Introduction
Use search engines to find and compare available antibodies
Match the antibody type to your application
Buy from companies that will work with you
Look for antibodies with complete validation data
Select antibodies that have been validated for your application
Check to ensure additives are compatible with your application
Review publications, but carefully scrutinize antibody data and references

Introduction

Antibodies are widely used for applications that range from flow cytometry and immunohistochemistry to western blotting and ELISA. Even though antibodies are central to basic research as well as drug development and diagnostics, quality concerns remain high, and finding an antibody that works well for a specific application is a formidable challenge.

One source of the antibody quality problem is that it is not easy to generate a high-performing antibody. Production of monoclonal and polyclonal antibodies relies on an animal’s immune response, which is unpredictable and can vary from animal to animal even when they have the same genetic background. Some proteins do not elicit a strong immune response, others are too immunogenic, and yet others share too much homology with non-target proteins to yield a highly specific antibody. As part of the Human Protein Atlas project, Berglund et al. (2008) quantified their antibody production success rate; 49% of their 9,000 internally generated antibodies failed validation.

Part I — selecting the best antibodies to test for your application

The first step to finding an antibody can be the most daunting — identifying antibodies that could work for your application. Product information and validation data can be difficult to decipher, and with hundreds of vendors to choose from it becomes difficult to know when a search has been exhaustive. The following guidelines are meant to simplify the process of identifying high-quality antibodies.

1. Use search engines to find and compare available antibodies.

Search engines, such as those available through Biocompare, SelectScience, UniProt, or NCBI, allow you to find and in some instances even compare antibodies from many different vendors. This saves valuable time that is otherwise spent visiting each vendor’s website and allows you to extend your search to vendors you may not be familiar with.

Use search engines to find and compare available antibodies.
2. Match the antibody type to your application.

Antibodies fall into three classes, polyclonal, monoclonal, and recombinant, each with distinct advantages and disadvantages.

Polyclonal Antibodies

A polyclonal antibody is a mixture of antibodies that all recognize different epitopes of the protein of interest. This makes these antibodies well suited for proteins that may have posttranslational modifications or heterogeneity in structure or sequence, proteins present at low concentrations, or applications that require fast binding to a protein of interest. Because polyclonal antibodies are generated in animals, they show relatively high batch-to-batch variability and are thus a poor choice for long-running studies that require repurchasing of the antibody or for applications that have low tolerance for variability. If your experiments have low tolerance for variability, but only polyclonal antibodies are available, ask the vendor to provide antibodies from only a single lot.

Monoclonal Antibodies

Monoclonal antibodies are generated by a single B-cell line and thus recognize only a single epitope of a protein of interest. This makes these antibodies highly specific and results generated with them more reproducible. Their high specificity makes them an ideal choice for immunohistochemistry applications, and the ability to generate immortal B-cell hybridomas ensures greater batch-to-batch homogeneity. Because these antibodies recognize a single epitope they can be more challenging to work with when looking at low-abundance proteins or proteins that show variability, such as those with posttranslational modifications, in the epitope recognized by the antibody.

Recombinant Antibodies

One caveat of monoclonal antibodies is that immortal B-cell hybridomas are not as eternal as their name implies; cell lines can die, not recover from frozen stocks, or even lose their antibody gene. Thus, for applications that have no tolerance for variability, recombinant antibodies are recommended. These custom synthetic antibodies provide an unlimited supply of identical antibodies, removing any batch-to-batch variability. Recombinant antibodies can be engineered to bind an epitope of choice with much higher affinity than that obtained in vivo. Because large libraries can be screened in a high-throughput manner, antibodies can be generated that distinguish similar compounds and bind their ligands only under desired conditions, such as a specific pH.

The high reproducibility and entirely animal-free production process has led the pharmaceutical industry to adopt recombinant antibodies as their preferred tool. Many academics, on the other hand, understandably consider recombinant antibodies a last resort due to their higher cost. However, particularly for long-term studies, recombinant antibodies should be seriously considered due to their batch-to-batch consistency and their guaranteed continuity of availability without any dependence on animal immunization.

Match the antibody type to your application.
3. Buy from companies that will work with you.

Choose a vendor who is willing to help you troubleshoot if an antibody does not perform as expected. If a vendor is unable or refuses to do so it may be a sign that they did not validate the antibody or that they are selling antibodies purchased from another vendor without additional quality control or the expertise to advise customers. Avoid vendors who provide only generic troubleshooting advice as this will be of little use if problems are encountered and it suggests a lack of technical expertise.

Also be careful of what may at first glance seem like generous exchange or return programs — letting customers test multiple antibodies for their target of interest can indicate poor quality and turns you, the customer, into an antibody testing tool.

Buy from companies that will work with you.
4. Look for antibodies with complete validation data.

Be wary of incomplete validation data; this is often a sign that an antibody is of poor quality and/or that the vendor will be able to do little to help you troubleshoot if the antibody does not perform as expected. Look for vendors who show the entire western blot image, provide detailed validation protocols, and validate their antibodies using multiple biologically relevant sample types or tissues. Not only do multiple sample types speak to the ability of an antibody to detect varying levels of expression, they often also reveal sample types that can be used as negative controls for your experiments.

