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Three factors to consider for IHC aptamers

When selecting the right IHC reagent make sure you consider these three factors and find out how IHC aptamers can help ensure success from the start.

Immunohistochemistry, IHC for short, detects and localizes a target of interest within a tissue sample. IHC is a technique used across diagnostics, prognostics, precision medicine, patient stratification, surgical pathology, drug development, and both applied and basic research.

IHC aptamers

IHC shows whether a biomarker is expressed, how much of a biomarker is expressed, and the spatial localization of the biomarker within the tissue.

Taken together, the information from IHC offers insights into disease stages, disease types, and the role of new biomarkers in tissues. However, like any powerful technique, IHC must be properly managed to ensure accuracy in the results.

Accurate labelling of specific biomarkers in IHC is vital, particularly when diagnostic and prognostic outcomes rely on the results. In each IHC assay the accuracy of the IHC result depends on the affinity reagent used and critically the characteristics of:

  1. Specificity
  2. Consistency
  3. Stability

Here, we look at each of these factors and how planning the right discovery methods can help deliver IHC aptamers for successful results.

Too tricky to tune IHC antibodies

Target specificity is crucial to make sure that the signal you are detecting in your assay is based on the biomarker of interest. This means that your IHC aptamers must bind to the target and not to other proteins or molecules in the tissue sample.

Cross-reactivity from antibodies in IHC has been highly cited in the literature, where antibodies bind to the required biomarker but also to other homologous targets in the tissue. In this case, your IHC assay measures more than your biomarker, giving false positive and unreliable results. Cross-reactivity of antibodies with a non-target biomarker is often the rule and not the exception. In one study, fewer than half of around 6,000 routinely used commercial antibodies recognized only their specified targets.

As antibodies are developed in vivo, controlling the discovery process is impossible and we rely on the immune system of the host animal to deliver suitable reagents for IHC. This means that cross-reactivity can only be engineered out of the binder in downstream development work, increasing time and cost, with no assurance of success. For many targets, relying on antibody generation in this way has failed, resulting in a lack of specificity, particularly to small changes, such as single amino acid mutations in a target.

Engineering specificity for IHC aptamers

Because of the molecular complexity of tissue, there are few other methods that challenge the specificity of an affinity reagent more than IHC, so designing the right discovery method to ensure the specificity of IHC aptamers is important.

Custom-developed aptamers use tailored discovery processes to increase specificity and functional performance. These steps include negative screening against multiple homologous targets where the aptamers that bind to these targets are removed from the selection process, to ensure specificity.

Inflammatory marker expression on immune cells in Crohn's disease tissue

Highly specific IHC aptamers can be developed through discovery programmes tuned for individual targets and specific tissue fixation processes. Left panel: Optimer labelling of immune cells expressing a specific inflammatory marker in FFPE colonic tissue from a patient with Crohn’s disease (Red = Optimer; Blue = DAPI). Right panel: Scrambled Optimer control labelling of FFPE colonic tissue from a Crohn’s disease patient shows no staining, highlighting the specificity of the Optimer binder (Blue = DAPI).

For IHC aptamers, the discovery processes can also include steps to screen aptamers in positive and negative tissues. Aptamers should bind to the positive target tissue with the expected localisation and show no binding to the negative tissue. With these steps included in the discovery process, you are assured that the aptamer recognises the target in tissue, whether that is in frozen or fixed samples.

Tissue fixation and antigen retrieval steps in IHC can change target structures in the tissue, so including tissue-based screening steps for IHC aptamers makes sure that the reagents bind their target in the end assay format and are functional in IHC assays.

If you know you want to use your aptamers in IHC, we can also incorporate fixed tissue screens into the aptamer discovery process. This ensures that the aptamer is not only specific for the right biomarker but also that it recognises the biomarker after the antigen retrieval steps performed for IHC, which can change the structure of your biomarker.

New IHC aptamer solutions to increase sensitivity

For many IHC targets, direct detection can be useful. It helps to cut down protocol steps, is quicker and easier. Also, no pesky secondary antibodies to control for. IHC aptamers can be employed for direct detection. However, for low abundance proteins or to incorporate your IHC aptamer into current automated workflows, you may need something more.

Low abundance targets can cause detection problems in IHC. If you suspect or know that your target is in low abundance, direct detection methods are probably not the favoured solution. To keep the specificity of your assay, using polyclonal reagents is not ideal. Instead using secondary detection reagents in your IHC allows signal amplification in the assay. Most IHC assays, particularly in larger labs with the use of automated IHC, include secondary antibodies for precisely this reason. The secondary antibody recognises the Fc portion of the primary antibody, with the ability to bind multiple secondary antibodies to each primary. As IHC aptamers do not have an Fc fragment region this signal amplification method isn’t possible.

However, we have worked with specialist across the industry to develop and validate the IHC aptamer solution to overcome this issue. Optimer-Fc is a new reagent solution for IHC. In Optimer-Fc we combine the high selectivity, tuneability and broad target range of our optimized aptamers, Optimer binders, with the Fc domain of an antibody.

Optimer-Fc new reagent solutions for IHC

Optimer-Fc reagents consist of an Optimer conjugated to the Fc fragment of an antibody, for the simple use of IHC aptamers in automated workflows.

IHC aptamers for reproducibility you can rely on

Aptamers, unlike most affinity reagents, are generated from oligonucleotides. The main method of oligonucleotide manufacture is solid-phase synthesis, which involves well-established, controllable, and scalable methods. This gives the highest batch consistency for aptamers compared to other affinity reagents.

Batch consistency is important for reproducible data. Reproducible data means that the same results are obtained every time an assay is performed and allows solid conclusions to be drawn from the assay results.  When talking about diagnostic or prognostic IHC assays for diseases, drawing the right conclusions enables patients to be correctly informed about their disease and receive the right medicines at the right time.

To prevent the loss of research and ensure the validity of data, it is sensible to begin IHC assay development with a reagent that you know will show long-term consistency and reproducibility.

Overcome your stability woes with IHC aptamers

Improper reagent storage can contribute to greater than 50% of IHC failures, particularly in laboratories that store refrigerated reagents. As protein-based reagents, antibodies are susceptible to infection and degradation without proper storage. This can lead to antibodies unable to effectively bind their targets, and false negatives in your IHC assay.

In contrast, IHC aptamers, being DNA-based reagents are highly stable. The stability of aptamers means that your reagent can be easily stored, shipped, and used without worrying about performance. Stored in a neutral buffer, at -20°C, DNA IHC aptamers have a shelf life of at least two years. Even at 37°C, stability is estimated for over six weeks. Studies have shown that fluorescently tagged aptamers, like used in direct IHC, have remained functional for over five years when stored in a lyophilised form.

High stability offers longevity for your IHC aptamer in your lab and assured performance every time you use your IHC aptamer reagent.

 

If you are looking for new reagent solutions to support your IHC, with new biomarkers or improved assays, get in touch today and speak with the team, to see how we can help.

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