Q&A Part 3: Industry Leaders Give Recommendations on Selecting and Validating Antibodies

While validation images and publication data are very important and must be reviewed when selecting an antibody, my key recommendation is that scientists also research the companies that they purchase antibodies from. Find out if they are a manufacturer-direct, a manufacturer that sells direct to their customers without any rebranding middle-man who often can’t or won’t take responsibility for the quality control of the product. A good antibody company will be transparent and show all validation images whether or not they were performed in-house or by another source. Buyer beware if a company does not disclose antigen information as the scientist is not purchasing from a transparent company and may have reproducibility problems with this product.

Today, more than ever, researchers must be able to discriminate high-quality antibody manufacturers from other providers that take risky validation shortcuts, or worse, do not perform validation of any type, especially in a global marketplace. Researchers should look for manufacturers that transparently convey antigen, testing and release data to the researcher, including in which assays the antibody is demonstrated to show acceptable performance. Manufacturers should report either on their website or on a certificate of analysis, the nature of the immunogen, screening criteria during antibody development, and lot-specific validation data. Researchers should consider any vendor not presenting this information as a red flag or warning concerning the potential performance of an antibody. Researchers must carefully review this information and, when necessary, independently validate an antibody before commencing with experimental research.

Manufacturers of high-quality antibodies participate in scientific meetings and interact with research scientists at every opportunity. This contact allows all manufacturers to adapt and change as the needs of researchers for novel antibodies and validation methods likewise change. Other companies that do not keep current with validation methods or data transparency may be providing astute users an insight into their approach to manufacturing, reselling and quality.

Always look for the inclusion of the appropriate positive and negative controls in validation experiments and discount the value of truncated data, for instance, the digital cropping of a Western blot image to only the molecular weight range of the target protein. When data of this type is presented the researcher has no way of knowing the degree of off-target binding for the antibody, which may be why the image was cropped.

When scientists are selecting antibodies to use in their particular experiment, a number of considerations must be taken into account:

• Try to find an antibody that fits the applications and species reactivity required. Often it is tempting to try to pick an antibody that does everything, but sometimes this does not provide an antibody that works the best in the most important applications.
• Over what period of time is the antibody going to be needed? If the antibody is going to be needed over many years with multiple purchases, then you may want to consider finding a monoclonal. While polyclonals can be excellent reagents, there is a slightly higher chance the antibody properties can change over time.
• How has the antibody been validated? Does the supplier show robust validation data verifying the applications for which they claim the antibody works?
• What experimental models have been used to validate the antibody? Have the appropriate cell line(s) been used? Are the proper experimental controls used?
• Has the antibody been used in publication? How many other people have used the antibody successfully to answer relevant biological questions?
• Has the antibody been confirmed to be specific? Has the supplier shown appropriate controls to confirm the antibody is detecting the correct protein of interest? Even better, has the antibody been shown to be specific by one of the IWGAV specificity pillars?

First, it is important to select an antibody that has been tested in the same application(s) the scientist is going to use it for, if possible. We recommend studying the validation data available on the supplier’s data sheets and the recommended protocols to evaluate which antibody best suits the intended purpose.

The product data available at Atlas Antibodies’ website includes both result images for each approved application, and we also present the exact immunogen used to raise each antibody. The immunogen sequence allows the end user to know where on the target protein the antibody binds and whether the antibody will recognize all isoforms or not.

The performance of an antibody depends on the assay used, the sample material, how the sample has been treated, if the target protein is in a native form or has been altered through, for example, antigen-retrieval methods. Therefore, it is necessary for the scientist to validate the antibodies in their specific analysis set up and include suitable positive and negative controls.

When selecting an antibody, one piece of advice is to understand the provenance of the antibody you use. There are estimated to be two million research antibodies out there, from several hundred sources. It is often not feasible to buy all antibodies for a target of interest and test them side-by-side. One common approach researchers take when selecting antibodies is to follow the literature. However, these reagents may simply be the first available, and may not actually be the best reagent to use. Many studies use improperly validated, low-quality reagents, or antibodies that are later shown to be nonspecific. Our advice would be to select antibodies from a reputable vendor that has been demonstrated to be fit-for-purpose in the application and sample type of interest. We provide over 17,000 sample size antibodies to enable economical testing.

After selecting an antibody, we recommend performing proper context-specific validation of the antibody for the research study in question. Following the vendor’s guidelines in antibody usage is a good start; it would surprise you how many researchers don’t follow the instructions provided. We would also recommend use of caution when employing an antibody in an application that is not recommended by the supplier. An antibody that has been shown by a vendor to work beautifully in Western blot, but has not been shown to work in other techniques, would require validation on the customer side for that new application. We recommend the use of proper positive and negative controls. There are different ways to validate an antibody depending on the research question and target type (e.g. transcription factor, cytokine or a PTM). Therefore, there isn’t a one-size-fits-all approach to antibody validation. Don’t forget that vendors like Bio-Techne provide expert technical support and you can have contact with the team or scientist who produced an antibody.

My recommendations are: first, when purchasing an antibody always go to the highest quality providers. If you are new to the field, ask your colleagues. Second, look for validation data supporting the use of an antibody in the specific application of interest (Western blotting, IHC, etc) provided by the vendor. Look at the data critically as if you were reviewing a manuscript. Third, look for data supporting the use of the antibody in the desired application within the peer-reviewed scientific literature. Publications are not perfect, but I believe well-published antibodies are a good place to start. Fourth, try to strictly follow the vendor’s protocol, if available, before you introduce experimental variations. Finally, if you end up buying an antibody for which no data is provided and there are no published citations available, beware. You should first determine its specificity using as many application–specific positive and negative controls as possible.

