyvinylidene difluoride
(PVDF) membrane for the detection of bound primary proteins
with antibodies specific to the protein of interest (Ness et al.,
Nonspecific sites in the membrane are then blocked with
BSA, non-fat milk powder, or casein. Finally, a labeled secondary
antibody is added for detection by chemiluminescence or
fluorescence (Lewis et al., 2016). WBA has been used for
the confirmation of presence of purified proteins produced
against various pathological antigens such as the lethal toxin of
Clostridium sordellii (Arya et al., 2013), shiga toxin Stx2f (Skinner
et al., 2013), Staphylococcus aureus alpha-hemolysin (alpha-
toxin) (Ladhani et al., 2001), Selenocosmia huwena huwentoxin-
IV (HWTX-IV) (Yu et al., 2014), and V. parahaemolyticus
thermolabile hemolysin (TLH) (Wang et al., 2015).
Dot Blot
Dot Blot (DB) assays are used to measure protein concentrations
semi-quantitatively. It is slightly different from the WBA.
Proteins in the sample are not separated by electrophoresis
but are spotted directly on a membrane and hybridized
with an antibody probe (Emmerich and Cohen, 2015). This
technique is cost effective and uses avidin-biotin technology
with diaminobenzidine as a chromogen. It is used for the
analysis and quantitation of 14-3-3 protein in cerebrospinal fluid
(CSF) samples from cases of Creutzfeldt-Jakob disease (CJD),
and for disease control of other neurodegenerative diseases
such as Alzheimer’s disease (AD) and Parkinson’s disease (PD)
(Subramanian et al., 2016).
Immunohistochemistry (IHC) is used for the detection and
identification of proteins and their localization in tissues. It
is essential to retain the morphology of tissues, cells and the
availability of antigen sites. Fresh, rapidly frozen tissue sections
are preferably used for IHC by chemically fixing tissues in
formalin and embedding in wax (Zhu et al., 2015). Additionally,
fixing crosslinks amino acids in the tissue that block access
to the epitope sites and prevent the action of any protein
specific antibodies. The exposure of hidden epitope regions is
performed by digestion with an enzyme or by heat treatment,
which removes endogenous peroxidase activity and non-specific
sites are blocked. A labeled antibody or an unlabeled primary
antibody specific to the protein of interest is used, followed by the
addition of a secondary labeled antibody specific to the primary
antibody (Diorio et al., 2016).
IHC has recently been used for determination and
identification of expressed proteins such as lysosome-associated
protein transmembrane-4 beta (LAPTM4B) associated with the
prognosis of several human malignancies (Xiao et al., 2017),
glucagon-like peptide-1 (GLP-1) against renal tubular injury
(Guo et al., 2017), and CD147 trans-membrane protein that
induces expression and activity of matrix metalloproteinases
(MMP) (Dana et al., 2016).
Immunoprecipitation (IP) is used for the study of protein-
protein interactions, specific enzyme activity, posttranslational
modifications, protein quantification, and determination of
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molecular weight of protein antigens. This technique includes
antibody/antigen purification complexes at conditions that
specifically bind antibodies. Rare proteins can be accumulated
up to 10,000-fold by IP (Dwane and Kiely, 2011). Luciferase
immunoprecipitation systems (LIPS) have been developed for the
rapid detection of antibodies against peste des petits ruminants
virus (PPRV) (Berguido et al., 2016), varicella-zoster virus (VZV)
(Cohen et al., 2014), zinc transporter (ZnT8) autoantibodies
(Ustinova et al., 2014), and pancreatic and duodenal homeobox
1 autoantibodies (PAA) (Donelan et al., 2013).
Flow Cytometry
Flow cytometry (FC) is used to study antibodies on the
cell surface and their related physiochemical properties. This
technique was developed over 40 years ago (Robinson et al.,
2012), and allows the rapid detection of various proteins on cells.
