Finding a Good Antibody:
Part I — Selecting the Best 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.

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.

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.

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.

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.

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.

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.