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Antibody production

Nowadays industrial production methods of antibodies involves the use of laboratory animals by exploding their immune systems ability to generate antibodies. Antibodies have a diverse set of applications and the ability to produce antibodies to fit a specific need is essential. Of the current techniques, the hybridoma technology is the most widely used method for producing monoclonal antibodies (mAb).

Antibodies are produced by B-lymphocytes of the adaptive immune system. They are highly specific proteins that remain surface-bound on the B-lymphocyte until it recognizes a foreign molecule like bacterial or viral proteins, generally termed an antigen. An antibody will recognize through binding a specific region of the antigen, called an epitope. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. Once the surface-bound antibody encounters an antigen to which it can bind, the B-lymphocyte will become activated. The activated B-lymphocyte will start to grow and proliferate, producing clones of itself. It can further differentiate into three types of cells, including the plasma cell, from which the antibodies will be secreted. Reference: Cowan MK. Host Defenses II - Specific Immunity and Immunization. Microbiology - A Systems Approach. 3 ed: McGraw-Hill; 2012. The antibody will be changed from a surface-bound receptor molecule to a soluble and secreted molecule by an alternative splicing mechanism of the pre-mRNA transcript of the antibody gene.
The activated B-lymphocyte produces just one type of antibody that has affinity for a single epitope on an antigen. The antibody structure consists of a constant part and a hypervariable part. The hypervariable part is the one responsible for specificity of an epitope of an antigen. The hypervariable part is generated by rearrangement of gene segments during development of B-lymphocytes and by RNA splicing. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. Furthermore protein processing contributes to diversity and affinity of the produced antibodies. Even a somatic mutation can greatly increase affinity for the antigen, a process called affinity maturation. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. Every B-lymphocyte goes through these processes differently, thus every B cell will produce different antibodies. This enhances the organism’s antibody repertoire and ensures the best possible protection from intruders. Antibodies are highly complex yet elegant and sophisticated molecules, designed to protect an organism against invaders. Its highly specific nature and ability to bind a target with high affinity can be exploited in many ways.

Applications of antibodies

The antibody will bind to the foreign molecule – the antigen, its target. This ability can be useful in many circumstances. Indeed antibodies can be used as antivenoms in treatment of snakebites. Reference: Chippaux JP, Goyffon M. Venoms, antivenoms and immunotherapy. Toxicon. 1998;36(6):823-46. Antibodies can also be used in a preventive manner through passive immunization of patients. This involves injection of ready antibodies instead of inducing production of them as in a normal vaccine. Reference: Clem AS. Fundamentals of vaccine immunology. J Glob Infect Dis. 2011;3(1):73-8. They can also be used to detect presence of a specific protein (its antigen) – which makes them useful in diagnostics (so-named Immuno-diagnostics). Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7. They are used in techniques such as Western Blotting, Enzyme-Linked ImmunoSorbent Assay (ELISA) Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7. and when conjugated to a fluorescent protein they can be used to localize proteins within cells (called immunohistochemistry, or specifically Immunolacalization), which is how images like this one is obtained! (insert picture of immunofluorescence).
The ability of antibodies to bind their target can be useful in treatment of cancer. Antibodies that bind and inhibit grow factors or blocking a hyperactive signalling pathway can potentially block tumor progression. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12(4):278-87. It has also been proposed to use antibodies as a drug delivery system by conjugating (linking) the antibody to a chemotherapeutic drug. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

BioPharma P. What Are Humanized Monoclonal Antibodies 2015 [cited 2015 08/08] (Link) As of now, only few antibody-based treatments are available. Cancer cells have antigens on their surface which can be cancer specific or present at a higher concentration than on normal cells which potentially can be targeted by antibodies. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30. The hope is that human-like antibodies against cancer antigens will be able to activate the immune system that in turn will eliminate the cancer cells like they were foreign material. It is no surprise why scientists, researchers and doctors are interested in this sophisticated and elegant molecule and why they would want to have antibodies produce at command to fit their needs. However, the available methods for producing antibodies are less sophisticated and elegant ethically speaking and stain the reputation of antibodies. The most widely used method for producing monoclonal antibodies is the hybridoma technology.

Industrial production of monoclonal antibodies using the hybridoma technology

Monoclonal antibodies are produced from one B-lymphocyte cell line (clones), and the antibodies produced will have affinity for the same epitope on the antigen. This means that they will recognize and bind the same region of the antigen. mAb are preferable to polyclonal antibodies in the field of research. The hybridoma technology was originally developed by Kohler and Milstein in 1975 Reference: Payne WJ, Jr., Marshall DL, Shockley RK, Martin WJ. Clinical laboratory applications of monoclonal antibodies. Clin Microbiol Rev. 1988;1(3):313-29. and relies on an animals ability to generate antibodies against a foreign molecule. The animal of choice in production of monoclonal antibodies is typically a mouse or a rabbit. This production method involves 6 general steps steps; Reference: Hnasko RM, Stanker LH. Hybridoma Technology. Methods Mol Biol. 2015;1318:15-28.

1. Immunization: Injection of the antigen into the animal continuously for several weeks. Often the animals are also injected with an adjuvant that enhances the immune response. Reference: Stills HF, Jr. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 2005;46(3):280-93.
2. Isolation: Euthanizing and extracting the blood (in polyclonal antibody production) and spleen from the animal and isolation of the antibody producing B-lymphocytes.
3. Fusion: Fusion of the B-lymphocytes from the spleen and a murine myeloma cell line (B-lymphocyte cancer cells), thus creating immortal and high-yield antibody producing hybrid cells.
4. Screening: Affinity based screening tests of the different antibodies to select the most effective one. This involves techniques such as ELISA and are followed by affinity chromatography purification. Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7.
5. Multiplication: Growing of selected hybridoma cell in a culture dish or transplantation of the tumor cells into a new animal for a higher yield. The latter is the typical procedure.
6. Harvesting: The hybridoma cells will mass produce their antibody and mass divide. This causes tumor development and accumulation of fluids in the abdominal cavities (ascites). The fluid can be drained and the antibodies harvested from it.

Antibodies generated in the murine model will have to be further modified before they can be used therapeutically in humans. The murine antibodies will be recognized as foreign in humans and will elicit an immune response against murine antibody. Before mouse antibodies (or any other animal antibody) can be used in clinical medicine, they have to be ‘humanized’. Humanized animal antibodies are also known as chimeric antibodies limited. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30. and are generated by combining the constant part of a human antibody with the variable (epitope-recognizing) part of the murine antibody. However chimeric antibodies are still about 30 % foreign for the human body, and therefore they elicit significant immunogenicity in humans. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30. Creation of chimeric and humanized antibodies is not as easy as it might sound. It requires extensive knowledge and characterisation of the antibody structure and effective methods are still limited. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.