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Several analytical techniques have been developed to detect and identify biomarkers associated with major human diseases. Among these, enzyme-linked immunosorbent assays (ELISA) stand out as a highly sensitive and specific tool for quantifying clinical biomarkers. Frequently, the limited availability of samples, combined with the demand for a reliable, cost-effective, and time-efficient method, makes immunoassays the preferred choice. ELISA technique is critically important in cell therapy research, playing a pivotal role in the evaluation, development, and quality control of cell-based therapies. Their importance stems from several key applications:
1. Biomarker Detection and Quantification
ELISA is a widely used technique to measure cytokines such as IL-2, IFN-γ, and TNF-α, which serve as key indicators of immune activity, inflammation, or therapeutic efficacy. Additionally, it is used for quantifying growth factors like VEGF and FGF, which help assess the potential of cell-based therapies for tissue repair and regeneration. ELISA also plays a role in detecting disease-specific markers relevant to cell therapy, such as cancer antigens in immunotherapy. Proteins and peptides in tissues and body fluids reflect an individual’s health status and can undergo changes in structure and quantity during disease. High-throughput proteomic analyses have been applied early on to diagnose therapy-related complications following allogeneic hematopoietic stem cell transplantation (HSCT). Various proteomic methods like ELISA are currently employed to predict or diagnose both acute and chronic graft-versus-host disease (GvHD).
2. Assessing Cell Functionality
ELISA is a powerful tool for assessing cell functionality in cell therapy research and clinical applications. It enables the quantification of secreted proteins, cytokines, growth factors, and other biomolecules that indicate whether therapeutic cells are active and effective. Below are key ways ELISA can be applied in evaluating cell functionality:
Measuring Cytokine and Growth Factor Secretion
Many cell-based therapies, such as mesenchymal stem cell (MSC) therapy or CAR-T cell therapy, rely on the secretion of specific cytokines (e.g., IL-2, IFN-γ, TNF-α) or growth factors (e.g., VEGF, EGF, FGF). ELISA can be used to measure the levels of these secreted molecules in cell culture supernatants or patient serum to assess whether the therapeutic cells are functioning as intended. For example, in CAR-T cell therapy, ELISA can quantify IL-2 and IFN-γ levels to confirm T cell activation and immune response against cancer cells.
Assessing Immunomodulatory Function
Some therapeutic cells, like regulatory T cells (Tregs) or MSCs, function by modulating the immune response through cytokine release. ELISA can evaluate whether these cells secrete anti-inflammatory cytokines such as IL-10 and TGF-β, which are crucial for therapeutic efficacy. If MSCs are used to treat inflammatory diseases, ELISA can determine whether they are secreting IL-10 and TGF-β, confirming their immunosuppressive function.
Evaluating Cell Viability and Stress Responses
When cells undergo stress or apoptosis, they release specific markers like HMGB1 (a damage-associated molecular pattern, DAMP) or LDH (lactate dehydrogenase). ELISA can detect these proteins in culture media or patient samples, indicating whether the transplanted cells are alive, stressed, or dying. In islet cell transplantation for diabetes, ELISA can measure insulin secretion in response to glucose to evaluate pancreatic beta-cell functionality.
Monitoring Engraftment and Persistence
To ensure that transplanted cells survive and remain functional, ELISA can measure specific biomarkers over time in patient blood or tissues. For example, detecting human albumin in a liver transplant model confirms hepatocyte engraftment and function. In stem cell-derived cardiomyocyte therapy, ELISA can measure BNP (B-type natriuretic peptide) as a marker of heart muscle function.
Validating Cell Potency Before Transplantation
Before clinical application, cells must be validated to ensure they express the expected functional proteins. ELISA can be used in quality control testing to confirm that the cells are producing key bioactive factors. For instance, in dopaminergic neuron transplantation for Parkinson’s disease, ELISA can measure dopamine levels in culture media to confirm cell potency.
