Antibodies are essential for many different applications. It is vital to choose antibodies carefully. This article provides a review of the main antibody types available to researchers and suggests some key factors to consider during antibody selection.

Polyclonal or monoclonal?

The functional difference between polyclonal and monoclonal antibodies is the number of epitopes within the target protein the antibodies will recognize. While monoclonal antibodies are specific to a single epitope, polyclonals are a heterogenous mixture of antibodies targeting multiple epitopes. This can provide signal amplification, increasing the likelihood of detecting scarce targets. Monoclonals however, harness their specificity to offer more consistent results across experiments.

The properties of each antibody type are summarized in the following table.

 Polyclonal antibodiesMonoclonal antibodies
Key features Host: rabbits, sheep, goats, or donkeys Host: mice, rats, or rabbits
  Purified directly from the blood Produced using hybridomas
  Source: Heterogeneous pool of B cells Source: Single pool of B cells
  Recognize multiple epitopes within the target protein Recognize just a single epitope within the target protein
Advantages Multiple epitope recognition can provide signal amplification Can select for the best-performing clones
  Likely to function across a broad range of applications due to epitope redundancy Improved specificity compared to polyclonal antibodies
  Often available sooner when a new target is first discovered (e.g., SARS-CoV-2) More consistent lot-to-lot performance
  Simpler production method may equate to lower cost No requirement for further animal use following initial production
    Renewable source of material
Disadvantages Available in only limited supply, corresponding to the lifetime of the host Small peptide antigens may not always generate a sufficient immune response in mice
  Exhibit lot-to-lot variability that can require re-optimization of experimental conditions May exhibit genetic drift over time
  May demonstrate unwanted cross-reactivities if not affinity purified  
  Require multiple rounds of animal use  
When to use When you wish to investigate a newly discovered target When it is important that your antibody binds a specific region of the target protein (e.g., a region far from a ligand binding site)
  When your protein target is scarce When it is essential to reduce the risk of off-target binding effects
  When your protein target has multiple configurations or post-translational modifications When lot-to-lot consistency is critical
  When epitopes may be masked by cross-linking (IP) or fixation (IHC) When you need a large quantity of antibody for your experiment, over an extended time
  When you would like to use the same antibody across multiple applications

 

Recombinant antibodies

Recombinant antibodies offer further refinement compared to naturally occurring polyclonal and monoclonal antibodies as their genetic sequence is both known and lab controlled. This allows researchers to enhance the specificity of the antibody with directed mutations. It also allows for mass-production of the antibody using traditional protein expression techniques without concerns for genetic drift. In recent years, recombinant antibodies have become increasingly popular for their reproducibility.

Antibody host species, cross-adsorption, and multiplexing

Host species is another important factor to consider when choosing antibodies, especially where secondary antibodies will be used for indirect detection. It is vital that secondary antibodies do not cross-react with the target tissue or the host species of the primary antibody, as this can yield false positive results. Cross-adsorbed secondary antibodies have been screened to remove those that would cross-react, making them an attractive option.

Antibody host species is also relevant to multiplexing, where samples are often stained with primary antibodies from different species (and/or of different isotypes) prior to detection with a cocktail of secondary antibodies. In this situation, it is recommended to avoid using antibodies from closely related species like mouse and rat or goat and sheep, and to confirm that all antibody reagents have been rigorously validated. Alternatively, using labeled primary antibodies both shortens workflows and eliminates the risk of secondary antibody cross-reactivities.

Modification-specific antibodies

Proteins frequently undergo post-translational modifications (PTMs) that regulate their function, such as phosphorylation, methylation, acetylation, or glycosylation. Often, these are detected with modification-specific antibodies (polyclonal or monoclonal) that do not recognize the unmodified target protein. By using antibodies specific to particular modifications, researchers can combine experiments with “total” antibodies to measure a ratio of modified to total protein levels. This enables semi-quantitative measurement of the many PTM marks that enzymes place on proteins.

Aviva Systems Biology offers an extensive collection of high-quality antibodies, including antibodies to many targets that are not commercially available elsewhere. To learn more, visit avivasysbio.com

About the Author

Emma Mason is the founder and director of Cambridge Technical Content Ltd, based in the U.K. Since graduating with a bachelor’s degree in biology from the University of Kent at Canterbury in 2000, she has gained extensive experience developing and running immunoassays within companies including Millennium Pharmaceuticals, AstraZeneca and Cellzome. She now produces a wide range of scientific content, including regular features for Biocompare.