Group 7 Project - Monoclonal Antibodies

From CellBiology
Basic structure of a Monoclonal Antibody


Monoclonal antibodies (mAbs) are identical antibodies that bind specifically to a single antigen[1]. The most common method used to generate mAbs is the creation of a hybridoma cell line, where an immune cell (producing a single antibody) is fused with a tumour cell (able to survive and proliferate indefinitely in cell culture) and a clonal “hybridoma” cell line results[2]. Using this and other methods, it is theoretically possible to generate mAbs against any antigen(shape) found in nature in unlimited quantities[3].

  • Overview of methodological theory:
  • They combined the properties of B-lymphocytes (extracted from rodent spleen or lymph node) that is, their ability to produce a single type of antibody that binds to a corresponding antigen, and the immortal property of myeloma cells. [4][5] [6]
  • They fused the two types of cells to produce hybridomas using Sendai Virus.
  • The hybridomas were left to grow and proliferate in the aim of generating substantially large numbers of single monoclonal antibodies against a desired antigen. [4][5].
  • Hybridoma cells go through careful selection procedures in order to eliminate hybrids producing no antibody or producing antibodies of wrong specificity or low affinity.[4].

The specificity of mAbs and the ability to combine them with other substances such as fluorescent dyes, enzymes and drugs makes it possible to use them for many applications outside the scope of conventional polyclonal antisera. [7] To date they have proved to be powerful research tools in many fields (including cell biology, microbiology, molecular biology and pathology) and a new source of drugs that can be designed from the ground up to be delivered to targets with high selectivity[7][8][9][10].

Interesting Facts

  • In 1982, Philip Karr, a victim of lymphoma, was successfully cured after treatment with a 'tailor-made' mouse anti-idiotype. This was the first indication that monoclonal antibodies had significant therapeutic potential.[11]
  • In early 1997, The American Anti-Vivisection Society (AAVS) petitioned, to the National Institutes of Health (NIH), for the prohibition of the use of an animal in the production of mAb. NIH asserted that the mouse method for producing mAb was scientifically required. The National Research Council formed the Committee on Methods of Producing Monoclonal Antibodies. It was concluded that the chosen method of producing mAbs must adhere to recommendations in the Guide for the Care and Use of Laboratory Animals. [12]
  • mAbs to CD3 and CD25 (IL-2 receptor) are the only licensed mAbs for organ transplantation in humans [13].
  • The first fully humanised mAb, Adalimumab, was made available for market in 2003 in the US.[14]
  • mAbs trialled for therapeutic use have an approval rate of approximately 20%, compared with 5% for new chemical entities (NCE) [15].
  • 30% of total biopharmaceutical production is made up of Monoclonal Antibodies, which are also the largest type of proteins currently undergoing clinical trials. [16].
  • Bacteriophage technology for the production of monoclonal antibodies resulted in the first fully human antibody subsequently approved as a treatment for rheumatoid arthritis.[5]
  • Sendai Virus was the original fusogen used to fuse the B-lymphocytes and myeloma cells, but the technology was subsequently simplified, replacing the virus with polyethylene glycol. [4].


In the late 19th century, the presence of effector molecules in the serum of animals in response to exposure to foreign pathogens (immunized animals) was discovered and termed Antibodies.[17] It was not until 1938 that significant advancements were made in the characteristics of these antibodies including the ability to classify them into different categories e.g. Immunoglobulin (Ig)-G, and, most importantly, the fact that they could bind to antigens with high affinity and specificity.[17][18] This advancement provided for the subsequent proposition by Paul Ehrlich in the 20th century that antibodies would be the ‘magic bullets’ that could selectively target an antigen and instigate a fatally toxic payload, and therefore be used as therapeutic options. [17]

Evidently, the late 1800s and early 1900s was a period where antibody therapy involved using sera from humans and animals for prophylaxis and therapy of viral and bacterial diseases, however, most of this serum therapy for bacterial infections was abandoned in the 1940s due to the advent of antibiotics becoming widely available.[17] Following this polyclonal antibody preparations proved to be clinically effective in many cases such as toxin-mediated infectious diseases. However, problems related to toxicity meant that a better solution was required (further explored in History.), this solution came from the work of Cesar Milstein and Georges Kohler, known as monoclonal antibodies from hybridoma cell line technology. [17]

The era of using antibodies as therapeutic drugs stemmed from their initial use as natural therapeutics extracted from the serum of immunized animals used to treat infectious diseases. [17] Although it took more than 100 years from this initial success for serum therapy to be approved by the US Food and Drug Administration (FDA) and become widely used for the treatment of human disease, the success of antibodies in this century will stem from the technological advancement that Milstein and Kohler made which would later lead to a Nobel Prize. [17][7]

Georges J.F.Kohler.Winner of the Noble Prize in Medicine in 1984 for his work in Hybridoma Cell Line Technology
Cesar Milstein. Winner of the Noble Prize in Medicine in 1984 for his work in Hybridoma Cell Line Technology

1975 signified a year of discovery when the journal, Nature, published a paper entitled ‘Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,’ by Milstein and Kohler.[17][2] [19].

