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Top 10 Tips for Choosing an Antibody

Hartmut Pohl
Tags: Successful Research Tips

Antibodies have transformed life sciences and many other scientific disciplines by allowing detection, quantification and capture of molecular targets that otherwise would remain unfathomable. Since Rosalyn Yalow and Solomon Berson developed the first immunoassay in 1960 to quantify insulin in blood plasma, a plethora of antibody-based applications have emerged and shaped scientific research. Antibodies have also drastically changed and continue to reshape the therapeutic landscape, from established diagnostic applications relying on antibody detection to modern di- or trispecific antibody-drug-conjugates for precise, targeted drug delivery. In fact, perhaps the very first use of antibodies was therapeutic, when Emil von Berg demonstrated in 1890 the use of serum therapy – a discovery that initiated the interest in serum components and paved the road to the discovery of antibodies and their modern day uses.

In essence, antibodies have become indispensable for state of the art research and represent an invaluable experimental tool. Yet, antibodies are also at the nexus of reproducibility issues plaguing modern life sciences. Though maybe not the leading cause, antibodies, and experimental reagents as a whole, are a common source of error and inaccuracy fueling the reproducibility crisis. It is therefore crucial to choose the right antibody from the start. For rare targets, it might be difficult to find an antibody at all, while for widely studied proteins, there might be thousands of antibodies to choose from. With an estimated total of 4.5 million commercial antibodies from 350+ suppliers available, it can be a daunting task to choose an antibody. However, time spent up-front to research the best product to buy can save time and resources down the line and substantially reduce frustration. Antibody comparison sites (e.g. Antibodies-Online, Antibodypedia, Antibody Resource, BioCompare, Labome) are a great way to get an overview, but you might still need to know what you are looking for.

Here are our top 10 tips to aid you in your decision:


  1. Define your target of interest

    Protein targets can be highly complex. For any given protein, a variety of names, abbreviations, isoforms, splice variants and in vivo modifications might exist, and sequence identity or homology with closely related proteins pose additional challenges in the form of potential cross-reactivity. Furthermore, cellular binding partners might mask the antibody’s binding site. To add to the confusion, sometimes the commonly used names for a given target vary across research fields. Make sure that you understand the biology of your target and the capabilities of any antibody in question to detect your target under the chosen conditions. Use genetic and protein databases, such as UniProt or GeneCards, to study your target and obtain unique identifiers. If your research depends on the antibody recognizing a specific portion of your target, make sure that the epitope the antibody was raised against is known and within the required domain.


  2. Ensure that the antibody suits your sample type and species

    Targets vary in their sequence and structure from species to species. Unless the datasheet specifies that the antibody has been validated for a species, there is no guarantee the antibody will work. Therefore, we recommend you use antibodies that have specifically been validated for your species of interest. For the commonly used research species, specific antibodies are generally available. However, if you’re using exotic species, you might be out of luck more often than not. But despair not. Often antibodies are raised against relatively preserved domains within target proteins, and even if sequence homology of the epitopes is as low as 75%, there is a decent chance that the antibody might recognize your target. You will just have to validate the antibody’s performance and specificity for the given species. Furthermore, expression levels of target and closely-related off-target proteins might vary with species, tissue or cell type, and alternative splice variants or different post-translational modifications might influence an antibody’s performance. Ideally, an antibody has already been shown to work for your sample types and conditions.


  3. Choose an antibody suited for your application

    It would be ill-advised to assume that an antibody that has been shown to detect a target in one application would do so under other circumstances. A given antigen might for example require that the target protein is present in its native, folded form or might only be accessible in a denatured protein. Thus, antibodies that work in western blot might not work in immunofluorescence of frozen sections and vice versa. Certain fixation methods might compromise an antigen, or permeabilisation or dissociation steps might be required to make an antigen accessible for an antibody. For example, an antibody detecting a membrane target in immunocytochemistry might not work in flow cytometry, simply because the target antigen isn’t on the cell’s surface.

    It is often preferable to buy a couple of antibodies with proven, outstanding performances for specific applications over a single antibody that supposedly does it all without any proof that backs up this claim.



    Figure 1: Common Applications for Antibodies.


  4. Carefully select host species, antibody type and clonality

    There’s good reason that a variety of antibody species, types and clonalities exist – they all offer different advantages and pitfalls. When choosing a host species, it might be worth it to have a look at available secondary antibodies as well as potential combinations for multiplexing. It is generally recommended to avoid antibody host species that are identical with the species of your target samples to avoid interference with immunoglobulins present in the sample – although workarounds do exist. Furthermore, the usage of different types of immunoglobulins (IgG, IgM, IgE etc.) might offer advantages in cross-reactivity and multiplexing.

    Polyclonal antibodies are a mix of different antibodies of varying sequences and recognize different epitopes on a given antigen. This offers benefits in signal strength and broadness of detection, but often limits specificity. Additionally, lot-to-lot variation might be considerable, depending on how the respective manufacturer defines a lot, and the availability of a polyclonal antibody in the future might be limited. Monoclonal antibodies are homologous and all antibodies share the same structure and target specificity, but the antibody’s sequence as well as the exact epitope it binds to might be unknown. A given clone identity will ensure comparability and consistency of the data. Recombinant antibodies cloned into and expressed in host cells are an additional step up in reliability, as the antibody’s DNA sequence is defined. There is no ideal type of clonality for all given applications, but monoclonal or recombinant antibodies are more often than not preferable.



