SPECIAL SECTION: MONOCLONAL ANTIBODIES
Scientific uses and advantages of rAbs and aptamers
Uses of rAbs and Aptamers:
As with traditional animal-generated antibodies, rAbs and aptamers can be used in blotting techniques (Western, Northern, Southern, etc.), immunostaining, flow cytometry, and enzyme linked immunosorbent assays (ELISAs). rAbs and aptamers are also used in the diagnosis of infectious diseases by detecting pathogens and can be used in protein arrays that analyze relative protein expression associated with disease or other biological change. Aptamers are frequently used as biosensors that convert molecular binding events into measurable signals. Aptamers have also been marketed to detect molecules, compounds, viruses, illegal or unsafe food contaminants, and diseased cells. Regulatory agencies have have also realized the utility of rAbs and aptamers in vaccine potency testing and in an on-site test kit that measures ground contaminants.
Advantages of rAbs and aptamers
As a result of the historical development of mAbs, many researchers have considered the mouse ascites method inexpensive and familiar. However, the ethical costs associated with animal use combined with the scientific limitations of antibodies produced in vivo have made the faster and more versatile recombinant approaches increasingly appealing.
There are several inherent limitations to the production of mAbs in vivo, including: (1) lack of immunogenicity of some protein antigens due to non-recognition by the host; (2) inability to raise antigens to molecules that do not activate an immune response (typically only proteins and some carbohydrates induce immune responses in vivo); (3) inability to raise antibodies against toxic molecules, and unpredictable instances in which only low-affinity antibodies can be generated against certain antigens. (4) In addition, many hybridomas cannot be raised to high concentrations (titres) by in vivo ascites production. (5) Antibodies that are created in vivo can also have experimental limitations during application, including non-specific binding that can lead to cross-reactivity and high background.
Benefits of recombinant antibodies and aptamers
rAbs and aptamers can be used in all applications in which traditional mAbs are used and both newer methods have inherent advantages over their animal-derived counterparts as well. Molecules created using recombinant techniques have several additional benefits:
Decreased time to produce rAb/aptamers: an antigen-specific antibody suitable for research purposes can be produced in as little as 8 weeks –significantly less time than the months required for hybridoma-based methods.
Exceptional avidity: in vitro selection for high affinity binding is high-throughput and robust, and can result in antibodies with affinities unobtainable in vivo
Reproducibility: batch-to-batch variation is avoided due to a tightly controlled chemical synthesis process
Specific Binding Recognition: recognition of modified versus unmodified protein is possible and precise with the use of aptamers and rAbs
Conditional Binding: a high degree of control is possible, making selection of aptamers and rAbs that bind in a particular pH, salinity, or in other specific buffer conditions precise and practical
Unlinked to Immune Response: the process is independent of the biological immune response
Isotype Switching Possible: once a desirable antibody fragment is found it can be easily converted into any antibody isotype (e.g. IgA, IgM IgG etc.) from any species by adding the appropriate constant domain, making these methods highly adaptable and very practical.
rAbs/Aptamers to highly toxic/non-immunogenic proteins possible: Antibodies to highly toxic or non-immunogenic antigens can be created using library methods, unlike animal immunization technologies.
No animal welfare concerns or costs related to animal care and use: eliminating the use of animals throughout the process also eliminates animal welfare concerns
Additional benefits of aptamers:
Regeneration: Aptamers can be stored denatured and then regenerated quickly.
Easy Transport: Aptamers can be transported in ambient temperatures without harm
Changeable selection conditions: binding conditions can be changed so that the aptamer has desirable qualities for differing assays (i.e., binding in non-physiological, extreme pH buffers)
Kinetic parameters changeable: on/off rates can be changed on demand
Reporter molecules: the use of reporter molecules can be used at precise locations without interfering with binding avidity
Exceptional target discrimination: recognition of chirality or the presence or absence of a single methyl group