  • This paper described the series of experiments the scientists conducted which led to the development of technology capable of producing cell lines that not only provided an unlimited source of identical cells but also secreted a single type of monoclonal antibody specific to a predetermined antigen; this technology was termed hybridoma cell line technology. [17][4]
  • Reason for development: Prior to the development of monoclonal antibodies, Polyclonal antibodies proved to be effective in therapeutics and treating infections.[17] Usually derived from immunized rabbit, sheep or goat, polyclonal antiserum was the conventional heterogeneous serum product of these animals [4] that is, this antiserum contained antibodies to different types of antigens, and therefore it was multi-specific with antibody composition varying between animals and batches[4]. These characteristics proved to be a downfall for polyclonal antibodies, due to its inability to provide the degree of specificity required to distinguish molecules a the individual epitope level in terms of fine structural differences and antigenic differences[4]. Hence, while polyclonal antibodies bound to a range of different epitopes, its disadvantages called for monoclonal antibodies from a single clone which would bind to a single type of epitope on an antigen[4].
  • Problems encountered with mAb production [20]: Hybridoma technology, like any other past and present innovations, required years of vigorous testing, and analysis in order to refine procedures and prevent errors. Some of the difficulties encountered in this decade included:
  • Errors resulting in poor growth parameters of hybridomas and antibodies of low-specificity and low-affinity.
  • Required considerable time/money/expertise/facilities
  • Relies on the use of animals.
  • Monospecificity signifies inexistent/poor antigen-precipitation properties when used as single reagents[4].
  • Yielding of rodent antibodies rejected by the human immune system
  • Application in Industry:
  • The first decade in the era of monoclonal antibodies saw a focus in using them for the treatment of cancer, with clinical investigations utilising monoclonal antibodies to target tumour cells with radiation or toxin. [11]
NOTE: The original plan was to use monoclonal antibodies to identify tumour-specific antigens and then destroy neoplastic cells without involving normal cells.[7].

In 1984 Cesar Milstein and Georges Kohler won the Nobel Prize in Medicine based on their aforementioned earlier work in pioneering the production of mAbs.[7].

1986- The first mouse monoclonal antibody, named OKT3-Orthoclone was approved for therapy such as to prevent cardiac and hepatic transplant rejection.[19].

However, during the 1980's attention focused on producing effective antibodies that were less immunogenic (as the murine mAb's would cause the development of systemic reactions or rapid clearance of the mAb from the bloodstream.)[17][21]

Timeline from 1897 to 2009 demonstrating the succession of murine monoclonal antibodies, to chimerized and humanized antibodies to fully human monoclonal antibodies.[22] Reprinted by permission from Macmillan Publishers Ltd:Nature(Weiner,L.M., Surana,R., Wang,S.(2010)Monoclonal Antibodies:versatile platforms for cancer immunotherapy.NatureReviewsImmunology.10:317-327.)2010 [1].
  • Various approaches were attempted at producing human-friendly antibodies [20] :
  • Chimeric Antibodies: Known as the 1st generation of humanized antibodies, recombinant DNA techniques were used to combine the DNA portion that encoded the binding site of mouse mAb’s with human antibody encoding DNA, resulting in hybrid immunoglobulin molecules. [20]
NOTE: This process could also result in humanized antibodies when only the complementary-determining regions from the murine mAb are retained [20] [23]
  • Human sourced hybridomas. Sourcing B-lymphocytes from humans would in theory overcome the thereapeutic hurdles encountered with murine (mouse) monoclonal antibodies. However this proved unsuccessful for reasons listed below. [24]
  • Genetically-engineered Mice: capable of producing antibodies with human antibody sequences. [20]

  • Unfortunately, despite the success in development that mouse monoclonal antibodies had, it proved difficult to develop technology for humanized antibodies as quickly or effectively.
  • Reasons for setbacks included: [21]
  • Suitable human myeloma fusion partners were in insufficient numbers.
  • Difficulty in establishing human myelomas as continuous cell lines.
NOTE: Two myeloma cell lines were widely available, with lymphoblastoid lines being utilized despite reported disadvantages.
  • Low numbers of antigen-specific human B-lymphocytes.
  • Only peripheral blood lymphocytes can be obtained from immunized individuals.[25]
  • Incapable of producing large-scale quantities.

