Choosing the right antibody for the intended application and sample type is critical to producing reliable results
Antibodies are essential tools for applications ranging from flow cytometry, immunocytochemistry (ICC), and immunohistochemistry (IHC) to Western blotting, ELISA, and immunoprecipitation (IP). Yet finding an antibody that works for a specific technique and sample type can be a daunting task. Not only must researchers assess the product offerings of hundreds of vendors, paying close attention to validation and quality control data, but they must also scrutinize the published literature to ensure the antibody they have chosen is the right one for the job. With the reproducibility crisis increasing pressure for high quality antibodies, vendors and researchers are united in their quest for greater transparency when it comes to reagent selection and use. This article shares some tips for choosing antibodies to help safeguard the accuracy and consistency of experimental results.
Sources of antibody variation
Antibodies are inherently variable in terms of how they will perform in a research setting. A main reason for this is that traditional antibody production methods harness an animal’s immune response, which differs significantly between individuals and will also be influenced by the choice of immunogen. While some proteins are incapable of eliciting a strong immune response, others are too similar to off-target biomolecules to yield a specific antibody reagent. Antibody performance also hinges on the types of validation and quality control carried out by the manufacturer. For example, where large quantities of purified protein are used in place of physiologically relevant material as positive controls, it is impossible to know how an antibody will behave when used with real samples. Although there are currently no enforced standards in place for verifying antibody performance, many vendors now adhere to the five pillars of validation, first proposed in 2016, as a means of ensuring reliable data in common research applications.
Tips for antibody selection
With so many antibody reagents available, it can be difficult to know where to begin when selecting a suitable product for your research. Here are a few suggestions to make things easier:
- Use search tools to find and compare different antibodies
Using a search tool for antibody selection can save vast amounts of time spent trawling individual vendor’s websites, as well as can introduce previously unfamiliar suppliers. FluoroFinder’s Antibody Search comprises over 3 million antibodies from all major vendors and includes an integrated spectra viewer to simplify experimental design where detection will be fluorescence-based. Simply input your target of interest, then refine your search based on factors including host species, antibody clone, validated applications, supplier, and available fluorochrome conjugates.
- Check that antibodies are validated for the chosen application
The only way of knowing whether an antibody will work in a particular application is to see proof. Antibody vendors’ product datasheets usually list validated applications, along with supporting images, which should each be accompanied by a comprehensive legend. When reviewing vendor data, it is important to check that relevant controls were used and that the validation dataset is complete –a detailed experimental protocol should be provided and images should not have been cropped or artificially manipulated. Likewise, when looking at published data, it is sensible to focus on comparable studies and to confirm that the authors verified antibody performance.
- Choose vendors who are willing to help troubleshoot any issues
A vendor who does not offer to provide technical support, if required, should ring alarm bells; this could indicate that they have either not validated the antibody in question or that they are selling another vendor’s products without performing any type of in-house quality control. In contrast, vendors who are willing to help troubleshoot if an antibody does not perform as expected are more likely to have deeper knowledge of a product and its antigenic target.
- Consider antibody clonality
Polyclonal antibodies recognize multiple epitopes, meaning they have traditionally been preferred for detecting scarce targets or proteins exhibiting heterogeneity in structure or sequence. Monoclonal antibodies instead bind only a single epitope and are often favored for their high specificity. Recombinant antibodies are defined by a specific protein sequence and are increasingly popular across all research applications due to their guaranteed consistency and capacity for engineering. These features and benefits of different antibody types should all be considered during reagent selection.
- Select antibodies from appropriate host species
Host species is a key consideration where labeled secondary antibodies will be used for indirect detection. In applications such as IHC, primary antibodies should be sourced from a different host species to the sample material to avoid secondary antibody binding to endogenous proteins. Cross-adsorbed secondary antibodies are recommended here, as well as for sandwich ELISA to prevent unwanted secondary antibody binding to the antibodies used for target capture. If antibodies from several host species will be combined in the same experiment, using secondary antibodies that are validated for detection of only a specific antibody class or subtype can increase panel flexibility.
- Check the antibody formulation
Antibody reagents are often formulated to include additives such as BSA, glycerol, or sodium azide. While these are not usually problematic, they should be avoided if the antibody will be labeled in-house using a commercially available kit. Some vendors offer additive-free versions of popular antibody products. Alternatively, additives can be removed via dialysis or using dedicated products; however, this approach can reduce the antibody’s concentration and limit its performance.
Supporting your research
FluoroFinder has developed a range of tools to simplify both antibody selection and panel design. Our Antibody Search function is linked to our Spectra Viewer, which allows you to quickly compare over 1,000 fluorophores from all suppliers in one intuitive platform, while our Panel Builder complements these by letting you view the fluorophore and antibody offerings of >60 suppliers in a single resource. And don’t forget, if you need additional guidance, our technical support team is always happy to help.
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