Antibodies are core reagents for a broad range of research and in vitro diagnostic (IVD) applications. They can be classified as either polyclonal or monoclonal, based on the method of production, or as primary or secondary reagents according to the type of target they recognize. Factors to consider when selecting antibodies include how rigorously they have been validated and whether they are available in sufficient quantities to meet the long-term needs of a particular project.
What are polyclonal and monoclonal antibodies?
Established methods for antibody production are initiated by immunizing an animal with a protein of interest. This is then processed by antigen presenting cells to enable its detection by circulating B lymphocytes, each of which expresses a unique antibody at its surface. Following target recognition, B lymphocytes undergo clonal expansion and secrete antibodies that can be purified from the serum. Antibodies produced in this way are described as being polyclonal since the resultant product comprises antibodies from multiple B lymphocytes that each recognize a distinct epitope.
As an alternative to this method, B lymphocytes can be isolated and used to generate hybridoma. By diluting these in appropriate culture media so that each well of a multi-well plate receives just one hybridoma, it is possible to test the cell supernatants and identify the best performing clones. Antibodies produced via this method are described as being monoclonal since every antibody in the final product is identical. An advantage of polyclonal antibodies is that they can provide signal amplification due to epitope redundancy, whereas monoclonals benefit from high specificity, consistent performance, and guaranteed long-term supply.
What are primary and secondary antibodies?
While primary antibodies bind directly to a specific antigen, such as a cytoskeletal protein or a cytokine, secondary antibodies are instead used for indirect detection. This means they bind primary antibodies only once these have recognized their target. Secondary antibodies are produced by immunizing an animal with an antibody from another species and are often used to provide signal amplification through binding of multiple secondary antibodies to each primary antibody.
Considerations for antibody selection
Factors to consider for antibody selection include whether to use a polyclonal or a monoclonal, whether direct or indirect detection is required, and what type of readout will be employed. While enzymes such as horseradish peroxidase (HRP) are typically used for colorimetric or chemiluminescent detection, fluorophores are preferred for multiplexed applications like flow cytometry or immunohistochemistry (IHC). Critically, antibody reagents should be proven to specifically recognize their target antigens without demonstrating unwanted cross-reactivities.
Why choose ICL?
With over 40 years’ experience in producing primary and secondary antibodies, right here in the USA, ICL is your trusted partner for both off-the-shelf and bespoke antibody products. Our customers include many of the world’s leading IVD manufacturers, pharmaceutical companies, and research institutions, who know they can rely on us for consistent high-quality products. With rapid customer service and troubleshooting available on demand, we’re here to help keep your project on track.