Academy of Diagnostics & Laboratory Medicine - Scientific Short

Are Aptamers Ready to Replace Immunoassays for Clinical Use?

Joe El-Khoury

The introduction of immunoassays into clinical laboratories in the 1960s was revolutionary, enabling clinicians and scientists to measure proteins in patient samples (1). This technology took advantage of the high specificity between an antibody and its target antigen to identify and quantify compounds of interest in biological fluids. A variety of immunoassay techniques have since been developed that employ different capture and detection mechanisms, like: radioimmunoassay (RIA), enzyme-multiplied immunoassay technology (EMIT), fluorescence-polarized immunoassay (FPIA), and enzyme-linked immunosorbent assay (ELISA), to name a few. As great as immunoassays are, they also have their shortcomings.

The development of antibodies for use in immunoassays is a very lengthy, laborious and expensive process that depends on the use of a biological system (like mice, goats or rabbits) (2). This requirement also makes the development of antibodies to toxins difficult and limits the optimization of antibodies to physiologic conditions. In addition, there are many factors that are difficult to control during development, such as: activity of antibodies from batch-to-batch, target site of the protein of interest, pharmacokinetic parameters, and modifications to the molecule. Also, antibodies themselves are temperature sensitive (irreversibly denature), have limited shelf-life, and can produce significant immunogenicity in the host. This last characteristic is why immunoassays are severely affected by heterophile antibody interference (commonly called HAMAs). Aptamers are an attractive solution to all of these issues.

First developed in 1990, aptamers are short oligonucleotides (usually 20-60 nucleotides of single stranded DNA or RNA) that, like antibodies, can bind to a specific target (2). However, their entire development is a chemical process that can be carried out in-vitro and target any protein under a variety of conditions. This makes the process of developing aptamers easy, cheap, and uniform (because of the highly controlled environment in-vitro). Moreover, the investigator can determine the exact target site of the protein of interest and can make a wide variety of chemical modifications to the aptamer in order to diversify its functions. On top of all of that, aptamers are resistant to temperature insults by returning to original confirmation after the insult, have unlimited shelf-life and are not immunogenic. All of these characteristics make aptamers ideally suited to overtake immunoassays in the clinical diagnostics space.

In 2012, the first aptamer-based assay was tested in biosamples and it can now measure over 1,300 protein targets simultaneously using only 50 µL of blood (3). This is remarkable throughput considering that multiplexed immunoassays can only measure 10-20 analytes in a single measurement while maintaining analytical validity. While extremely promising, aptamer technology is still not validated for clinical use. There are currently no US FDA-approved/cleared aptamer assays (Pegaptanib is the first therapeutic aptamer approved in 2004), but there are around 40 aptamer-based companies actively engaged in diagnostics and therapeutics research (4). In a recent study, our group compared the performance of one type of aptamer assay, called slow off-rate modified aptamer (SOMAmer), with immunoassays for the measurement of biomarkers of acute kidney injury in urine and plasma collected pre-and post- operation from adult patients undergoing cardiac surgery (5). Overall, out of the 33 biomarkers we compared, 27%, 23% and 50% showed strong, moderate and poor correlation between assay types (aptamer vs immunoassay), respectively. This is not surprising considering most of the markers are research-use only, with a few (like Cystatin C and NGAL) that are clinically used and showing strongest correlation between different assay types. The analytical performance of biomarker measurements used in clinical research varies widely and is often not adequately reported, which explains the poorer correlation between assay types for these markers (6). This can produce conflicting results and hinder the translation of reliable assays into clinical practice (7). Aptamer-based technology should be evaluated further against existing clinical immunoassays and mass spectrometry to assess its potential and validity for use in the clinical research and diagnostics laboratory.

References

  1. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960;39:1157–75.
  2. Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249:505-10.
  3. Gold L, Walker JJ, Wilcox SK, Williams S. Advances in human proteomics at high scale with the SOMAscan proteomics platform. N Biotechnol 2012;29:543–9.
  4. Kaur H, Bruno JG, Kumar A, Sharma TK. Aptamers in the Therapeutics and Diagnostics Pipelines. Theranostics. 2018;8: 4016–4032.
  5. Kukova LZ, Mansour SG, Coca SG, et al. Comparison of Urine and Plasma Biomarker Concentrations Measured by Aptamer-Based versus Immunoassay Methods in Cardiac Surgery Patients. J Appl Lab Med. 2019;4:331-342.
  6. Sun Q, Welsh KJ, Bruns DE et al. Inadequate Reporting of Analytical Characteristics of Biomarkers Used in Clinical Research: A Threat to Interpretation and Replication of Study Findings. Clin Chem. 2019;65:1554-1562.
  7. Bunch DR, El-Khoury JM. Emerging Biomarker of Kidney Disease: suPAR or Subpar? Clin Chem. 2018;64:1545-1547.

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Fellows of the Academy use the designation of FADLM. This designation is equivalent to FACB and FAACC, the previous designations used by fellows of the National Academy of Clinical Biochemistry and AACC Academy. Those groups were rebranded as Academy of Diagnostics & Laboratory Medicine in 2023.