Immunoassay Troubleshooting

When an immunoassay produces unsatisfactory results, researchers often blame primary antibody reagents. But, in many cases, primary antibodies are not at fault—even the very ‘best’ primary antibodies can struggle to perform if they are not handled correctly. Here, we share five top tips for troubleshooting common immunoassay problems.

Antibody specificity, affinity, and selectivity underpin immunoassay success

According to Dr. Tobias Polifke, Co-Founder and Managing Director at CANDOR Bioscience, an immunoassay can only produce reliable results if primary antibodies bind their target analytes specifically and with high affinity.

“The rationale behind any immunoassay is that an antibody will bind its target—and only its target—in a crude matrix,” he explains. “If the antibody binds to other biomolecules, the results cannot be trusted. Likewise, if the antibody has low affinity for its target, it may not bind all of the target molecules that are present, and there is an increased risk of cross-reactivity. In these situations, researchers are correct to blame primary antibodies for unusual results.”

Jan Voskuil, Director and Founder of Aeonian Biotech, adds that selectivity is another core antibody characteristic in determining immunoassay success. “Selectivity is related to the uniqueness of the epitope and is thus maximal when that epitope is found only on the target protein,” he says. For a deeper dive into specificity, affinity, and selectivity, Aeonian Biotech has published a blog and Cell Signaling Technology an article covering the main points.

Critically, highly specific primary antibodies offer improved assay sensitivity by increasing the signal-to-noise ratio. “Non-specific binding will generate higher baseline noise to reduce the assay window,” comments Anne Sloan, Ph.D., Senior Scientist at Cell Sciences. “Although noise can be decreased by assay optimization—such as through testing different concentrations of antibody components—using antibodies that exhibit high specificity for their target analytes is what underpins the sensitivity of any immunoassay,” she says.

Troubleshooting tips

So, what can you do to overcome common immunoassay problems such as weak or no signal, high background, or data that are inconsistent with a hypothesis? Here are our five top tips:

Troubleshooting tip #1—review the protocol

When faced with immunoassay problems like those just described, the best first step is to review the protocol carefully in comparison to that recommended by the vendor, including the suggested antibody concentration,” reports Katie Crosby, Sr. Director, Antibody Applications and Validation at Cell Signaling Technology. “Even seemingly small protocol differences could be responsible for poor results, which speaks not to the quality of the antibody, but to the complex nature of the factors that influence antibody:epitope interactions.

Troubleshooting tip #2—understand the target biology

To fully appreciate what is going on in your immunoassay, it is essential that you understand the target biology, especially since unexpected results can often be some of the most interesting. “Western blot is an obvious case in point, where post-translational modifications, target cleavage, splice variants, and multimers can all be genuine reasons for detecting bands of an unexpected molecular weight,” comments Crosby.

“A main advantage of western blot is that it allows issues such as non-specific binding and cross-reactivity to be seen more easily and sooner than with other applications,” notes Voskuil. “Where such problems go undetected, the consequences can be far-reaching. For instance, The Antibody Society’s recent antibody validation webinar series describes how using cross-reactive antibodies for measuring the breast cancer biomarker estrogen receptor beta led to spurious clinical trial results.”

Troubleshooting tip #3—rule out non-antibody-related problems

Problems attributable to non-antibody reagents include contaminated buffers, expired detection reagents, and unsuitable washing solutions. These should be investigated using appropriate positive and negative controls to support any findings. It is also important to consider external factors such as the ambient temperature and the type of lighting available in the lab, which can impact enzyme and fluorophore performance, respectively.

Troubleshooting tip #4—always include relevant controls

Depending on the nature of your immunoassay and the chosen readout, controls can take many different forms. “For assays leveraging fluorescent detection, an unstained control is useful to understand baseline autofluorescence,” says Crosby. “Secondary only controls should be deployed routinely in order to understand what signal, if any, may result from that reagent and not the primary antibody. This is especially important when working with mouse monoclonal antibodies and mouse-derived samples. Well-characterized reference controls are also a key consideration. In IHC assays, for example, particularly when dealing with diseased tissues that may no longer express proteins in the same way as the tissue of origin, cell pellets serve as excellent controls.”

In addition to these types of controls, spiking experiments can help determine how an antibody will perform in a particular sample matrix. “Spiking experiments involve testing primary antibodies with a purified form of the protein target in a simple solution, as well as in a complex biological mixture that closely represents your samples,” explains Sloan. “Following this, you should validate the antibody with your chosen sample type. If your immunoassay will include a standard curve, the matrix used for diluting the reference should always match the sample matrix to ensure any concentrations are accurately calculated. Here, it is crucial to ensure that the target protein concentration of your samples falls within the dynamic range of your assay.”

Troubleshooting tip #5—employ multiple testing strategies

If you experience unsatisfactory immunoassay results, cross-checking experiments can help narrow down the source of the problem. Popular strategies include running the same application with multiple antibodies (each targeting a different epitope) to see if similar staining is observed; performing a different immunoassay, such as investigating unexpected immunocytochemical staining by producing a western blot using lysates from the same cells that have been treated in a similar manner (provided the antibody is validated for both techniques); and comparing antibody-derived results to orthogonal data, such as that produced using mass spectrometry or RNA sequencing.

Improving immunoassay performance with modern reagents

Modern reagents are designed to overcome common immunoassay problems. Examples include blockers such as The Blocking Solution  and SmartBlock, which provide more complete surface coverage than BSA to reduce immunoassay variability (%CV), and antibody diluents like LowCross-Buffer^® or Hispec Assay Diluent (Bio-Rad), which function to reduce cross reactivity, non-specific binding, and matrix effects. The latter can be used to rule out low affinity antibodies, which will produce a measurable signal when diluted in PBS/BSA/Tween, but not when diluted in LowCross-Buffer or Hispec Assay Diluent.

Factors to consider when selecting primary antibodies

1. Check whether the antibody has been used in the application of interest—vendor-provided recommendations are a good starting point; publications demonstrating antibody use in well-controlled experiments are also helpful

2. Be critical when reviewing product data—does the vendor show results using more than one cell or tissue model? Do the data align with what you understand about the target? Are clear and detailed protocols provided that will enable you to replicate the results? Does the vendor offer technical support if you should encounter any issues?

3. Understand the vendor's approach to ensuring antibody performance is consistent across different lots

4. Confirm whether the antibody recognizes a native or denatured form of the protein target

5. Never be afraid to ask vendors for guidance