  • Fortunately, there have been successes including:
  • The second generation of humanized antibodies- antigen-binding loops of the murine mAbs were incorporated into a human immunoglobulin, with molecular modelling techniques resolving the issue of specificity and subsequently resulting in successful reshaped antibodies capable of use for clinical purposes.[7].
  • A solution to the lack of a suitable human myeloma cell line as a possible fusion partner by fusing human B cells with mouse myelomas to generate hybrid cell lines.[25]
  • Technology allowing for murine proteins to be readily transformed into humanized forms, considerably less recognizable by the human immune system [26] was developed.
NOTE: During this decade a range of murine mAbs were produced against blood cell antigens, some of which were used in blood typing[25].

These successes are demonstrated in the Timeline of 100 years since the discovery of the magic bullet, with the years 2007 and 2009 producing fully human monoclonal antibodies, including Ofatumumab and Panitumumab.[22].

Mode of action of therapeutic monclonal antibodies. Reprinted by permission from Macmillan Publishers Ltd:Nature(Chatenoud,L.(2003).CD-3-Specific Antibody-Induced Active Tolerance:From Bench to Bedside.3:123-132.2003.[2]

Despite the fact that by 1994 only one monoclonal antibody had been licensed for clinical use, by the turn of the century numerous monoclonal antibodies demonstrated promising clinical results, and 8 more has been approved by the FDA,[11]. including:

  • - Anti-cancer antibodies- used to harness cancer patients immune response more effecitvely or deliver toxic agents. [19]
  • - Anti-immune system antibodies- antibodies directed against the immune cells e.g. t-cells, that make up the immune response. [19] A summary of the way that therapeutic t-cell monoclonal antibodies function is given in the following picture.[27]
  • - Anti-viral antiboides- used to kill viruses or block their ability to infect normal host cells.[19]
  • - Anti-idiotype antibodies- used in tumor suppresion by activating specific t-helper cells and to stimulate persons immune system against their own antibodies as well as possible tumour cells. [19]

Two innovative methods that proved central to the modernisation and success of monoclonal antibody production techniques were:

  • messenger RNA display technologies- a selection technique that utilizes the translation-terminating antibiotic puromycin to allow for the preparation of polypeptide libraries with greater complexity than what has been possible with phage display. The puromycin is covalently attached to the 3' end of a messenger RNA (mRNA), which results in a link between a polypeptide and its encoding mesage, and therefore the ability to subject these mRNA-peptide fusions to purification and in vitro. [28]
  • phage display technology- a technique which involves a protein being displayed on the outer surface of a bacteriophage in order to enable selection of the phage containing DNA coding for that protein; widely used and well-established for the production of human antibodies as it allowed for the selection of ligands with high affinity.[28][19][7][29]

Furthermore, a number of methods have been recently developed in order to decrease the immunogenicity of mAbs, including repertoire cloning (utilizing human cDNA libraries as a source of human complementary-determining regions for antigen targeting) and using Epstein-Barr virus-tranformed human B cells. [21][23]

However, the process of generating mouse mAbs practically has not changed since the early 1980’s, and Kohler and Milsteins myeloma fusion partner is still the gold standard in hybridoma development. [20]

Methodology: Hybridoma Cell Lines


Summary of Hybridoma Method of mAb production

Hybridoma cell lines make it possible to mass-produce a pure monoclonal antibody. With the necessary resources, hybridoma cell lines can be set up to manufacture a mAb indefinitely- there is no theoretical limit to the quantity of antibody that can be made [2].

The procedure involves fusing two cell types in the laboratory to create an artificial ‘hybrid cell’. Hybrid cells allow you to combine the desired traits of two cell types- meaning it is possible to generate lines of cells unlike anything found in nature [3].

The mammalian immune system generates antibodies to bind to foreign antigens as part of the immune response. The cell type responsible is the B-lymphocyte—which upon exposure to the antigen matching its B-cell receptors—differentiates into a plasma cell [30]. Plasma cells devote their resources to generating and secreting large quantities of their one specific antibody. This system is an ideal starting point as it already has most of the characteristics that are needed to produce mAbs in the laboratory- however it is specifically designed to work in the body as a temporary response to infections. It is not ideal for laboratory production of mAbs because:

  • Mature plasma cells do not divide- making it difficult to raise a whole line of cells to produce a significant amount of antibody [3]
  • B-lymphocytes & plasma cells have a short lifespan in cell culture [31].

These hurdles can be overcome by making hybrid cells. The second type of cell used is actually that found in cases of myeloma- malignant neoplasia (cancer) of plasma cells [31]. Myeloma cells are capable of surviving and multiplying in cell culture indefinitely, a quality they can pass on to B-lymphocytes in hybrids. In a sense, the very traits that make myeloma a serious disease are what make a powerful cell biology technique- the large scale production of monoclonal antibodies- possible. Hybrids involving myelomas are termed “hybridomas".

