Immunohistochemistry (IHC) is a widely used technique to analyze the anatomy of a tissue of interest and to visualize the expression, localization, and intensity of a specific antigen. Although IHC is a well-established laboratory method with a long-standing history, there is a wide range of factors influencing its outcome. The quality of the staining may be influenced by several variables that need to be considered to produce reliable and consistent results, for instance, antibody selection.
Antibodies are indispensable for laboratories and represent an invaluable tool with multiple applications. Yet, antibodies are also at the nexus of reproducibility issues plaguing laboratories.1 While maybe not the leading cause, antibodies, and 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-plus suppliers available, it can be a daunting task to choose an antibody. However, time spent researching the best product to buy can save time and resources down the line and substantially reduce frustration. In this article, we offer guidance on how to select the right antibody according to the multiple variables.
Define your protein 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 protein target vary across science fields.
As such, make sure that you understand the biology of your protein 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.
Ensure the antibody suits your sample
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, it is recommended that you use antibodies that have been validated specifically for your species of interest. For the commonly used species, specific antibodies are generally available.
However, if you are 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 percent, there is a decent chance that the antibody might recognize your target. In these instances, it is important to validate the antibody’s performance and specificity for the given species.
Choose a compatible antibody
It would be ill-advised to assume that an antibody that has been shown to detect a target in one application will 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 denaturated protein. Thus, antibodies that work in Western blot with denaturated proteins might not work in immunofluorescence of frozen sections where antigens are present in their naïve conformation – and vice versa. Certain fixation methods might compromise an antigen, or permeabilization 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 is not 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.
Carefully select host species
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 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.
Understand how clonality can affect results
Monoclonal antibodies are produced by hybridoma cell lines, immortalized, cloned B-cells from immunized animals that are all genetically identical and, therefore, generate only one specific antibody of a fixed sequence and structure. Common host animal species include rabbit and mouse. The resulting antibodies display greater specificity towards one particular antigen epitope. Hybridoma cell lines may provide less variation in reactivity between different lot productions.
An advantage of using monoclonal antibodies is the greater likelihood of reproducing results as a consequence of less variation between different lot productions. Greater mono-specificity also provides decreased reactivity with other antigens, resulting in reduced background staining. However, a natural consequence of this mono-specificity is a less robust sample signal compared to polyclonal antibodies. Protocols that result in subtle shifts in epitope structure can potentially have drastic effects on staining.
Choose a compatible antibody
It would be ill-advised to assume that an antibody that has been shown to detect a target in one application will 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 denaturated protein. Thus, antibodies that work in Western blot with denaturated proteins might not work in immunofluorescence of frozen sections where antigens are present in their naïve conformation – and vice versa. Certain fixation methods might compromise an antigen, or permeabilization 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 is not 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.
Carefully select host species
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 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.
Understand how clonality can affect results
Monoclonal antibodies are produced by hybridoma cell lines, immortalized, cloned B-cells from immunized animals that are all genetically identical and, therefore, generate only one specific antibody of a fixed sequence and structure. Common host animal species include rabbit and mouse. The resulting antibodies display greater specificity towards one particular antigen epitope. Hybridoma cell lines may provide less variation in reactivity between different lot productions.
An advantage of using monoclonal antibodies is the greater likelihood of reproducing results as a consequence of less variation between different lot productions. Greater mono-specificity also provides decreased reactivity with other antigens, resulting in reduced background staining. However, natural consequence of this mono-specificity is a less robust sample signal compared to polyclonal antibodies. Protocols that result in subtle shifts in epitope structure can potentially have drastic effects on staining.
Polyclonal antibodies in contrast are extracted directly from the blood of the host animal, and not from cultured B-cells. As multiple different B-cell clones become activated, the resulting polyclonal antibodies are a mix of antibodies of different structure and binding affinities and specificities, including off-target antibodies. Downstream purification steps can increase the specificity of a polyclonal antibody mix (see below). The advantage is that antibody production is streamlined, and a wider variety of host species can be used, as the immortalization and culturing of B-cells can be omitted. These host species include horse, donkey, pig, mouse, and rabbit, but also less-common animals – from chicken and guinea pig to camel.
Due to polyclonal antisera – including multiple antibodies from multiple B-cell clones – there is a greater potential to recognize differing epitope configurations. Subsequently, this also means that one should expect greater variation between different lot productions. An inherent advantage of using polyclonal antibodies is greater signal detection due to the multiple epitope configurations that can be recognized. There is also a greater tolerance for changes in experimental conditions that could possibly induce changes to epitope structure. However, greater epitope recognition may also result in more non-specific staining that results in higher background signal. Polyclonal antibodies may show lower chances of reproducing experimental results as a consequence of greater lot-to-lot variation.
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, consisting of other immunoglobulins and proteins might be detrimental and purified antibody products are generally preferred. Polyclonal antibodies require more thorough purification. Due to their production methods, monoclonal or recombinant antibodies are 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. The 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 is affinity-purified antibodies that have been selectively purified for their ability to bind the target antigen.
Look for (independent) validation
An antibody should ideally be validated by the supplier for at least your protein target, species and general application type, but it is impossible for any supplier to validate all conceivable applications and protocols. 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.
Consider detection method and system
All immune-mediated techniques rely on the means to detect the antibody that was used to detect and identify the protein target of interest. A variety of detection methods and systems are available but should be carefully chosen. The 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.
Technical support and return policy can save the day
After you have run into severe experimental challenges, a good technical service might be invaluable. The supplier’s experts might also be able to assist you should you face difficulties deciding which antibody to buy or whether a given antibody might suit your needs. Do not hesitate to contact the supplier’s tech support before buying an antibody. This is a way to not only get invaluable help and insight but also to separate the wheat from the chaff of suppliers.
Be prepared to validate on your own
Even the best-characterized antibody that is beloved by the scientific literature and has enriched many publications is not guaranteed to perform in all settings flawlessly. You 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.2 An increasing number of journals not only require detailed information on the antibodies used in a study (such as name, clone, supplier, catalog and even lot number) but also encourage 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 user’s responsibility to use proven experimental tools.
References:
- Baker, M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521, 274–276.
- Bordeaux, J. et al. Antibody validation. Biotechniques. 2010; 48(3), 197–209.
