Here we leave you a summary with the 5 key factors when producing an antibody as it works or selecting the commercial antibody that best suits our assays.
Key Factors For Antibody Production
1.- Selection And Obtaining Of The Antigen
It is essential to analyze the target against which we want to obtain the antibody:
- Antigen type (protein, peptide, small molecules, carbohydrates …)
- Availability of sufficient quantity for immunizations and validations or possibility of obtaining it
- Homology with other proteins that you do not want to detect
2.- Boost The Immunogenicity Of The Antigen
The antigen must be capable of arousing an immune response intense enough in the animal to obtain the antibodies of interest. Immunogenicity can be increased by:
- Carriers or large molecules (KLH, BSA …) bound to small antigens (peptides)
- Adjuvants that act as catalysts for the immune response, increasing its magnitude. These adjuvants allow to increase the antibody titer in a relatively short period of time. The most common is Freund’s adjuvant, but other alternatives such as aluminum salts can be used on certain occasions.
3.- Selection Of The Host Or Animal In Which The Antibodies Will Be Produced
This selection will determine the type of antibody to be obtained:
- Rabbits : they produce an intense and very fast immune response, which is why it is usually the species of choice for obtaining polyclonal antibodies .
- Mice : It is one of the species that generates the best performance of immune cells for the development of hybridomas, which is why it is usually the species of choice for obtaining monoclonal antibodies .
- Goat : Like rabbits, the immune response is intense, with the addition that, since it is a larger animal, the volume of antibody that can be obtained is higher.
- Camels / Llamas : They produce the so-called “Single domain Antibodies”, significantly smaller in size than IgGs, with greater ease of binding to small molecules.
- Chickens : They produce a special type of immunoglobulin, IgY, and allow antibodies to be obtained against highly conserved mammalian proteins.
4.- Type Of Antibody That You Want To Obtain
- Polyclonal antibodies : blood will be drawn from the animals and the antibodies will be titrated during the immunization process, to ensure that the final bleeding is done at the time of highest titration and thus obtain the greatest amount of antibody possible. It should be borne in mind that the immune response experiences a different dynamic in different animals, so the time at which the peak of the titration occurs may vary from one species to another.
- Monoclonal Antibodies : Once the immune response has developed, the spleen cells of the animal will be extracted from which the hybridomas will be obtained. The production of antibodies from these can be done in cell culture (in vitro) or in ascites fluid (in vivo).
5.- Test In Which The Antibody Will Be Used
The same antibody can work perfectly in one technique, and give a diffuse signal in another. We remind you of these tips to optimize your ELISA, Western Blot and Immunohistochemical tests.