3. Quality Control and Validation
The advent of biotherapeutics has significantly advanced the treatment of various chronic diseases. However, the immune system may identify these biologic agents as foreign, leading to the production of anti-drug antibodies (ADAs), including neutralizing antibodies (nADAs). Such immunogenic responses can alter the pharmacokinetics and pharmacodynamics of the drug, potentially impacting its safety and efficacy. ELISA is one of the most effective techniques for validating and measuring ADAs and NAbs. ELISA is also essential for verifying the purity of cell preparations and ensuring they produce the expected therapeutic proteins or factors. It plays a key role in ensuring batch-to-batch consistency in manufacturing cell therapies. Furthermore, it helps detect contaminants such as endotoxins or unwanted cytokines that could compromise the safety of cell-based treatments. ELISA is used to detect endotoxin contamination in mesenchymal stem cell (MSC)-based therapies before clinical use. Endotoxins, produced by Gram-negative bacteria, can trigger severe inflammatory reactions and compromise patient safety.
4. Immunogenicity and Safety Assessment
In cell therapy, ELISA is commonly used to monitor host immune responses, such as the production of antibodies or inflammatory cytokines. It also aids in identifying elevated levels of pro-inflammatory markers, helping predict potential adverse events or toxicities. In MSC or CAR-T cell therapy, it is essential to evaluate immunogenicity, which refers to the ability of the therapeutic cells to trigger an immune response. Unwanted immune activation can lead to therapy rejection or adverse effects. ELISA is used to measure anti-drug antibodies (ADA) or anti-CAR antibodies in patients receiving CAR-T cell therapy to assess the risk of immune rejection.
5. Preclinical and Clinical Studies
ELISA is instrumental in preclinical models to measure therapeutic outcomes, such as reduced inflammation or enhanced tissue repair, by detecting relevant biomarkers. In clinical trials, it helps track biomarker changes in patients, allowing researchers to evaluate therapy effectiveness and adjust treatment protocols accordingly. In preclinical and clinical research, ELISA is used to measure circulating cancer antigens such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), and cancer antigen 125 (CA-125) in patients undergoing immunotherapy.
6. High Sensitivity and Specificity
One of ELISA’s major advantages is its high sensitivity and specificity, which allow for the detection of even low-abundance proteins. This level of precision is critical in understanding the subtle effects of cell therapies on biological systems. Reducing sample volume requirements while maintaining detection accuracy saves costs in expensive reagents and biological samples. Preventing false positives/negatives reduces the need for retesting or confirmatory essays, increasing efficiency and lowering the costs. High sensitivity and specificity ensure that measured concentrations remain consistent, leading to reliable conclusions in biological research with lower costs as well. The high sensitivity and specificity of ELISA play a fundamental role in biological research, clinical diagnostics, and assay validation. They ensure accurate, reproducible, and reliable measurement of analytes, which is essential for making valid scientific and medical conclusions.
Applications in Specific Cell Therapies
In CAR-T cell therapy, ELISA is used to measure cytokines like IL-6 to monitor efficacy and potential risks such as cytokine release syndrome. In stem cell therapy, it helps quantify factors like TGF-β and BMPs, which influence differentiation and tissue regeneration. For immune modulation, ELISA assesses regulatory cytokines such as IL-10, produced by MSCs or regulatory T cells, to evaluate their anti-inflammatory properties.
Advantages of ELISA in Cell Therapy
ELISA is a cost-effective technique, making it more affordable compared to advanced tools like mass spectrometry. It is scalable and can be easily adapted for high-throughput screening in both clinical and research laboratories. Furthermore, it is an established and widely validated technology, making it suitable for regulatory and academic purposes. While MS provides detailed insights into protein composition, its higher cost and complexity often make ELISA the preferred choice for routine applications in cell therapy research.
Limitations
Despite its advantages, ELISA has some limitations. It is typically designed to detect only one analyte per assay, which may limit its usefulness when studying complex interactions. Additionally, it may not always capture the full spectrum of concentrations in complex samples due to its dynamic range. The technique also requires careful optimization and handling to ensure reliable results, making it somewhat labor-intensive.
