Producing Monoclonal Antibodies

How are Monoclonal Antibodies Produced

What is monoclonal antibody production? Monoclonal antibodies are a series of identical antibodies produced by a single clone of B cell. In 1975, monoclonal antibodies were first generated by Milstein and Köhler. This method for production of monoclonal antibodies is called hybridoma technology. Since then, monoclonal antibodies have been widely used as an essential tool of biomedical research and therapeutic applications.

Another method of manufacturing monoclonal antibodies is by using phage display which was discovered by G. Smith in 1985. And it has become one of the most effective techniques for producing large amounts of peptides, proteins and antibodies.

Based on hybridoma and phage display technology, Sino Biological has the capability to offer custom monoclonal antibody services as well as large scale antibody production and purification. Whatever your specifications are, Sino Biological can propose tailored solutions and its expertise to successfully complete each project.

What are the advantages of monoclonal antibodies?

• Same quality of the antibody is maintained amongst the different production batches.
• Highly reproducible and scalable, unlimited production source.
• Speed and sensitivity and specificity of assays.
• Can produce antibodies when needed.
• No need to worry about maintaining the animals.
• Antigen or immunogen need not be pure.
• Selection helps to identify the right clones against the specific antigen.

Steps to Produce Monoclonal Antibodies in Mouse

Some assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To achieve this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs). Production of monoclonal antibodies involves several critical procedures as follows.

Animal immunization   Fusion & Selection   Antibody purification   Quality control
Fig 1. How to generate monoclonal antibodies

1) Animal immunization

The very first step in hybridoma technology is to immunize an animal (usually a mouse), with appropriate antigen. The antigen, along with an adjuvant like Freund's complete or incomplete adjuvant is injected subcutaneously (adjuvants are non-specific potentiators of specific immune responses). The injections at multiple sites are repeated several times.

This enables increased stimulation of B-lymphocytes which are responding to the antigen. Three days prior to killing of the animal, a final dose of antigen is intravenously administered. The immune-stimulated cells for synthesis of antibodies have grown maximally by this approach. The concentration of the desired antibodies is assayed in the serum of the animal at frequent intervals during the course of immunization.

When the serum concentration of the antibodies is optimal, the animal is sacrificed. The spleen is aseptically removed and disrupted by mechanical or enzymatic methods to release the cells. The lymphocytes of the spleen are separated from the rest of the cells by density gradient centrifugation.

2) Fusion & Selection

The thoroughly washed lymphocytes are mixed with HGPRT defective myeloma cells. The mixture of cells is exposed to polyethylene glycol (PEG) for a short period (a few minutes), since it is toxic. PEG is removed by washing and the cells are kept in a fresh medium. These cells are composed of a mixture of hybridomas (fused cells), free myeloma cells and free lymphocytes.

When the cells are cultured in HAT medium, only the hybridoma cells grow, while the rest will slowly disappear. This happens in 7-10 days of culture. Selection of a single antibody producing hybrid cells is very important. This is possible if the hybridomas are isolated and grown individually. The suspension of hybridoma cells is so diluted that the individual aliquots contain on an average one cell each. These cells, when grown in a regular culture medium, produce the desired antibody.

3) Antibody purification

Monoclonal antibodies may need to be purified before they are used for a variety of purposes. Protein A affinity chromatography is used to purify monoclonal antibodies. It's the golden standard for monoclonal antibody (mAb) purification, and a technology that has gained high interest because of its great performance and capabilities. If customers need higher purity, further purification methods can be performed.

4) Quality control

After purification, a series of quality control tests are performed to ensure the quality of monoclonal antibodies. Antibody concentration is evaluated by absorption at 280 nm (A280). The purity of monoclonal antibody is checked using SDS-PAGE. To estimate the monoclonal antibody titer, an ELISA test was the most suitable method.

References

1. Schook, L. B. (Ed.). (1987). Monoclonal antibody production techniques and applications. Dekker.
2. Groth, S. F. D. S., & Scheidegger, D. (1980). Production of monoclonal antibodies: strategy and tactics. Journal of immunological methods, 35(1-2), 1-21.
3. Pandey, S. (2010). Hybridoma technology for production of monoclonal antibodies. Hybridoma, 1(2), 017.
4. Liu, J. K. (2014). The history of monoclonal antibody development–progress, remaining challenges and future innovations. Annals of Medicine and Surgery, 3(4), 113-116.
5. Bazan, J., Całkosiński, I., & Gamian, A. (2012). Phage display—A powerful technique for immunotherapy: 1. Introduction and potential of therapeutic applications. Human vaccines & immunotherapeutics, 8(12), 1817-1828.

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