Creating Hybridoma Cell Lines

(adapted from [31] [2] [32] & [33])

Immunisation and Harvesting of B Cells

  • A mouse is immunised with the antigen against which mAbs are to be raised.
  • B-lymphocytes in the mouse with B-cell receptors matching the antigen will be activated. The highest level of antibody and highest number of B-cells are found after mouse is immunised with 2-3 separate small doses of the antigen. The second and third exposure produce a greater response because memory B-cells will be present, however with exposures further to this immune tolerance begins to develop [34].
  • The spleen- an organ with a very high concentration of lymphocytes, is removed from the immunised mouse, and cells (splenocytes) taken from it. Many B-lymphocytes will be extracted, only some of which will produce antibodies to the antigen of interest—and of these, different cells will produce antibodies directed at different epitopes on that antigen.
Selection of Hybridomas using HAT Medium

Hybridoma Generation

  • The B cells are suspended, along with myeloma cells, in liquid with a fusing agent (such as inactivated viruses or polyethylene glycol). The fusing agent will cause changes to the plasma membrane necessary for the next steps.
  • The suspension is mixed, some cells fuse to form heterocaryons- that is, cells with two nuclei inside.
  • During mitosis, nuclear envelopes are dissassembled. This is also the case in heterocaryons, which is significant because in the daughter cells, all of the chromosomes from both nuclei will be packaged together in a large single nucleus.
  • In numerous wells, cells are grown on a medium specially designed to be selective for heterocaryons. The medium used is HAT medium- standing for Hypoxanthine Aminopterin Thymidine. Aminopterin blocks normal DNA synthesis- preventing cell division, unless certain enzymes are present which allow the normal pathway to be bypassed-with a so called “salvage pathway”. B-lymphocytes have the enzymes for the salvage pathway, but do not survive and divide in culture, so B-lymphocytes will not grow in HAT medium. The Myeloma cells used do not have the enzymes of the salvage pathway, so they will not grow in HAT medium due to the action of aminopterin. Only hybrids formed from both cell types are able to replicate their DNA and proliferate.

Screening and Isolation of Desired mAb Producing Line

Isolation of Hybridoma Pure Cultures
  • The supernatant of each well with growth is tested for the presence of the desired antibody using ELISA (Enzyme linked immunosorbance assay). Only where the desired hybridoma is present will the antibody be present.
  • If an incubation is positive for the antibody, it is re-cultured with single cells distributed to separate wells.
  • Each separate well is tested for the presence of the desired antibody in the same way.
  • With further rounds if necessary, a pure culture of clones is obtained. This hybridoma cell line is maintained and will continue to produce large quantities of the monoclonal antibody.

Isolation of the monoclonal antibody itself

Even with the hybridoma cell line set up, extraction and purification of the monoclonal antibody may be quite labour intensive, particularly if the antibody is to be used clinically.

  • Supernatant is harvested from the cultured hybridoma cell line. This supernatant will contain the monoclonal antibody.
  • The supernatant is centrifuged and filtered to remove debris and other contaminants larger than antibody size, then the remaining solution concentrated by circulation through a hollow fibre membrane cartridge.
  • The monoclonal antibody must then be purified using chromatography methods in the steps below:
    • Capture step. antibodies are separated from non-antibodies, eg: by protein A chromatography.
    • Intermediate Step. Further purification by separating the mAb from items of similar size and biochemical properties. Hydroxyapatite chromatography is an example of a method.
    • Cleaning step. Trace contaminants are removed- eg: endotoxin removal gel chromatography ensures no bacterial endotoxins remain in the solution.
  • Finally, quality control tests can be done to confirm a pure preparation of the mAb has been produced. Quality control procedures may include:
    • Comparing UV absorbance to the known properties of the mAb.
    • Checking the migration pattern is consistent with the mAb, using isoelectric focusing electrophoresis.
    • Analysis of the purity of the mAb using size exclusion chromatography.
    • Testing binding of the preparation to the target epitope to give information on specificity and potency.

Video of method

Producing Human Monoclonal Antibodies

While the above method has proved robust and is still widely used to generate monoclonal antibodies, it has limited clinical applications [24]. Because the B-lymphocytes and myeloma cells used are sourced from mice, the antibodies generated are from the mouse’s genome (called “murine antibodies”). The mouse genes coding for antibodies are different to the human genes, so the end product murine mAbs will have different amino acid sequences to antibodies produced naturally in a human. For many applications this is not significant and murine mAbs are still the antibody of choice—eg: mAbs used in research to investigate protein localisation within cells, mAbs used in serotyping bacteria [35], [9]. However murine mAbs have very limited clinical uses because they are immunogenic in humans—the human immune system recognises the murine mAbs as foreign antigens and mounts an immune response (Called a HAMA respose- human anti-mouse antibody response [24]). Murine mAbs put into the body are removed from circulation fairly quickly, and in some cases an allergic reaction also occurs [36]. Furthermore, the constant region of murine mAbs- the part immune cells recognise- does not interact normally with the receptors on human cells responsible for the action of the antibody [24]. The bottom line is that murine mAbs do not produce the desired therapeutic effects and are potentially unsafe. This has led to efforts to make mAbs more human so they may be clinically useful. A simple solution would seem to be to use human B-lymphocytes rather than mouse B-lymphocyes, however this does not work because:

  • Hybridomas from human cells are unstable
  • B-cells in the blood are less suitable than those obtained from the spleen
  • Human B cells will not produce antibodies against human tissues
  • There are safety and ethical issues that go with immunising humans with some antigens [24]

A variety of methods have been used to overcome the obstacles above in an effort to produce clinically useful mAbs. Using techniques outlined below, three key classes have been developed- chimeric, humanized, and fully human mAbs.

