- The ability of animal immune systems to produce antibodies capable of binding specifically to antigens can be of use to develop probes for the detection of molecules of interest in a variety of research and diagnostic applications.
- Nearly all medical or cell biology researchers use immunochemical techniques for molecular analysis.
- Usually, all immunochemical methods depend upon the utilization of antibodies, and their effectiveness relies on the quality of the antibodies used.
- The antibody production process involves the preparation of antigen samples and immunization of laboratory or farm animals to trigger the expression of antigen-specific antibodies within the serum at a high level, which then can be recovered from the animal.
- Successful antibody production depends upon cautious planning and implementation concerning several steps and considerations.
Steps and Considerations of Antibody production
- Synthesis and purification of the target antigen
- Selection of an appropriate immunogenic carrier protein
- Conjugating the antigen and carrier protein to make an immunogen
- Immunization of animals using appropriate schedule and adjuvant formula
- Screening of serum or hybridoma for antibody titer and isotype
There are two major classes of antibodies utilized in immunochemistry:
Monoclonal antibodies v/s Polyclonal antibodies
|Monoclonal antibodies||Polyclonal antibodies|
|Monoclonal antibodies are epitope specific for an antigen.||Polyclonal antibodies are antigen specific.|
|Derived from one specific clone of B cell, that recognizes one particular epitope on an antigen.||Derived from many B cell clones, that produce antibodies recognizing different epitopes of an antigen.|
|Produced by animal cells artificially in tissue culture using hybridoma technique.||Produced by host animal immunized with the substance of interest usually three or four times.|
|Expensive to produce and generally produced in mice.||Cheap to produce and generally produced in rabbits and guinea pigs for small amounts, and sheep or goats for large amounts.|
- Monoclonal antibodies are antibodies that are made by identical immune cells, clones belonging to a single parent cell.
- Monoclonal antibodies have monovalent affinity and bind to one particular epitope of an antigen.
- Monoclonal antibodies can be produced in specialized cells through a method now referred to as hybridoma technology.
- Kohler and Milstein in 1975, were the first to fuse lymphocytes to generate a cell line, both immortal and a producer of specific antibodies. They won the Nobel Prize for Medicine in 1984 for the development of ‘hybridoma’.
- The value of hybridoma was not well acknowledged before 1987, around which the production of monoclonal antibodies in rodents for usage in diagnostics started.
- B cells can mutate into tumor cells that result in a type of cancer called myeloma. Myeloma cells are immortal and grow indefinitely in culture. The fusion of a single activated B cell and a myeloma cell creates hybridoma that can grow continuously in culture and produce antigen-specific antibodies.
Production steps of Monoclonal Antibody:
- Mice are immunized with an antigen, and their blood is screened for antibody production.
- The antibody-producing splenocytes are extracted for in vitro hybridoma production.
- Myeloma cells are made ready for fusion.
- Myeloma cells and isolated splenocytes are fused, creating hybridomas in the presence of polyethylene glycol (PEG). PEG causes the fusion of cell membranes1.
- The hybridoma clones can be selected using HAT medium. Clones are screened and selected based on antigen specificity and immunoglobulin class.
- Each positive clone is confirmed, validated, and characterized (Isotyping).
- Positive clones are expanded, scaling up the production of the desired antibodies.
Figure: Production of monoclonal antibodies
HAT (hypoxanthine, aminopterin, thymidine) selection:
- HAT medium contains drug aminopterin that blocks the de-novo synthesis of DNA nucleotides.
- This makes the cells depend upon another pathway that needs HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) enzyme for DNA synthesis.
- Myeloma cells which do not fuse with the B cells cannot grow in HAT medium, since they are HGPRT negative2.
- B cells that are not fused with the myeloma cells also die as they have short life-span.
- Therefore, the HAT medium allows the selection of hybridoma cells with the HGPRT gene and immortal property.
- Polyclonal antibodies are antibodies that are secreted by different B cell lineages within the body.
- They are a collection of antibodies that react against a particular antigen, each binding to different epitopes.
- Polyclonal antibodies are produced in the appropriate donor animals.
- Usually, antigens are conjugated with an adjuvant before immunizing the animals.
- Adjuvants are substances that increase the immunogenicity of the antigen, reducing the amount of antigen required as well as stimulating specific immunity to it.
- Freund’s complete adjuvant (FCA), alum, bentonite, and Bacillus pertussis are some of the adjuvants that can be of use.
Production steps of Polyclonal Antibody:
- Preimmunize blood samples are collected to produce baseline IgG levels.
- The first two immunizations are done within 14 days.
- Later immunizations are spaced at intervals of 4-6 weeks to maximize the antibody production.
- Blood samples are collected 10 days after the completion of immunization program.
- The serum screened for presence of antibodies with specific activity to antigen. Method such enzyme-linked immunosorbent assay (ELISA) can be used for the activity testing.
Figure: Production of polyclonal antibodies
1. Wojcieszyn J, Schlegel R, Lumley-Sapanski K, Jacobson K. Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes. J Cell Biol. 1983;96:151-159. doi:10.1083/jcb.96.1.151
2. Martínez P, Iborra A. Antibody Synthesis in Vitro. In: ; 2006. doi:10.1038/npg.els.0001115