Immunohistochemistry has helped to make surgical pathology the ‘gold standard’ for tumour diagnosis. Antibodies have been used extensively as the molecular probes; however, there have been issues with their specificity, which is the most critical determining factor in IHC.
Previous reports have suggested that 40-50 % of diagnostic inconsistencies are related to poor antibody selection, cross reactivity, variability with antibody purity and batch-to-batch variation leading to discrepancies in staining intensity. Moreover, it has been reported that half of the IHC failures results from improper antibody storage in laboratories.
Aptamers as an alternative molecular probe for IHC
There is no question that aptamers can offer benefits over antibodies, as they have been demonstrated as excellent probes for histochemical staining of frozen and formalin fixed along with paraffin embedded (FFPE) tissues. Aptamers have unique properties that make them advantageous in clinical settings by overcoming the limitations of antibodies. These include:
- Isolated in vitro
- Smaller size which allows faster tissue diffusion
- Significantly lower batch to batch variation
- Reduced cost and faster production
- Potentially increased stability and convenient storage
Moreover, in direct comparison with antibodies in IHC, aptamer probes provide:
- Optimal staining with shorter reaction times
- Less restricted antigen retrieval conditions
- Reduced time to perform the aptamer histochemistry technique
- Lower background staining (negligible non-specific binding)
The following researchers have reported their findings where they have used aptamers in histochemistry thereby proving their potential to revolutionalise the field of histopathological diagnostics.
Aptamer identifies human epidermal growth factor receptor (HER2) in breast cancer cells and tissues
HER2 plays a significant role in the development and progression of certain aggressive types of breast cancer. Therefore, HER2 has become an important biomarker and therapeutic target for most of the breast cancer patients. In this regard, Liu et al., 2012 developed a novel 86-nucleuotide DNA aptamer called HB5 that could target the extracellular domain of HER2 protein with a Kd of 316 nM and showed minimal reactivity with albumin or trypsin. The aptamer also exhibited specific binding to HER2 positive breast cancer cells, with minimal binding to HER2 negative cells.
This aptamer was further evaluated by another researcher Chu et al. in 2015 to identify the expression profile of HER2 in breast cancer tissues. HB5 aptamer showed strong membrane staining in HER2 positive tissues as compared with commercial anti-HER2 antibody (Figure 1). Moreover, the HB5 staining signal was further enhanced by a newly developed rolling circle replication (RCA) technique involving a process of unidirectional nucleic acid replication that could rapidly synthesize multiple copies of circular molecules of DNA. This method overall was shown more effective and specific than the FDA-approved commercial kits.
Figure 1. Evaluation of HER2 with the aptamer HB5 in breast cancer cells and tissues. (a) and (b) Binding profiles of the aptamer HB5 as compared to anti-HER2 antibody and random DNA to HER2-positive (SKBR3) or HER2-negative (MDA-MB-231) breast cancer cells. (c) Examples of IHC staining for HER2 in invasive ductal carcinoma where with a commercial anti-HER2 antibody or the aptamer HB5. (d) HB5 staining signal enhanced by RCA technique with stronger expression of HER2 in situ.
Aptamer identifies toxic dimer of amyloid protein aggregates in Alzheimer brain tissue.
Amyloid fibrils of senile plaques in Alzheimer’s disease (AD) consist of amyloid β – proteins (Aβ40 and Aβ42) which are produced from the Aβ precursor protein. These toxic oligomers, mainly Aβ42 can induce neuronal death and cognitive dysfunction and therefore, specific amyloid inhibitors are in higher demand. In this view, Murakami et al., 2020 developed RNA aptamers with potent affinity for the toxic protofibril (PFs) dimer model of Aβ42 protein (E22P-V40DAP-Aβ42 dimer) with Kd of 20 nM and further evaluated the application of the RNA aptamers in histochemical staining of AD mouse brains.
The selected RNA aptamer (E22P-AbD43) specifically recognized diffuse premature aggregates originated from toxic PFs derived from Aβ in the cerebral cortex and hippocampus regions whereas; an anti-Aβ-N-terminus antibody (82E1) mainly stained senile plaques with dense cores (Figure 2). Considering the preferential recognition of the pre-matured aggregates in AD mice, this RNA aptamer showed potential for early diagnosis or positron emission tomography (PET) imaging of amyloidogenesis for determining the progression of AD.
Figure 2. Comparison of histochemical analysis of AD mouse brains using RNA aptamer (E22P-AbD43) and an antibody (82E1). Red arrowheads represent diffuse aggregates and blue arrows represent senile plaques.
Aptamers and their use in rapid IHC protocols
Overall, aptamers have consistently demonstrated promising results in both peroxidase and fluorescence based IHC techniques, often at greatly reduced incubation times. The findings suggest that aptamers can provide a robust and cost-effective tool to translate biomarker discovery into pathological diagnostic practice. This will assist in the development of rapid IHC protocols that can potentially be used in both research and clinical settings, including intraoperative diagnostics where speed and accuracy are paramount.
Aptamer group technology
At Aptamer Group Ltd, we start with the end in mind by designing the aptamers according to the customer specific requirements, so that they are optimized for end applications. For such IHC applications, we perform initial few rounds of selection against for target protein until we see a greater signal to ratio as compared with the negative target (Figure 3). This is followed by the selection against cells / tissues of interest; after they have undergone all the necessary treatments (fixation, embedding, antigen retrieval etc).
Figure 3. Schematic representation of the overall aptamer selection for end IHC application.
Taking this approach means that we isolate the aptamers against the antigen, as it will be presented in the final application. This helps to ensure that the aptamers are fit for purpose faster than traditional technologies. If you want more information on exploring applications of aptamers in IHC, please contact us using the form below.
Bauer M, Macdonald J, Henri J, Duan W, Shigdar S. The Application of Aptamers for Immunohistochemistry. Nucleic Acid Ther. 2016;26(3):120-126. doi:10.1089/nat.2015.0569
Chu M, Kang JR, Wang W, et al. Evaluation of human epidermal growth factor receptor 2 in breast cancer with a novel specific aptamer. Cell Mol Immunol. 2017;14(4):398-400. doi:10.1038/cmi.2015.31
Liu Z, Duan JH, Song YM, et al. Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro. J Transl Med. 2012;10:148. Published 2012 Jul 20. doi:10.1186/1479-5876-10-148
Murakami K, Obata Y, Sekikawa A, et al. An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer’s disease. J Biol Chem. 2020;295(15):4870-4880. doi:10.1074/jbc.RA119.010955