Types of monoclonal antibodies

Chimeric mAbs

Chimeric mAbs have a mouse variable region (which recognises and binds antigen) and a human constant region. A hybridoma cell line generating murine antibodies against the desired epitope can be used as the starting point [37]. Reverse Transcriptase PCR is then used to generate many copies of DNA complementary to the mRNA coding for the variable region of the murine mAb. Recombinant DNA techniques allow this cDNA for the variable region to be combined with DNA coding for the human constant region. When this recombinant DNA is introduced into a myeloma cell line using expression vectors, the chimeric mAb is expressed [37]. Because these have the original murine variable region, they will bind to the same epitope, and because they have a human constant region, they will be less immunogenic and interact with human receptors to elicit their therapeutic effects. Rituximab is an example of a chimeric mAb that is used clinically to treat non-Hodgkins lymphoma [24]. Unfortunately, even the murine variable region can still be recognised as foreign—leading in many cases to the same limitations as murine mAbs [24].

Humanized mAbs

Humanized mAbs are similar to chimeric mAbs but with further exchange of human sequences for murine sequences within the variable region—to further reduce immunogenicity. The “complementary determining region” or CDR is the part of the variable region that binds antigen. Humanized mAbs have been made with only the CDR remaining from the murine mAb—however unfortunately these usually loose their binding activity. Ideally, a minimum number of murine residues will be left in the variable region in order to produce functional but non-immunogenic humanized mAbs. Transtuzumab is a humanised mAb used in treating breast cancer [24].

Fully Human mAbs

To eliminate the immunogenicity issues of all the above classes of mAbs, mice are genetically engineered to have B-lymphocytes with totally human genes for all parts of antibodies (“XenoMouse” strains.) When these mice are immunised, their immune response will consist of fully human antibodies. Therefore hybridomas produced from B-lymphocytes of these mice, using the standard hybridoma technique, will result in fully human mAbs [24].

Type of mAb Composition Example
Murine Entirely murine amino acid Muromonab
Chimeric Murine variable (V) + human constant (C) regions Rituximab
Humanised Murine complementarity-determining regions (CDRs) + Human Ig scaffold Alemtuzumab
Human Entirely human amino acids Adalimumab

Table adapted from [15].

Variations to Traditional Hybridoma Method

The hybridoma method of generating monoclonal antibodies is a time consuming, labour intensive and expensive exercise. Therefore modifications are continually being developed to reduce the time, labour and financial cost of generating mAbs. Some examples are given below.

Immunisation Strategy Variations

  • Singe Step Immunisation- special adjuvants added to the antigen injection make it a sustained release preparation- so that the one dosage has the same effect as two usually would (Examples include microparticles & liposomes). This results in a quicker and more efficient immunisation schedule [34].
  • Genetic Immunisation- Rather than protein antigens, DNA expression vectors are delivered to the mouse [38]. The protein will then be expressed in vivo- complete with any post translational modification that may not be achievable in the lab, and B-cells respond to the proteins as normal. This technique is suitable for protein antigens which are difficult to isolate in their natural conformation, and is faster than conventional immunisation [38]. In cases where the protein antigen can be purified, the response can be further improved if the protein is injected after the DNA vector (“Prime-boost strategy”[34]).
  • Multiplex Immunisation- this involves the injection of multiple antigens into single mice, with a view to generate multiple hybridoma lines [34]. When multiple cell lines are required this method is more efficient- decreasing not just the number of animals needed but also the amount of tissue culture, number of supernatants to test etc.

Hybrid Generation Variations

  • Semi-automated somatic fusion- the fusing of B-lymphocyte and myeloma cells as well as the subsequent steps in isolating desired hybridomas is done with the help of robotic machines and customisable software programs [34]. Automation can make the process less labour intensive by assisting in fusing the cells, culturing and plating the cells, exchanging the media in which the cells grow, and harvesting and screening supernatant.