Proteins produce fluorescence or scattered light when passed
through the machine sensing point to generate quantitative data
on a large number of cells. Labeled antibodies are used for the
detection of target protein molecules or antigens on the surface
of cells (Álvarez-Barrientos et al., 2000). FC has been used for
the visualization of pulmonary clearance (Zhou et al., 2015),
fluorescence decay lifetimes, control sorting (Houston et al.,
2010), quantification of DNA-end resection in mammalian cells
(Forment et al., 2012), microsphere-based protease assays, high-
throughput screening (Saunders et al., 2010a), and botulinum
neurotoxin type A light chain protease inhibitors (Saunders et al.,
Fluorescence Activated Cell Sorting
Fluorescence Activated Cell Sorting (FACS) is used for the
detection of specific cells from a mixed population of cells based
on their distinctive fluorescence or light scattering characteristics
(Yilmaz et al., 2010). The technique allows the isolation of cells
by flow cytometry. FACS has been used to isolate high-lipid
strains of Tetraselmis suecica (Montero et al., 2011), high-lipid
mutants of Nannochloropsis (Doan and Obbard, 2011), E. coli
O157, (Ozawa et al., 2016), high-lipid Chlamydomonas mutants
(Terashima et al., 2015), and Chlorella (Manandhar-Shrestha and
Hildebrand, 2013). In addition, the technology has been used for
quantification of the cellular uptake of cell-penetrating peptides
and mRNA (Date et al., 2014).
Enzyme Linked Immunospot
Enzyme linked immunospot (Elispot) assay is used for
monitoring cellular immune responses in humans and other
animals (Whiteside et al., 2003). It involves a polyvinylidene
difluoride (PVDF) assisted microtiter plate pre-coated with
antibodies specific to the antigen of interest. A capture antibody
binds to the analyte of interest under precise conditions. Then, a
biotinylated antibody specific to the analyte of interest is added
to detect the original antibody after a wash to remove cell debris.
Finally, an enzyme labeled conjugate is added after a second wash
to remove unbound antibody and to visualize a colored product.
The product is typically a black spot representing a single cell
that produces the antigen of interest (Janetzki et al., 2005). This
technique was used in the development of a coxsackievirus A16
neutralization test (Hou et al., 2015) and the determination of
rotavirus infectivity (Li et al., 2014). Diagnostic applications
include diagnosing sensitization to house dust mites (Chang
et al., 2016), pulmonary tuberculosis (Pang et al., 2016), pleural
tuberculosis (Adilistya et al., 2016), smear-negative tuberculosis
(Li et al., 2015), spinal tuberculosis (Yuan et al., 2015), and
cytomegalovirus infection (Nesher et al., 2016).
Lateral Flow Test
The lateral flow immunochromatographic test (LFT) is a simple
and cost effective device used to detect the presence or absence
of a target antigen. It is widely used for medical diagnostics at
home (pregnancy test) or in a laboratory testing. The technology
involves the transportation of fluids (e.g., urine) through capillary
beds, fragments of porous paper, microstructured polymers, or
sintered polymers (Hansson et al., 2016).
The technique comprises various components and steps. A
sample pad acts as a sponge and absorbs the fluids. Then,
fluids migrate to a conjugate pad containing protein conjugates
immobilized on the surface of bio-active particles in a salt-sugar
matrix that reacts with the antigen. Next, sample fluids dissolve
the conjugate salt-sugar matrix and antibody-particles. The fluid
mix flows through the porous structure causing the analyte to
bind with particles while migrating further through the third
capillary bed. Finally, there is a third capture molecule in striped
areas that binds to the fluid mix containing analytes and the
particle consequently changes color (Yu et al., 2012). LFTs can
be used as a competitive or sandwich assay. Latex (blue color),
nanometer-sized particles of gold (red color), or fluorescent or
magnetic labeled particles are also used (Seydack, 2005). The
technique is qualitative, nevertheless, the quantity of analyte in
a sample can be measured by the intensity of the test line color.
It is usually carried out by optical and non-optical lateral flow
readers or biosensors (LFBs) such as complementary metal-oxide
semiconductor (CMOS) or a charge-coupled device (CCD) and
magnetic immunoassay (MIA) (Gui et al., 2014).
Diagnosis and Cure
Progress in hybridoma technology and the production of
highly specific mAbs has revolutionized the therapeutic use
of antibodies for the diagnosis and cure of infections, the
development of vaccines, antigenic characterization, and genetic
manipulation. Antibodies have widespread applications in
diagnostics, therapeutics and targeted DDS against potent
pathogens, cancer, and physiological disorders (Tiwari et al.,
They are used for the diagnosis of lymphoid and myeloid
malignancies, tissue characterization, ELISA, radiolabeled
immunoassay, and serotyping of microbes. In addition, they
are used in the diagnosis of immunological interpolation with
passive antibodies, anti-idiotype suppression, or magic bullet
treatments with cytotoxic agents coupled to anti-mouse specific
antibodies (Keller and Stiehm, 2000). Similarly, recombinant
DNA technology (rDNA) has revolutionized the reconstruction
of mAbs by genetic engineering using chimeric antibodies,
humanized antibodies, CDR grafted antibodies for therapeutic
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