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SPECIAL SECTION: MONOCLONAL ANTIBODIES

Recombinant antibodies (rAbs) produced without the use of animals

Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than using mice.  Advances in molecular biology have lead to the ability to synthesize antibodies de novo in vitro – completely without the use of animals.

These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.  Recombinant antibodies are translated from recombinant DNA and displayed on the surfaces of cells or phage particles.  In 1990 John McCafferty demonstrated that variable regions from antibodies could be displayed on the surface of a filamentous phage. In 1990 John McCafferty demonstrated that variable regions from antibodies could be displayed on the surface of a filamentous phage. Since then, various antibody display platforms using yeast, bacteria, mammalian cells, and ribosomes have been developed.  

General production methods for the manufacture of non-animal recombinant antibodies can be broken down into five general steps: (1) creation of an antibody gene library; (2) display of the library on phage or cell surfaces; (3) isolation of antibodies against the antigen of interest; (4) modification of the isolated antibodies; and (5) scaled up production of selected antibodies in a cell culture expression system:

1.      Antibody Gene Libraries: Antibody gene libraries consist of microorganisms that have been transformed with the genes encoding the variable regions of different antibodies. Each variable region gene is spliced into a vector (vehicles that transfer foreign genetic material into another cell), and the vector is taken up and integrated into the genome of a microorganism.

2.      Library Display: Genes encoding antigen-binding variable domains of antibodies are fused to phage genes that encode coat protein, allowing for the phage coat to express, or “display”, the antibody fusion protein. A collection of recombinant phage that display unique antigen binding domains on their surfaces is known as a phage display library.  Display on the cell or phage surface of the cloned antibody sequence allows for rapid, high throughput selection of recombinant antibodies that bind a specific antigen.

3.      Antibody Isolation: Once the rAbs are displayed, tools such as paramagnetic beads, fluorescence-activated cell sorting (FACS), and/or Enzyme-Linked Immunosorbent Assays (ELISAs) can be used to isolate individual antibodies that bind to a specific antigen target.  Library members are incubated with the target molecule and unbound library members are washed away.  The result is a library enriched for target-binders.  This process can be repeated as needed to achieve the desired specificity.  This process is called Exponential Evolution of Ligands by Systematic Enrichment or SELEX. 

4.      Modification: Bacteria, yeast or phage encoding the selected antibodies are grown in greater quantities and are put through the selection process again to enrich for the strongest binding (highest specificity) candidates. If the affinities of the lead candidates are not strong enough, antibodies with higher specificity can be generated through "maturation" by random or rational mutagenesis.

5.      Antibody Expression: Once an antibody is selected, the genes for that antibody are transferred into an expression system—bacteria, yeast, or mammalian cell lines specially designed for the expression of foreign proteins. The choice of vector and expression system depends on the type of antibody that is to be produced.

It is possible to perform all of these techniques in a standard molecular biology lab or the process can be outsourced to a variety of facilities specializing in the generation of rAbs.

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Progress in Refinement: Enhancement of Scientific Integrity and Animal Well-Being
November 30, 2107
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December 1, 2017
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Advances in Cell and Tissue Culture
May 21-23, 2018
Cardiff University, UK
 
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August 23-24, 2018
Rio de Janeiro, Brazil 
 

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