Antibodies are proteins produced by the immune system as part of the body's defense against pathogens. Each antibody is specific to one molecule or a small number of molecules, known as antigens. Antibodies are commonly used in research to understand disease processes. However, care must be taken when handling and storing these proteins.
Kateryna Kon | Shutterstock
How are antibodies used in research?
Examples of the use of antibodies in research are:
- Protein purification, where antibodies are used to pick up a specific protein from a mixture of proteins;
- Enzyme-Linked Immunosorbent Assay (ELISA), where an enzyme is added to the antibody, which allows for quantification of the antigen;
- Western blot, where proteins are first separated by electrophoresis before being transferred to a membrane, which is then incubated with antibodies and can show relative differences in the level of the protein antigen, or the size of the protein which may have changed after translation;
- Immunohistochemistry, which indicate the presence of specific proteins within a tissue sample.
Common antibody challenges
The main challenge when using antibodies in research is quality. This issue came to light when certain landmark studies using antibodies could not be reproduced. However, this may not be completely surprising, since it is estimated that more than two million antibodies are sold by over 300 companies for research purposes.
Worryingly, when 6,000 of these commercial research antibodies from 26 companies were tested, 75% were found to be not specific, or did not actually work. Also, 50% of the 5,000 antibodies tested by the Human Protein Atlas consortium were not able to be used as intended. This brought about a conversation about the need for validation of these antibodies prior to commercial use.
While this is a good approach, for companies it may not be practical nor commercially attractive to validate their extensive antibody catalogue, since validation needs to go beyond testing. For example, testing would be the presence/absence of a signal from immunohistochemistry, while validation would need to be carried out in specific tissue, and then seeing whether the signal is generated from the correct place.
Manufacturers are, however, validating an increasing number of their antibodies, which would be of great benefit to research and perhaps address the reproducibility issues highlighted earlier. However, antibodies are made in batches, and there is a possibility that there will be extensive differences between batches. This would mean that the validation is not accurate for this particular batch - even though previous batches were properly validated.
Why do antibodies need specific storage conditions?
In their natural environment (i.e. in the host) antibodies have a stable condition in which they live in; the pH and the temperature are stable, and there are other molecules and proteins which aid in keeping the antibodies intact.
However, once out of this host environment the antibodies become less stable due to changes in the temperature and pH, and the purification process will remove the other molecules/proteins which help in stabilizing the antibody. What this means is that antibodies used in research are not stable, therefore, they have to be stored and handled carefully.
Generally, antibodies should be divided into smaller aliquots and stored between -20°C and -80°C. When required, an aliquot should be thawed and kept at 4°C rather than re-freezing, as this will damage the antibody. However, at 4°C it will not be stable beyond a few weeks, hence the remainder of the aliquot may not be usable. Dilution will mean that there is less antibody present, and this then affects stability of antibodies. In short, once diluted, antibodies should not be kept at 4°C for more than one day.
Another consideration is the potential for the stored antibody to thaw. Frost-free freezers will periodically raise the temperature in order to defrost the ice build-up, and this could subsequently damage the antibody. Opening of the freezer is likely to cause a change in temperature at the front part of the freezer, therefore it is not an ideal place to store antibodies.