We are like them; we are strong advocates for proof of performance, whether it is through a peer-reviewed journal or data provided by a supplier with a reputation for quality and customer service. When in doubt, one should be able to ask an adviser who can help. Organizations with a strong and engaging technical support staff are a good source for purchasing the right product the first time.

The scientist should definitely confirm when they are purchasing an antibody from a commercial vendor that the antibody has been validated for the intended application. It is always advisable to check the data on the vendor’s site and when in doubt one should request further validation data by the vendor. Once bought, the antibody might need further validation depending upon the context of the researcher’s application. The recently published article in Nature methods and the upcoming guidelines from GBSI could be a good start to validate the antibody on the user’s/researcher’s end.

Scientists should first critically review any data provided by the supplier for the applications in which they intend to use the antibody. In the case of Western blotting data, scientists should look for antibodies that detect the endogenous target protein in the appropriate samples (i.e., only in cell or tissue lysates that are expected to express the target protein). No matter how good the data looks, scientists should then retest the antibody in their lab using both positive controls and negative controls. In order for scientist to easily replicate the supplier’s experiments, scientists should look for suppliers that provide sufficient information on the antibody, including physical properties (e.g., host species, clonality, purification) what target it recognizes and the protocol used to test the antibody.

When selecting an antibody from a commercial source, there are some basic practices to follow:

• • Source antibodies from a reputable company with a well-established quality system.
  • Look for example data generated with real samples.
  • Make sure the product you are buying passed a functional QC assay, in addition to the more common biochemical checks (e.g. purity, MW, concentration).
  • Carefully compare the demonstrated performance of the antibody in relevant assays to that shown for other available antibodies. If no data is shown, then ask the vendor to provide some. If they can’t readily provide data, then you should be very wary.
  • Look for publications that utilized the particular antibody. Companies should try to make these readily available.
  • Look at reviews from independent third parties. Many websites, such as Biocompare, SelectScience and AntibodyResource post product reviews by end-users.

When validating an antibody:

• Always follow manufacturer’s recommendations.
• Employ a relevant model system to test the antibody.
• Use ample controls, both for the target (e.g. negative cell line) and for the reagent (e.g. isotype controls that are modified in the same fashion as your antibody).
• Compare to other similar antibodies if available.
• If something looks amiss, contact the supplier’s Technical Service and share your data with their scientists. We consider having expert scientists to assist customers with trouble-shooting or experimental design to be a key part of helping our clients achieve success.

When looking to purchase an antibody, scientists can increase their chances of buying the best product for their needs if they follow some simple suggestions. First, try not to let price dictate choice. Antibodies are expensive reagents, but low-priced antibodies often have little supportive data on company websites. And though it is always wise to shop around, it is generally better to spend more for a well-validated antibody as it will likely prove more economical in the long run by minimizing waste of researcher time, materials and money, not to mention by eliminating artifactual results. Second, researchers should carefully read the datasheet. Antibodies are frequently application- and species-dependent. This type of information is almost universally listed on datasheets. Researchers should verify that the application and species of interest to them have been directly tested. If one doesn’t see data supporting use in one’s intended application or species, a call to the company should be placed. In addition, there is usually information about the sequence of the immunogen used to generate the antibody. This can be crucial when trying to determine how specific an antibody will be for one member of a protein family with high sequence identity among its members. If it is not listed on the datasheet, a call to technical support can often help identify the immunogen sequence. Third, most scientists learn of reliable reagents through publications and word-of-mouth. It is advisable to check publications to see what antibody is being used to detect a given target. There are often specific monoclonal antibody clones or polyclonal antibody products that are widely accepted for detection of particular targets. Finally, always carefully review the vendor data and look for antibodies whose specificity has been validated through knockdown or knockout technologies.

The presence of a single band at the expected molecular weight on a Western blot is a great start but it is not sufficient for antibody validation. Combining several validation strategies is the most effective way to demonstrate antibody specificity and validate performance. For example, it is important to demonstrate antibody specificity and selectivity for endogenous levels of target protein expression; purified or overexpressed target protein should not be used for validation. You can incorporate knockdown or knockout strategies to decrease the abundance of your target, or treat cells with growth factors or chemical compounds that induce or inhibit expression of the target. A blocking peptide that contains the epitope recognized by your antibody can be used to prevent binding to the target protein. And you should always record the antibody name, source, catalog or clone number, lot number, host species, immunogen, and other information that may be needed later.

Selecting the right antibody is a complex process and we recommend careful review of the following considerations based on your experiment:

• The species of the sample: Human, mouse, rat, virus or other;
• The nature of the sample: Protein/peptide, whole organisms: cell or bacteria. You need to know as much as possible about the target protein expression and cell type, distribution, and quantity;
• The species of primary antibody host: Normally, the antibodies are raised from mouse or rabbit. Then a mono- or poly-clonal antibody corresponding with the experiment requirements is chosen;
• The applications verified by the supplier: Because of the complex nature of biological samples, one antibody can not be used for every technique. We can only guarantee the applications approved for each product.
• The secondary antibody: Antimouse or antirabbit secondary antibodies are raised from different species than the primary antibody. Note that these anti-IgGs should not cross-react with the sample;
• The proper fluorochrome and chromogen labels: Scientists should take into consideration the mechanism and settings of equipment detection.

There are many tips for antibody-based experiments. Choosing the right antibody, following good protocols, and paying close attention to detail can make antibodies a reliable tool for biological research.