Screening Variations

  • Automation of ELISA assay- robotics are used to test the supernatants of the single isolated hybridomas, making the process less labour intensive. [34].
  • Automated Protein microarray- this technique is also automated, using a robot microarrayer, but it is also more sensitive than ELISA so when it is used to screen for antibodies, less supernatant and less reagents are needed, saving time and money [39]

Alternative Methods

Bacterial Expression Systems

Recombinant DNA techniques make it possible to introduce genes coding for specific antibody proteins to bacteria such as E. coli[24]. When promoters are included with the introduced genes, it is possible induce expression of these antibody genes at high levels. This system is simpler, faster and cheaper than the hybridoma method, however bacteria do not have the same array of cellular machinery found in the endoplasmic reticulum of mammalian cells [40]. This machinery is necessary to carry out protein re-folding, post translational modification and the assembly of complete functional antibodies, therefore the products of bacterial expression are antibody fragments which often require alteration to make them soluble[24]. While bacterial expression is not appropriate for making complete functional antibodies, it can be used to produce ‘functional fragments’ of antibodies—which may then be fused to other molecules such as metal-binding proteins, drugs, cytokines etc [40].
MCB- general principle of bacterial expression systems

Phage Display Libraries

This alternative to the hybridoma method uses filamentous bacteriophages (viruses that infect bacteria) and bacterial hosts to produce antibody fragments [24]. As for bacterial expression systems, complete functional antibodies cannot be obtained but this method. However compared to the hybridoma method, phage display is quicker, allows high production of mAb (fragments) with less investment of resources, and allows selection for mAb fragments with high binding affinity[41].
Similar to hybridomas, the starting point is B-lymphocytes of an immunized animal- however in this case they may be from any animal[42]. This means human mAb fragments can be produced- or mAb fragments for use in studies involving mice (eg: mouse gene knockout studies)[42]. RNA is isolated from B-lymphocytes, and that coding for the variable (antigen binding) parts of the antibody are amplified to produce a large amount of complementary DNA by reverse-transcriptase PCR[42]. This is incorporated into expression vectors and transformed into E. coli cells, which are then infected with ‘helper phages’. Specially chosen strains of E. coli will only be infected by one bacteriophage per cell, meaning only one specific antibody fragment will be produced (this is due to removal of the F-pilus from the bacterial membrane when a phage has infected it. The phage recognises and uses the F-pilus to infect the E. coli cell) [42].
E. coli will be expressing and releasing phage particles, which will contain both the antibody protein and its encoding DNA, linked together, on their surface. A diverse library of phages will be present due to the random recombination of heavy and light chain fragments that make up the antibody fragments[41]. Selection carried out in the form of several rounds of ‘panning’, which will result in selection not just for mAb fragments recognising the desired antigen, but those that recognise it with high affinity[41]. Put simply, panning involves exposing the phages to the antigen then washing, such that only those that bind the antigen will remain. These can then be re-infected into E. coli, reamplified, and panned again. Single clones producing monoclonal antibody fragments binding the desired antigen are obtained, and the mAb fragments can be purified from them[42].
Immunobiology- phage display production of mAb fragments

Hybridoma free method

In this method, immune cells are taken from genetically modified mice and there is no need for creating hybrids [43]. The transgenic B-cells have a temperature sensitive gene & promoter that at 33°C produces monoclonal antibodies in culture. These transgenic cells are more genetically stable than hybridomas, and simpler to generate mAbs from. However, they grow at lower density and need long periods in cell culture to generate comparable results to hybridomas.

Modification of Monoclonal Antibodies

Much of the power of monoclonal antibodies, both clinically and in the lab, comes from the ability to combine them with other molecules. The great specificity of mAbs can be used to deliver molecular parcels selectively to predetermined sites—opening up an array of possibilities limited only by the imagination, a couple of which are given below.

  • When labelled with fluorescent dyes, mAbs can be used in conjunction with fluorescence microscopy to visualise the location of the matching antigen- eg: within cells [35]. This is the key principle of indirect immunocytochemistry. Often secondary antibodies directed against the primary, target antigen-binding antibody are used to amplify the effect [31]. MBoC- indirect immunocytochemistry
  • When linked with colloid gold spheres, mAbs can label target antigens in a simlar way but for electron microscopy [31]
  • enzymes may be linked to mAbs. When the antibody remains bound to the antigen, its presence can be easily detected by the action of the enzyme, removing the need for special imaging[31]. This is the principle behind ELISA. The enzyme alkaline phosphatase is commonly used, as it can catalyse the production of a large amount of inorganic phosphate, visible as a coloured precipitate. With specifically targeted mAbs, this technique has been used to test for pregnancy or infection [31]. Biochemistry- ELISA
  • tumoricidal drug molecules can be linked to mAbs directed against receptors overexpressed in forms of cancer, allowing direct delivery of cancer-fighting drugs where they are needed[8].
  • radioactively labelled metal can be joined to monoclonal antibody fragments produced by E. coli. DNA coding for the part of the antibody that specifically binds antigen can be combined with DNA coding for a protein that is known to tightly bind the radioactively labelled metal. The isolated antibody can then be chemically fused to the radioactive metal, and used to detect the antibody fragment bound to antigen in conjunction with special imaging equipment. [44].

