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A 3D render of a monoclonal antibody (mAb or moAb), which is an antibody made by cloning a unique white blood cell.
While antibodies are used prevalently, their use has been linked to false scientific results, causing a reproducibility crisis in science.
iStock, Naeblys

How to Make Your Antibodies Work for Your Lab

Not all antibodies work. Learn how to optimize them

Andy Tay, PhD

Antibodies are a common lab consumable used in diverse applications including flow cytometry, immunohistochemistry, and western blot, and as clinical therapeutics. They are produced by B lymphocytes upon exposure to molecules known as antigens, such as proteins and carbohydrates, like glycans. While antibodies are used prevalently, their use has been linked to false scientific results, causing a reproducibility crisis in science. A study in 2008 found that fewer than ~6,000 routinely used commercial antibodies are specific to their targets. Poorly characterized antibodies might have also led to poor replication of landmark preclinical studies, costing the biomedical research community an estimated $530 million per year in the United States. 

When an antibody is not specific to its target, a cell type could be wrongly identified by its surface protein expression, causing a misreporting on its cellular functions. In addition, when an antibody binds to multiple targets in a non-specific manner, it can also lead to confusion on mechanisms that adversely affect downstream drug discovery. Here, we will first discuss some common applications of antibodies before sharing tips to optimize them for use in labs.

Application of antibodies

Antibodies can be used to label a variety of biomolecules. Western blot is a technique that is used to detect the presence and compare relative amounts of a target protein in a protein mixture. The protein mixture is first loaded into polyacrylamide gel and separated using electrophoresis by their molecular weights. Primary antibodies that bind to the target proteins are added before adding fluorescence molecule- or enzyme-conjugated secondary antibodies that bind to the primary antibodies. Fluorescence molecule- or enzyme-conjugated secondary antibodies subsequently emit and amplify signals, such as through breaking down substrates for detection via methods like fluorescence, colorimetry, or radioactivity.

Antibodies are also useful for immunohistochemistry such as in situ tissue staining to localize the spatial distribution of specific cell types or proteins. For such application, tissues are first frozen and fixed before adding in primary and secondary antibodies. To promote diffusion of antibodies into densely packed tissues, electric fields and physical swirling can be introduced to improve the migration and uniformity of antibody binding.

“While incredibly valuable in scientific research, antibodies may not always work effectively due to reasons like poor specificity and denaturation.”

Antibodies are also widely used as therapeutics. For instance, monoclonal antibodies are used to inhibit actions of proteins such as programmed cell death 1 receptor (PD-1) that is implicated in cancer. PD-1 signaling negatively regulates T cell-mediated immune response and an antibody that blocks PD-1 has been shown to enhance anti-tumor immunity and prolong survival. There are already three US Food and Drug Administration-approved monoclonal antibodies against PD-1 including atezolizumab, durvalumab, and avelumab.

While incredibly valuable in scientific research, antibodies may not always work effectively due to reasons like poor specificity and denaturation. Below are some tips to optimize your antibodies.

Check for host species and clonality

Antibodies are typically used in pairs: the primary antibody binds to the antigen of interest, such as a protein, and the fluorescent molecule- or enzyme-conjugated secondary antibody binds to the primary antibody for signal detection and amplification. The host species of the primary and secondary antibody should be different from the antigen species to prevent cross-reactivity and unwanted background staining or interference. 

The antibody clonality is also an important factor. Monoclonal antibodies are more specific as they bind only to a single antigen, but they cannot be used across multiple species. On the other hand, polyclonal antibodies bind to multiple epitopes with greater tolerance of antigen changes (such as those unintentionally induced by sample treatment and preparation), but they suffer from greater batch-to-batch variability.

Talk to other users

Before attempting to perform antibody-related experiments, it pays to read scientific literature to check which antibodies are being used by researchers in the same field. While there is a possibility that everyone might be using a non-specific antibody, the pooled risk is lower. Users can also consider attending webinars by The Antibody Society to learn tips on choosing antibodies.

Purchase validated antibodies

Different manufacturers can adopt widely different standards for antibody manufacturing, which affect material quality. It is advisable to check databases of validated antibodies such as Antibodypedia, which contains data on 19,000 human protein targets (~95 percent of human genes). It is also recommended to read the guidelines by the International Working Group for Antibody Validation on techniques to validate antibodies and outline which applications each antibody is suitable for. 

Scrutinize quality analysis documents from manufacturers

Reputable manufacturers will provide detailed information about their antibodies, such as the antigen sample, host species, and cross-species reactivity, unless there are proprietary reasons. Bordeaux at al. reported three levels of antibody validation efforts from companies. The first is “low/no validation” where there is minimal information except for a brief description on the target and recommended applications, without examples of references that have successfully used the antibodies. The second is “moderate validation” where manufacturers include at least one example of the antibody being used to correctly identify targets through western blots. The third is “high validation” where companies include western blot data for multiple cell sources with high batch-to-batch consistency. Users should try to find suppliers that share antibody manufacturing and quality analysis data, which is useful in troubleshooting.

Adopt good techniques to handle antibodies

Being proteins, antibodies are vulnerable to extreme environmental conditions like heat and acidity that can cause denaturation and affect binding specificity to targets. Some malpractices include repeated freezing and thawing of the same antibody samples. Users should aliquot the antibodies into smaller quantities and thaw only when needed. Users can also read protocols from suppliers and publishers to know the ideal temperature to handle antibodies, duration for incubation with antibodies, and various troubleshooting tips. 

Incorporate experimental controls

As much as possible, users should include positive and negative controls in their experiments to validate antibodies. Positive controls can be lysate or recombinant proteins that are known to bind to the specific antibody. Negative controls can include genetic knockout samples that no longer produce the antigens. Users can also omit a primary antibody to understand whether secondary antibody can cause unwanted background interference.

Antibodies are popular and valuable materials in labs and for use in clinics. Nevertheless, how they are manufactured and handled can lead to poor reproducibility. To fully optimize antibodies for use, tips such as sharing and learning from your peer community, choosing only reliable manufacturers, and incorporating good handling practices will greatly benefit science done in your lab.


For additional resources on antibodies, including useful articles and a list of manufacturers, visit www.labmanager.com/antibodies