Enzo Life Sciences has extensive experience in designing, producing, and commercializing affordable ELISA kits for use in research, particularly in the discovery stages of cell therapy. Some of their widely used ELISA kits include:
IL-6 (human), high sensitivity ELISA kit
ENZ-KIT178
Most sensitive (0.057 pg/mL) colorimetric kit for IL-6 quantification
| Application | Colorimetric detection, ELISA |
|---|---|
| Assay Time | 3 hours |
| Sensitivity | 0.057 pg/mL (1.56 – 50 pg/mL) |
IL-2 (human), ELISA kit
ADI-900-118A
The IL-2 (human), ELISA kit is a colorimetric immunometric enzyme immunoassay kit
| Application | Colorimetric detection, ELISA |
|---|---|
| Assay Time | 3 hours |
| Sensitivity | 6.6 pg/ml (range 7.81 – 500 pg/ml) |
IL-10 (human), ELISA kit
ADI-900-036
The IL-10 (human), ELISA kit is a colorimetric immunometric enzyme immunoassay kit
| Application | Colorimetric detection, ELISA |
|---|---|
| Assay Time | <3 hours |
| Sensitivity | 3.75 pg/ml (range 7.81 – 500 pg/ml) |
TNF-α (human), ELISA kit
ADI-900-099
Highly sensitive ELISA for measuring TNF-α, the primary mediator of immune regulation.
| Application | Colorimetric detection, ELISA |
|---|---|
| Assay Time | 4 hours |
| Sensitivity | 8.43 pg/ml (range 15.63 – 1,000 pg/ml) |
Our ELISA kits can be a great help in preclinical cell therapy research, providing critical insights into cell function, therapeutic potential, and quality control. ELISA remains indispensable in cell therapy research, enabling scientists and clinicians to assess efficacy, safety, and quality throughout the development pipeline. While more advanced techniques such as multiplex assays and flow cytometry provide additional insights, ELISA continues to be a cornerstone method due to its simplicity, reliability, and precision. Its role in ensuring the success and scalability of cell-based therapies cannot be overstated.
References
- Farina Silveira C R et al. (2022). Cytokines as an Important Player in the Context of CAR-T Cell Therapy for Cancer: Their Role in Tumor Immunomodulation, Manufacture, and Clinical Implications. Front Immunol, 12(13):947648. https://pubmed.ncbi.nlm.nih.gov/36172343/
- Nicole C. Japp, C N et al. (2021). Tumor Biomarker In-Solution Quantification, Standard Production, and Multiplex Detection. J Immunol Res, 9942605. https://pubmed.ncbi.nlm.nih.gov/34514003/
- S.F.H. de Witte et al. (2017) Cytokine treatment optimizes the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease. Stem Cell Res Ther. 8, 140. https://pubmed.ncbi.nlm.nih.gov/28595619/
- U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, & Center for Veterinary Medicine. (2011, January). Guidance for industry: Process validation: General principles and practices. https://www.fda.gov/media/71021/download
- U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, & Center for Veterinary Medicine. (2018, May). Bioanalytical method validation: Guidance for industry. https://www.fda.gov/media/119271/download
- Weissinger, E. M., Basílio-Queirós, D., Metzger, J., Bieling, L. M., & Ganser, A. (2020). Proteomics for hematopoietic stem cell transplantation. Expert Review of Proteomics, 17(3), 201–206. https://doi.org/10.1080/14789450.2020.1748501
- Mojtahed Poor, S., Ulshöfer, T., Gabriel, L. A., Henke, M., Köhm, M., Behrens, F., Geisslinger, G., Parnham, M. J., Burkhardt, H., & Schiffmann, S. (2019). Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab. Clinical and Experimental Immunology, 196(2), 259–275. https://doi.org/10.1111/cei.13261
Rozbeh Modarresi, PhD
Senior Scientist – Linkedin
Rozbeh Modarresi received his Ph.D. in 2005 and subsequently joined Weill Medical College of Cornell University as a postdoctoral researcher. During his time there, he was honored with two Young Investigator Awards from the Conference on Retroviruses and Opportunistic Infections in 2007 and 2009 for his research on the side effects of drugs in HIV patients, contributing to numerous publications in the field. In 2013, he joined Alpha 1 Biologics, where he published a paper on the feedback regulatory pathway between LDL and alpha-1 proteinase inhibitor. Since 2022, he has been serving as a Senior Scientist in the ELISA group at Enzo Life Sciences.