Selected Current Applications

Cells stained with mAbs for GAPDH
mAbs against yeast proteins

Tracking of Specific Intracellular Proteins

Monoclonal antibodies can be used in cell biology research to mark selected proteins within cells, allowing their abundance and distribution to be precisely tracked over time (among thousands of other protein types that may be present in the same cell) [31]. This may be accomplished by the labelling of the mAbs with fluorescent dyes for fluorescence microscopy. To illustrate with an example, Kamma et al.'s group (2001) tagged two alternatively spliced nuclear RNA binding proteins hypothesised to be involved in autoimmune disorders and cancer- heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and hnRNP B1, with different monoclonal antibodies. This allowed direct comparison of their intracellular distribution and functional properties over time in the context of the cell cycle[45].

mAbs and Metastatic Breast Cancer

Slamon and his colleagues (2001) investigated the effects of chemotherapy and the use of a mAb on patients with breast cancer that overexpresses human epidermal growth factor receptor (HER2). HER2 is a growth factor receptor gene that is found in high levels in the malignant cells of 25-30% of breast cancers [8]. It is known that women who overexpress HER2 experience a more aggressive form of the cancer. Consequently, the chance of survival is shortened. This study found that the most effective antibody to help suppress the problem of overexpression is Trastuzumab. It works by inhibiting the proliferation of the cancer cells that over-express HER2. When used in combination with first-line chemotherapy, Trastuzumab improves overall survival by decreasing disease progression, slowing down the duration of the response, and increasing the rate of the response.

Examples of mAbs used for the treatment of cancer

mAbs and Parasitic Infections

Chuang and his colleagues (2010) investigated the effects of 'anti-CCR3' mAb towards Angiostrongylus cantonensis (parasite) infection by injecting the anitbody into infected ICR mice. It was found that the use of the mAb reduced:

  • Infiltration of eosinophils
  • The severity of eosinophilic meningitis in mAb-treated mice relative to infected but untreated mice,
  • Levels of CCL11 (eotaxin)in the peripheral circulation,
  • Expression of the Th2-type cytokine interleukin-5 (IL-5) in the brain.

mAbs and Allergic Asthma

Omalizumab is '...a humanised IgE-specific mAb developed to target free IgE and membrane-bound IgE' [15]. On the other hand, it was designed to not target IgE that is bound to IgE Fc Receptors on mast cells, and, therefore, to not trigger mast-cell degranulation Cite error: Closing </ref> missing for <ref> tag. Two human monoclonal antibody Fab fragments (HMab) (cloned before the emergence of S-OIV) were found to neutralise H1N1 strains. The human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection. These HMabs can:

  • help with the understanding of vaccine trial data in the shortest possible time
  • constitute the basis, alone or in a combination with other monoclonal antibodies, for a new class of

drugs to be used in the treatment and in the prophylaxis of this disease.

Simmons and his colleagues research in the H5N1 Influenza strain proved that cross-reactive mAbs helped prevent and treat the infection in mouse models. As a result, they highlighted the potential of mAbs in the treatment of human cases of H5N1 influenza.

IAA localisation in Strawberries

mAbs and Strawberries in the Horticultural Industry

The use of mAbs has shown to be a useful tool in the area of horticulture, especially to accurately and rapidly detect pathogens infecting plants. In 2004, one study [3], published under The Journal of Horticultural Science & Biotechnology, used a specific mAb to localise indole-3-acetic acid (IAA)in developing field-grown strawberries. This study provided evidence for IAA distribution in fleshy fruits. The researchers identified certain areas of the plant that had the greatest localisation of IAA.

Other studies have also used the IAA mAb for further research into the location of the production of IAA, and the transportation of this acid to certain areas. For example, in 2005, Hou & Huang found that IAA in the shoot apex is likely to be produced in young leaves and transported to the vascular tissues to certain functional areas of the plant [46]

mAbs and Alzheimer's Disease Research

Amyloid-beta (Abeta) peptides accumulate forming extracellular 'amyloid plaques' in the brains of patients with Alzheimer's disease[47]. Labelled monoclonal antibodies directed against different isoforms of Abeta have been valuable in studying the pathogenesis of Alzheimer's, because they allow light to be shed upon the distribution and prevalence of slightly different forms of Abeta. The great specificity of monoclonal antibodies allowed differentiation between two very similar isoforms- Abeta-40, and Abeta-42- which has an additional two amino acids at the C-terminus but is otherwise identical[48]. From this research it is now known that Abeta-42 is the more widespread and toxic isoform found in Alzheimer's disease affected brains. This is an example of mAbs providing unique insight that is foundational for better understanding of a disease, and helpful in the search for treatments and diagnosis.