    Figure 2: Commonly used, naturally occurring immunoglobulin types for experimental settings, immunoglobulin fragments and recombinant antibody types.



  5. Check formulation and purification

    Antibodies come in a variety of formulations and various degrees of purification. The simplest presentations are probably neat serum, ascites fluid or cell culture supernatant. While they are easily produced, readily available and often of low cost, their content of other immunoglobulins and proteins might be detrimental and purified antibody products are generally preferred. Polyclonal antibodies require more thorough purification. Monoclonal or recombinant antibodies are due to their production methods of higher purity, but even those may without purification steps contain unwanted immunoglobulins, if the cell cultures contained undefined serum.

    Antibodies might be purified by a variety of methods. The simplest method is some form of physiochemical fractionation to isolate a protein fraction that is highly enriched in immunoglobulins, but may contain other proteins. Most common is probably a purification step for immunoglobulins, regardless of their specificity, such as using the specific binding of protein A to immunoglobulins. The gold standard are affinity-purified antibodies that have been selectively purified for their ability to bind the target antigen.


  6. Look for (independent) validation

    An antibody should ideally be validated by the supplier for at least your target, species and general application type, but it is impossible for any supplier to validate all conceivable applications and protocols. Thus, chances are high that your desired experimental settings are not covered precisely. Literature references are a great source of validation. Finding the right antibody can be difficult and costly. Suppliers may offer solutions should you want to challenge an antibody with new applications or end up with unsatisfactory results. Enzo Life Sciences offers a no-risk antibody trial program. Evaluate our antibodies in new applications or species not listed on our data sheet. If the antibody does not give you usable results, we will issue you a credit!


  7. Consider detection method and system

    All immune-mediated research techniques rely on means to detect the antibody used to detect and identify the target of interest. A variety of detection methods and systems is at the researcher’s disposal, but should be carefully chosen. Most commonly used is indirect detection with conjugated secondary antibodies. Here, a polyclonal antibody that reacts with immunoglobulins of the host species of the primary antibody is used for detection. The secondary antibody is covalently conjugated with molecular means of detection. These might be fluorochromes for fluorimetric detection, enzymes that can be used to catalyze chromogenic or luminescent reactions, or molecular binding moieties such as biotin for subsequent binding with (strept-)avidin-based detection reagents. The complexity of detection systems is basically limitless. Exotic conjugates include gold particles for electron microscopy or radioisotopes.

    But indirect detection, while often drastically increasing sensitivity and allowing a significant degree of flexibility, might not be suited for all applications. Sometimes a direct link might be preferred, for example in flow cytometry or immunoprecipitation. Flow cytometry relies on fluorescently labeled primary antibodies, which often limits the selection of suitable antibodies considerably.


  8. Think about suitability for multiplexing

    There are two independent factors dominating the suitability of a given antibody. For direct detection, the conjugate is paramount. In flow cytometry for example, the fluorescent conjugate and its spectral specificity defines if two or more primary antibodies might be used in combination. If indirect means of detection are being used, the species and sometimes the class of antibody are the defining factor. Primary antibodies raised in distinct species can often be easily identified and their signal be separated by respective secondary detection systems. Smart combination of species and/or immunoglobulin classes will enable sophisticated multiplexing.


  9. A supplier’s technical support and return policy can save the day

    Not only after the milk is spilled and you’ve run into severe experimental challenges might a good technical service be invaluable. The supplier’s experts might also be able to assist you, should you face difficulties to decide which antibody to buy or whether a given antibody might suit your needs. Don’t hesitate to contact tech support before you buy. This way you might not only get invaluable help and insight, but you might also be able to separate the wheat from the chaff of suppliers.


  10. Be prepared to validate on your own

    Even the best characterized antibody that is beloved by the scientific literature and has enriched many a publication isn’t guaranteed to perform in your settings and your hands flawlessly. And you would limit your resources severely if you only consider antibodies validated exactly for your needs. You should always plan to validate an antibody on your own to some extent. Include the appropriate controls in your experiment to analyze the specificity of an antibody in your precise circumstances. A useful guide for very thorough antibody validation has been compiled by Bordeaux and colleagues. An increasing number of journals not only requires detailed information on the antibodies used in a study (such as name, clone, supplier, catalogue and even lot number), but is also encouraging proof of validation. It is in the interest of us all that scientific data generated with the help of antibodies is reliable and reproducible, but ultimately it is the researcher’s responsibility to use proven experimental tools.

Enzo Life Sciences offers a comprehensive portfolio of antibodies, with thousands of monoclonal, polyclonal or recombinant antibodies. We provide a broad variety of antibodies for immunology, neuroscience, cancer and many other fields. Our antibody range covers most applications including flow cytometry, immunohistochemistry and Western blot and are also worry-free. Additionally, we offer an expansive selection of ready-to-use immunoassays and ELISAs. For further insights into antibodies or the methods they are applied in, please have a look at our other TechNotes on successful research tips or immunohistochemistry. Or contact our Technical Support Team for further assistance.

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