Apoptosis Detection Using ELISA Kits

How can ELISAs be used as a detection method for apoptosis

The ELISA (enzyme-linked immunosorbent assay) is an antibody-based technique for detecting specific antigens of interest, including biomarkers of apoptosis. Apoptosis, or programmed cell death, plays an important role in many biological processes including cell turnover, development, immune response, and cancer development. Apoptotic biomarkers include proteins that play central roles apoptotic pathways, such as BCL-2 family proteins and caspases. Using the ELISA method, these biomarkers can be measured in a variety of biological samples, including tissue, cell cultures, blood, and other bodily fluids, to evaluate the apoptotic process.

While other methods can also be used to detect apoptosis, such as Western blotting, flow cytometry, TUNEL assays, and caspase assays, ELISA kits offer several distinct advantages, such as scope, sensitivity, simplicity, and throughput.

Advantages of ELISA kits for assessing apoptosis

Wide scope of protein targets - Many apoptotic proteins have been identified and more are being studied to fully elucidate their role in related pathways. Likewise, many ELISA kits have been developed that target a wide variety of unique proteins. The scope of available targets across different ELISA suppliers is so wide that researchers have access to the immunodetection of nearly any apoptotic protein of interest. Researchers interested in a particular protein who wish to study it in the context of apoptosis will more than likely be able to find a dedicated ELISA kit for that target.

Specificity, sensitivity, and quantification - ELISA kits are highly specific and sensitive to their target antigen. Specific antibody binding enables the detection of minute amounts of antigens, making them suitable for samples with even low amounts of apoptotic proteins. Kits are available that can measure at concentrations as low as the picograms per milliliter range. This sensitivity can also enable the accurate quantification of apoptotic proteins. As the ELISA can determine the precise concentration of the target antigen present in the sample, researchers may also draw meaningful conclusions on the level of protein expression.

Simple workflow and high throughput - ELISA kits generally contain all the necessary supplies to set up an experiment, including specific antibodies, pre-coated multi-well plates, buffers, and detection reagents. Detection is completed using an ELISA-compatible plate reader, which is a fairly common laboratory instrument. In contrast to Western blots or flow cytometry, an ELISA procedure can be performed in as little as two hours with fewer steps using less specialized equipment. As a plate-based assay, ELISAs can also be done under high-throughput conditions, which can be ideal when many apoptotic proteins are being studied. To learn more, read our Guide to Choosing the Right ELISA Kit.

Common protein markers of apoptosis

Apoptosis is a tightly regulated process involving several complex mechanisms. The intrinsic pathway, driven by mitochondrial instability, involves activation of pro-apoptotic proteins leading to the formation of the apoptosome. Proteins involved in this pathway include APAF1, cytochrome c, caspase 9, IAP family proteins, and BCL-2 family proteins, including Bcl-2, Bad, Bax, and Bcl-XL. Smac/DIABLO, APAF1, cytochrome c, caspase 9, IAP family proteins, and BCL-2 family proteins, including Bcl-2, Bad, Bax, and Bcl-XL.¹

The extrinsic apoptotic pathway involves receptor-mediated interactions that leads to the formation of a death-inducing signaling complex (DISC) and caspase 8 activation. Proteins in this pathway include cytokines such as TNF alpha, FasL, TRAIL, TWEAK, and TNF receptors such as TNFR1, TNFR2, FAS (CD95), DR3, and DR5.¹

Caspases are a conserved family of cysteine proteases with a well-understood central involvement in inflammation and apoptosis. Initiator caspases, caspase -8, -9 and 10, function to initiate apoptosis by activating executioner caspases, caspase -3, -6 and -7.²

Other commonly used markers for apoptosis capitalize on cellular changes. Recombinant annexin V binds strongly and specifically with translocated phosphatidylserine residues present on the outer membrane on the surface of dying cells.

Differentiating between apoptosis and necrosis

An added benefit of using protein-specific methods such as ELISAs is the ability to distinguish apoptosis from other cell death processes such as necrosis, autophagy, and pyroptosis. For example, translocation of phosphatidylserine, detected by annexin V, can be found on both apoptotic and necrotic cells due to cell membrane damage.³ Activities of key proteins, however, such as initiator and executioner caspases, are unique hallmarks of apoptosis, and can be used as more definitive markers.

Limitations of ELISAs in apoptosis detection

It is prudent to understand that ELISAs will have certain limitations when being used to study apoptosis. Traditional ELISA kits measure only one target at a time, unlike flow cytometry or multiplex assays, which can analyze multiple apoptotic markers simultaneously. This can be time-consuming in studies requiring the analysis of several markers to fully understand the apoptotic process. The single-protein focus of ELISA assays also lack cellular and tissue context. Unlike microscopic methods such as TUNEL staining or immunocytochemistry, ELISA does not provide information on the spatial distribution of apoptosis within tissues or among cell populations. If such context is necessary, users may consider supplementing ELISA results with microscopy-based experiments. Beyond ELISAs, other methods for studying apoptosis are further outlined in Choosing an Apoptosis Detection Assay.

Applications of ELISA kits in apoptosis research

The use of ELISA kits to investigate apoptotic processes has been documented in published studies. Some examples are highlighted below:

• In a study of antiepileptic compounds in a murine model, ELISA kits for Bax, Bcl-2, and cytochrome c were used to assess apoptosis in rat hippocampus tissue.⁴

• In a study of anti-cancer effects of an antioxidant compound in a colon cancer cell line, ELISA kits were used to quantify the levels of the apoptotic proteins, BAX, Caspase-3, and BCL-2.⁵

• A study testing the effects of trastuzumab/AT-101 combination therapy used a Cell Death Detection ELISA kit to evaluate apoptosis in human breast cancer cell lines.⁶

ELISA kits are useful tools for immunodetection in many research areas beyond apoptosis. To learn more, visit our comprehensive ELISA resources page.

References

1. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516. doi:10.1080/01926230701320337

2. Van Opdenbosch N, Lamkanfi M. Caspases in Cell Death, Inflammation, and Disease. Immunity. 2019;50(6):1352-1364. doi:10.1016/j.immuni.2019.05.020

3. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun. 2005;73(4):1907-1916. doi:10.1128/IAI.73.4.1907-1916.2005

4. Al Omairi NE, Albrakati A, Alsharif KF, et al. Selenium Nanoparticles with Prodigiosin Rescue Hippocampal Damage Associated with Epileptic Seizures Induced by Pentylenetetrazole in Rats. Biology (Basel). 2022;11(3):354. Published 2022 Feb 23. doi:10.3390/biology11030354

5. El-Atawy MA, Hanna DH, Bashal AH, et al. Synthesis, Characterization, Antioxidant, and Anticancer Activity against Colon Cancer Cells of Some Cinnamaldehyde-Based Chalcone Derivatives. Biomolecules. 2024;14(2):216. Published 2024 Feb 12. doi:10.3390/biom14020216

6. Bulut G, Atmaca H, Karaca B. Trastuzumab in combination with AT-101 induces cytotoxicity and apoptosis in Her2 positive breast cancer cells. Future Oncol. 2020;16(3):4485-4495. doi:10.2217/fon-2019-0521