It is also important to carefully scrutinize a vendor’s validation data. If the positive control is merely purified protein, keep in mind that the specificity of the antibody remains unknown, since you are not looking at a complex biological sample. Make sure the vendor specifies how much protein was loaded and compare this amount to that expected in your sample. If your protein of interest is present in much lower amounts, you may still be able to use the antibody for some applications by enriching for your protein of interest through fractionation or IP.

Look for antibodies with complete validation data.
5. Select antibodies that have been validated for your application.

Whenever possible choose an antibody that is recommended by the vendor for your species and application. If such an antibody does not exist, contact the antibody vendor; in some cases the antibody may have failed validation for your application, while in other cases the vendor may not have tested it. You can also look to validation data in published studies to evaluate antibody performance. If no validation data are available for your application, choose a trusted vendor rather than one who simply states that antibodies have been validated for all applications. If you must use an antibody for non-recommended applications, be prepared to rigorously validate the antibody and to optimize vendor-suggested protocols for your specific experimental conditions.

Select antibodies that have been validated for your application.
6. Check to ensure additives are compatible with your application.

Vendors often include additives that stabilize and extend the shelf life of antibodies. For most applications this is unproblematic, but there are some notable exceptions. For example, sodium azide can interfere with HRP-conjugated antibodies, antibody conjugation, and staining of live samples. Similarly BSA should not be added to antibodies that you will conjugate because it competes with the antibody for your label and can reduce conjugation efficiency. Another common additive, glycerol, lowers the freezing point to below –20°C, preventing freeze-thaw damage at –20°C because the antibody does not freeze. This cryoprotection does not extend to –80°C; at this temperature even antibodies stored in glycerol will freeze and will thus be subject to freeze-thaw damage (Johnson 2012). If you are adding glycerol to an antibody yourself, be sure to use sterile glycerol as it is easily contaminated with bacteria.

When an antibody is not available without the interfering additive you may have to take steps to remove the additive or work with the vendor to see whether they can supply the antibody without the interfering additive. Additives can be removed through dialysis or by using commercially available kits. Keep in mind that these steps can reduce the antibody’s concentration and impact its performance.

Check to ensure additives are compatible with your application.
7. Review publications, but carefully scrutinize antibody data and references.

Journals like Nature and JBC are now starting to enforce guidelines for publishing antibody data, but this was not true in the past. When reviewing the literature, trust an antibody cited in a publication only if appropriate positive and negative controls are included. A new antibody should have validation data as well. When references are provided in place of validation data confirm that the authors of the original study performed and published the required validation experiments. If validation data are not presented in the original study, contact the authors to request this information. If authors cannot provide validation data, use the antibody only with the highest degree of caution and be sure to thoroughly validate the antibody before using it for your experiments.

Focus your literature search on studies similar to yours. An antibody that performs well for flow cytometry may not be a good choice for immunoprecipitation, and host specificity can vary greatly. As you review the literature, be wary of antibodies that show discrepancies, such as an antibody detecting proteins of different molecular weights or showing different protein expression patterns in the same tissue types in different studies. If an antibody detects a protein with an unexpected molecular weight, look for controls that validate that the protein detected is actually the target protein.

If authors show cropped western blots, contact them to request the full blot before you purchase the antibody. And if you struggle with a published antibody, don’t hesitate to contact the authors as they can often provide valuable troubleshooting information.

Review publications, but carefully scrutinize antibody data and references.

Tune in in June for Part II to learn how best to validate antibodies after you have purchased them and in July for Part III to see what information to include in publications to ensure that antibody quality and results can be evaluated by the reader.

For further reading on the issues facing researchers and what they and antibody suppliers can do to ensure proper antibody validation, read Validating Antibodies — the Good, the Bad, and the Necessary.

Bio-Rad’s Solution for Better Antibodies

Learn more about Bio-Rad’s solution for better antibodies with antibody validation

References

Berglund L et al. (2008). A genecentric Human Protein Atlas for expression profiles based on antibodies. Mol Cell Proteomics 7, 2,019–2,027.

Bradbury A and Plückthun A (2015). Reproducibility: Standardize antibodies used in research. Nature 518, 27–29.

Elliott S et al. (2006). Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood 107, 1,892–1,895.

Johnson M (2012). Antibody shelf life/how to store antibodies. Mater Methods 2, 120.

Prassas I and Diamandis EP (2014). Translational researchers beware! Unreliable commercial immunoassays (ELISAs) can jeopardize your research. Clin Chem Lab Med 52, 765–766.

Rosenblatt M (2016). An incentive-based approach for improving data reproducibility. Sci Transl Med 8, 336ed5.

Veronique Neumeister, Department of Pathology, Yale University School of Medicine, New Haven, CT
Poulomi Acharya and Anna Quinlan, Bio-Rad Laboratories, Inc., Hercules, CA

First published as: Acharya P et al. (2017). The ABCs of finding a good antibody: How to find a good antibody, validate it, and publish meaningful data. F1000Res 2017 6, 851.

Bio-Rad is a trademark of Bio-Rad Laboratories, Inc.

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