Like other sectors of the global economy, the current COVID-19 pandemic, caused by the human SARS-CoV-2 virus, has disrupted life sciences research and development. With increased awareness and utilization of personal protective equipment, laboratories have gradually resumed activity, but with limited staff working shorter shifts. As a result, researchers are exploring ways to reach experimental endpoints in less time while achieving pre-pandemic productivity.
By relying on advances in technology, laboratories can reduce hands-on time across multiple workflows. New, time-saving approaches include walk-away automation and miniaturization, higher throughput formats, and efficient protocols. Similarly, the adoption of advances in bioassay screening workflows reduces turnaround time with applications such as cytokine panel evaluation in emerging areas of cell therapy.
Cytokines, a diverse family of low molecular weight proteins secreted by cells of the immune system, are essential biomarkers to monitor disease state, assess prognosis following treatment, and evaluate cytokine release syndrome (CRS) in models for CAR T-cell (Chimeric Antigen Receptor T-cell) therapies. CRS is caused by increased activation of immune cells, resulting in the release of proinflammatory cytokines. In comparison, a "cytokine storm" from a SARS-CoV-2 infection leading to COVID-19 results in a high level of IL-6 and subsequent depletion of lymphocytes (Tang et al., 2020).
From now, when developing new therapies for cancer, autoimmune disorders, and inflammatory diseases, or following up on SARS-CoV-2 infections, laboratories or clinicians rely on cytokine analysis to explore the mechanism of diseases and measure treatment response.
Cytokine measurements or profiling is performed to evaluate inflammatory responses, assess infection status, or monitor responses from cell therapy or other therapeutic strategies. A single cytokine, or, more commonly, an entire panel of cytokines is evaluated due to the complexity of dynamic and interrelated immune pathways in vivo. Historically, radioimmunoassays (Yallow and Berson, 1959) with radioactive isotopes were used for cytokine and biomarker detection. Following its development as a non-radioactive alternative, ELISA (enzyme-linked immunosorbent assay) has been widely used (Crowther JA, 2001). However, several limitations are associated with conventional ELISA.
The assay workflow requires many steps that span over 12 hours. Reliability strongly depends on reagent quality and end-user handling techniques. Although next-generation ELISA technologies offer shorter workflows, faster turnaround times, and enable the evaluation of multiple cytokines in a single experiment, the technique is hampered by a narrow dynamic range and limited sensitivity compared to newer multiplex assay formats. For samples with a broad range of cytokine concentrations, ELISA adds a preparatory step for the dilution of other related biomarkers.
Newer, higher throughput assays offer rapid workflows with a wider dynamic range and greater sensitivity. Accurate results can be obtained in an hour or two with simple steps facilitating walk-away automation, freeing up resources to enable faster experimental turnover. These benefits are particularly important during the current limitations to hands-on work in the pandemic environment. In addition, these new assay formats provide superior data quality due to reduced background noise, interference correction, and a brighter signal allowing for higher sensitivity and wider dynamic range. Thus, drug discovery and development scientists can make confident decisions with reliable data.
Cytokine or biomarker detection platforms leverage the specific interaction of analyte (substance or macromolecule being identified) and antibody in two ways:
- Competitive binding of an unlabeled or labeled analyte to the antibody (homogeneous assays)
- The labeled, unbound analyte is washed away and the remaining labeled bound analyte is detected and quantitatively measured (heterogeneous assays)
Contemporary heterogeneous immunoassays have evolved over traditional ELISA to deliver better performance, multiplex capabilities, and higher throughput to cover a wider range of applications, but still involve a washing step within the workflow. This makes them well suited for complex samples such as blood. A number of multiplex assay technologies are commercially available in different formats. Time-resolved fluorescence (TRF) methodologies extend the acceptor emission to create long-lived fluorescence with a lanthanide, such as Europium cryptate.
The TRF assays offer higher sensitivity and a wider dynamic range, enabling less reagent use and improved detection of low levels of analyte, eliminating the need to dilute samples. With extended fluorescence, TRF reduces sample interference, making it relatively easy to automate.
The advantages of homogeneous assays include a no-wash, all-in-one-well workflow requiring fewer steps that can be completed in under two hours, resulting in significant increases in efficiency and productivity. The homogeneous assay platforms provided by PerkinElmer offer greater sensitivity, wider dynamic range, and robust performance amenable to miniaturization and automation.
For example, the Alpha (Amplified Luminescent Proximity Homogeneous Assay) utilizes a pair of antibodies to the analyte.