Identification of bacteria using a mAb


  • mAbs have made it possibel to identify the 'autoantigen' (molecule in the body that is the target of the host's own immune system) in research of autoimmune diseases. For example, myosin in the heart and glomerular basement membrane proteins in rheumatic fever stemming from bacterial infection [10]
  • mAbs can be used in 'serotype' identification of bacterial strains, which is quicker and cheaper than PCR [9].
  • Fluorescently labelled mAbs make it possible to visualise intracellular pathogens such as Legionella pneumophilia, which cannot be detected using light microscopy alone [49].


There are some limitations with the use of mAbs. Aside from being expensive, mAbs may also:

  • Cross-react with other antigens other than their initial target. By replacing antisera with mAbs, researchers have reduced but not eliminated the chances of cross-reactions[50]. The mAb may potentially bind to a minor determinant that is co expressed on other nearby homologous molecules, causing the cross-reactions.
  • Not suit the intended application if not carefully chosen. Depending on the class and subclass of the mAb, its biological activity will vary. For example, an antibody of IgG subclass will not fix complement or bind to macrophage Fc receptors [50]
  • Increase the risk of immune reactions in clincal use(e.g.acute anaphylaxis)- especially when murine monoclonal mAbs are used.[15]
  • Cause autoimmune conditions (e.g. Thyroid disease[15].
  • Contribute to tumor progression [15]
  • Cause cardiac dysfunction. For instance, Trastuzumab has been identified as a mAb that may cause Cardiotoxicity. [15]
  • Like all antibodies, mAbs have the potential to effect [15]:
    • antibody-dependent cell-mediated cytotoxicity
    • complement-dependent cytotoxicity
    • antibody-dependent cellular phagocytosis

Although, the first two can be minimised by replacing the Fc region of the mAb with IgG4. Hansel et al. (2010) outlined that the recruitment of Complement or effector cells can be minimised by modifying this Fc region, for example, by removing carbohydrates[15].

Hansel and his colleagues (2010), in their article 'The Safety and side effects of Monoclonal Antibodies', summarised the side effects of licensed monoclonal antibodies into a table. Abstract of their article

Future Directions

Like all other scientific inventions, mAbs are continually being refined and developed for novel purposes. Though subject to some limitations, mAbs continue to contribute to many areas of science, providing new avenues and techniques for research and clinical practice. Some of the future directions of mAbs are listed below.

  • The risks associated with the use of specific mAbs are not fully known, and therefore require more research. Identifying and characterising these risks lays the foundation for developing strategies to overcome them.
  • The development of new techniques and concepts promise to make mAb production more efficient, increasing their availability and therefore their clinical impact. For example, anti-TNFα mAbs have been proven to be clinically effective for Rheumatoid arthritis, but is too expensive to produce, and their role in clinical practice is yet to be determined [51].
  • The use of mAbs for histopathological diagnosis of human malignancy is an important improvement. However, the potential value of this finding to the clinical setting is yet to be determined.[52]
  • More research must be performed in order to fully understand the diagnostic and therapeutic potential of mAb 14C5 for Pancreatic Cancer. [53]


Adjuvant: material added to an antigen to increase the immune response generated when it is inoculated [3]

Antibody: An immunoglobulin that is specifically reactive with an immunogen (or antigen).[5]

Antigen: Anything that is capable of inducing an immune response.[5]

BacteriophageViruses that are capable of infecting bacteria.[54]

Chimeric antibodies antibodies containing antigen-binding regions derived from mouse genes and constant regions derived from human genes, therefore they are essentially antibodies encoded by genes of multiple species.[22]

Cytotoxic the ability to have a toxic killing effect on cells. [55]

ELISA: Enzyme Linked Immunosorbant Assay- a technique allowing measurement of how much of a molecule present in solution, using antibodies with enzymes attached. When the antibody-enzyme complex binds to its substrate, a reaction product is produced which can then be detected- often in the form of a colour change [1].The substrate can be another antibody [3]

Epitope The portions of the antigen that are recognized by the immune system. [56].

HAT medium Hypoxanthine Aminopterin Thymidine Medium, which is selective for hybrid cells due to the presence of aminopterin. Aminopterin blocks normal DNA synthesis- preventing cell division, unless certain enzymes are present which allow the normal pathway to be bypassed. Only hybrids formed from both cell types are able to replicate their DNA and proliferate [2]

Heterocaryon a cell containing a number of nuclei that are genetically different.[57]

Humanized antibodies mouse antibodies genetically engineered by modifying the protein sequence in an attempt to have both their constant and variable regions more human-like.[22][58]

ICR (Imprinting Control Region) mice a strain of mice that express a gene only from one of the two alleles (the other having been silenced). Usually, gene expression occurs equally from both the maternal and paternal alleles.[59].

Monoclonal antibody antibodies that are specific to a single epitope by having uniform variable regions.[22].

Murine antibodies antibodies derived from rodent species [24]

Phagocytose when a cell ingests and destroys particulate substances.[60]


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2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer