Like searching for the needle in the haystack, antibodies have a phenomenal ability to select a single target, against the odds...
In nature
Antibodies are produced by the immune system in preparation for an invasion by foreign materials or ‘antigens’, marking them for destruction or blocking their ability to cause harm. An antibody and its target (the antigen) behave like a lock and key. Due to a highly diverse structural arrangement at the antibody binding site, an antibody can be specific for only one target. When an invasion is encountered, the highly specific antibody finds its target, is selected and its production is increased to fight the invasion.
The unique ability of antibodies to single-out antigens has been exploited by the scientific community making them a valuable resource in investigative research, diagnosis and as therapeutic treatments.
Animal derived antibodies
Antibodies against a specified target are traditionally produced in animals by injecting the antigen into the animal and initiating an immune response. They are then extracted and used in scientific applications to bind to that same antigen encountered elsewhere. For example: antibodies can be used to identify a particular disease by detecting the upregulation of a particular molecule or ‘marker’ that is characteristic of that disease; or can interfere with the function of a protein that is key in the development of a disease, blocking its activity and preventing further progression; or can detect a potentially harmful contamination in a food product.
Immunization: A typical immunisation protocol for polyclonal antibody production requires two immune-compromised animals (rabbits, guinea pig, rat, mouse or larger animals). Initial injection and subsequent booster injections (usually 3) are subcutaneous at multiple sites, combining the antigen of interest in emulsion with an adjuvant (Complete or Incomplete Freund's Adjuvant) to boost the immune response. Animals also undergo test bleeds and final exsanguination.
Hybridoma: Following the immunisation protocol, antibody producing cells are harvested from the spleen of the animal. These isolated cells are then fused with immortal cancer cells. Only one immortal, antibody producing cell, specific for the antigen of interest, will be selected and therefore antibodies produced by this method will be ‘monoclonal’
Ascites method: hybridoma cells are injected into another animal, in the peritoneal cavity surrounding the gut initiating the development of tumours and secretion of an antibody-rich fluid called ascites fluid. This procedure has been banned in several European countries and is restricted in the EU. However, 42,000 uses of the ascites method were reported for 2020 by the European commission, almost entirely by one member state. Restrictions do not apply outside the EU and ascites produced antibodies may be imported as, for example, research reagents or in diagnostic kits. Further,
Polyclonal antibodies: Following the immunisation protocol, antibodies may also be collected from the serum, where they are secreted by different antibody producing cells. Therefore antibodies produced by this method will be ‘polyclonal’
Animal friendly affinity-reagents (AFAs)
Affinity reagents are antibodies, peptides, nucleic acids, or other small molecules that bind specifically to a molecule of interest in order to detect, capture or modify its mode of action.
Animal friendly affinity-reagents (AFAs) are recombinant (or genetically recombined) antibodies and non-antibody binding reagents that are generated in vitro using synthetic gene manipulation techniques. They are not derived from immunized animals and do not require the use of animals at any stage of production. Some recombinant antibodies are still derived from immunized animals, since antibodies derived from animals that have been immunized against a particular target will have a higher proportion of antibodies specific to the target (producing ‘immunized phage display libraries’). However, a higher specificity can also be achieved without immunization or using animals, producing ‘naïve phage display libraries’, by using various non-animal techniques such as ‘affinity maturation’,
AFAs are selected in vitro by phage, ribosome or yeast display. Not limited to antibodies, AFAs also include other affinity binding molecules including DARPins, affibodies, monobodies, anticalins, aptamers, affimers, (naive) nanobodies and molecularly imprinted polymers (MIPs), all of which can be produced without animal use.
Phage display is the technique used to create the AFA. Viruses typically can only survive by using the DNA of, and replicating within the living cells of a host. Phage (bacteriophage) are small viruses that infect and complete their lifecycle within a bacterial host. This natural process has been adapted for scientific use to express proteins of interest such as antibodies. The gene sequence for the protein is inserted into a special circular piece of DNA known as a plasmid vector. The vector also contains the gene sequence for a protein that will become one of the phage's coat proteins and the antibody sequence is fused to this. The vector is subsequently transformed into the bacterium. When the phage infects the bacterium, it adopts the vector DNA during replication. When the phage progeny are released from the bacteria, the antibody is expressed as a protein which is ‘displayed’ on the surface of the phage, fused to the coat protein. The antibody is now accessible for functional applications. A copy of the antibody gene sequence is retained within the phage and thus is accessible to infect further bacteria, for further analysis or modification. In molecular biology applications, a phage display library is made which consists of millions (10^6-9) or billions ((10^9-12) or more bacteria, each containing a vector with a unique antibody sequence. These unique antibody sequences may be copied from the natural immune repertoire from any mammalian species or produced synthetically by using targeted randomisation processes. Once expressed as a protein, the antibody has the potential to recognise and bind to a unique target. The number of unique antibody sequences describes the diversity of the phage display library.
By immobilizing a protein target (the antigen) to a surface and incubating it with phage expressed from the phage display library, phage displaying antibodies that recognise and bind to the antigen will be retained whereas non-binding phage can be washed away. The process is repeated several times to achieve an increasingly enriched antibody population. The repeated cycling of these steps is referred to as 'panning', in reference to the enrichment of a sample of gold by removing undesirable materials.
Alternative display systems for protein evolution in vitro include mammalian display, ribosome display, bacterial display, and mRNA display.