One biotinylated antibody is bound to the streptavidin-coated donor bead while its counterpart is linked directly to the acceptor bead. When the pair binds to the analyte, the beads are brought in close proximity and respond to laser excitement with chemiluminescent emission. Its wide dynamic range makes this assay well suited for complex matrix samples like blood, plasma, saliva, and CNS fluid. In the homogeneous time-resolved fluorescence (HTRF) assay, however, the fluorescent signal is generated via the proximity of an acceptor (short-lived, longwave fluorescence) and a donor (long-lived, shortwave fluorescence, such as Europium cryptate or Terbium cryptate).
|ELISA||Multiplex Immunoassays||Time-Resolved Fluorescence (TRF)||Alpha||HTRF|
|Plate format||96 well||96-384 well||96-384 well||96-1536 well||96-1536 well|
|Time to results||4-24 hrs.||1 hr.||4-6 hrs.||1-2 hrs.||1-2 hrs.|
|Dynamic Range||2 log||≥ 3.5 log||5 log||5 log||4 log|
|Sensitivity||10 pg/mL||Single digit pg/mL||10 pg/mL||1 pg/mL||10 pg/mL|
|Signal Stability||Up to one hour||Over 24 hours||Up to 24 hours||24 hours to days|
|Multiplexing||No||Yes (up to 50)||Yes (up to 3)||Yes (up to 3)||No|
Compatible Plate Readers for Immunoassays
Compatible plate readers are needed to detect and analyze the assay signal. Most plate readers, such as PerkinElmer’s EnVision™ and Victor Nivo™, can read multiple assay formats and offer enhanced security software that enables compliance with GMP 21 CFR Part 11. Furthermore, some software applications also offer secure off-site access for remote work. When plate readers support compliance in the GMP environment, the instrument, as well as the subsequent methods developed and validated, can be used in the manufacturing and QA/QC functions for drug product release testing.
Cytokines are important prognostic biomarkers that are central to monitoring metabolic and disease pathways. These low molecular weight mediators play a critical role in immune system networking in normal or disease states as well as a signal via transcriptional mechanisms in controlling the biology of cell growth, differentiation, development, apoptosis, and survival. Therefore, monitoring cytokines via sensitive and robust technology platforms is critical for various preclinical and clinical studies. As discussed above, an array of tools has been developed to perform immunoassays to evaluate single cytokines through a broad panel of cytokines in a physiological context.
Conventional ELISA techniques can measure single to several cytokines in a single assay; however, next-generation technologies offer multiplex capabilities. Typically, for basic research goals, screening a wide panel of cytokines provides insight into physiological networks.
Subsequently, upon selection of relevant biomarker(s), downstream assay development and bioanalytical studies tend to leverage platforms that offer higher throughput, enhanced sensitivity, broader dynamic range, as well as robustness, which is critical across projects. Hence, a total workflow solution and systems approach enable optimized and reliable study.
- Tang Y, Liu J, Zhang D, Xu Z, Ji J, Wen, C. Cytokine storm in COVID-19: The current evidence and treatment strategies. Frontiers in Immunology. 2020; 11.
- Yallow RS, Berson SA. Assay of plasma insulin in human subjects by immunological methods. Nature, 1959; 184 (Suppl 21):1648-1649.
- Crowther, JA. The ELISA Guidebook. Humana Press; Totowa, NJ: 2001.
- Peters CD, Jespersen B, Nørregaard R. AlphaLISA versus ELISA-based detection of interleukin 18 in healthy subjects and patients with end-stage renal disease. Scandinavian Journal of Clinical and Laboratory Investigation. 2012; 72(8):583-592. DOI: 10.3109/00365513.2012.713175
- Degorce F, et al. HTRF: A technology tailored for drug discovery—a review of theoretical aspects and recent applications. Current Chemical Genomics. 2009; (3):22-32. https://pubmed.ncbi.nlm.nih.gov/20161833/
- Cox KL, Devanarayan V, Kriauciunas A, et al. Immunoassay Methods. 2012 May 1 [Updated 2019 Jul 8]. In: Markossian S, Sittampalam GS, Grossman A, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences. Available from: https://www.ncbi.nlm.nih.gov/books/NBK92434/
- Chowdhury F, Williams A, Johnson P. Validation and comparison of two multiplex technologies, Luminex® and mesoscale discovery for human cytokine profiling. Journal of Immunological Methods. 2009; (340):55-64. https://www.sciencedirect.com/science/article/abs/pii/S0022175908003116?via%3Dihub