Cytokines and Cytokine Assays
Cytokines are signaling proteins that help to regulate inflammatory and immune responses in our body. Cytokine assays enable the detection and cytokine quantification in biological samples such as serum, blood, plasma, tissues, and others. These assays are crucial in diagnosing and monitoring diseases since cytokines are biomarkers of several diseases and can play a pivotal role in the progression or regression of pathophysiological conditions.
Different Types of Cytokine Assays
Depending upon the methods and protocols involved in the testing, Cytokine Assays can be of different types, such as bioassays, protein microarrays, high-performance liquid chromatography (HPLC), and antibody assays such as sandwich enzyme-linked immunosorbent assay (ELISA), bead-based multiplex immunoassays (MIA), and mesoscale discovery (MSD).
Bioassays for Cytokines
Bioassays focus on the activity of the cytokines and use this as their readout. The biological activity of cytokines, irrespective of their form (as soluble protein, cytokine-serum protein complexes, or cytokine receptor complexes), is determined using sensitive cell lines. These assays involve the neutralization of cytokine-specific antibodies to identify the cytokine present in the sample. Bioassays are highly sensitive (picogram range) but have relatively poor specificity and are semi-quantitative, time-consuming, and labor-intensive. Poor specificity of bioassays can be attributed to the responses of cell lines to different biomolecules present in the sample, leading to interference in data. Due to the limitations, bioassays are mainly used for validation rather than cytokine identification and quantification.
Protein Microarrays for Cytokines
Protein microarrays allow the analysis of interaction, function, and biological activities of cytokines on a large scale. These assays use protein chips with different capture proteins immobilized on their surface. The capture proteins can include antibodies, aptamers, or affibodies based on the target cytokine in a sample. Fluorescent-tagged probe molecules are added that interact with the capture proteins, resulting in a fluorescence signal read by the detector. These interactions provide valuable information on expression levels, binding affinity, and specificity of the proteins in the sample. Protein microarrays are rapid, sensitive, high-throughput, and economical. These assays allow the quick identification of several cytokines. Protein microarrays are extensively used as a platform to detect cytokine biomarkers. However, they are not without limitations. The primary challenge involves manufacturing chips with stabilized capture proteins with intact naive structures so that they can interact freely and display their biological activity. Furthermore, the shelf life of protein arrays is short due to the easy denaturation of proteins. In addition, technical challenges exist for identifying and isolating capture proteins for the vast range of proteins available within the human proteome.
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High-performance Liquid Chromatography for Cytokines
This platform operates based on specific weight, hydrophobicity, ligand affinity, ion exchange, and protonating abilities of proteins. Thus, HPLC-based cytokine assays utilize a method based on the specific chemistry of each analyte to identify and separate the compound. This method allows the quantification and purification of cytokines by passing a mixture of compounds through a solid-phase separating column under pressure. The separation of the compounds is governed by the solvent (mobile phase) conditions used and interactions between the compounds and column particles (stationary phase), which leads to a partition of the compounds between the stationary and mobile phases. This method has reportedly purified cytokines such as IL-1 secreted by macrophages and epidermal cells. Despite the notable abilities, HPLC-based cytokine assays have drawbacks, including complex methodology, high cost, risk of co-elution for molecules with similar chemical structure and polarity, and irreversible adsorption of compounds on the stationary phase that goes undetected.
Immunoassays
These are antibody-based assays such as the ELISA, MSD, and MIA. While ELISA and MSD are plate-based immunoassays, MIA is a bead-based immunoassay. The underlying principle of these immunoassays is the sandwiching of a cytokine between two antibodies (capture and detection antibodies). The detection antibody is tagged with a fluorescently labeled reporter molecule for the signal readout corresponding to the cytokine capture. Immunoassays are popular due to their high specificity, sensitivity, and reproducibility; simple methodology; rapid detection; high throughput; and multiplexing capabilities.
However, before using an immunoassay, one must remember that the performance of an immunoassay depends upon multiple factors. These include (i) the quality of the capture and detection antibodies used, (ii) the risk of detecting biologically inactive cytokines or their fragments, if the epitopes are recognized by the antibodies, (iii) the possibility of obtaining negative results if the primary structure of the antibody has been modified chemically, e.g., through amidation, glycosylation, etc., and (iv) non-specific binding of cytokines to the assay matrix, leading to high background signal, thus making it difficult to interpret data. Other drawbacks include the high cost of reagents.
In addition to these assays, droplet digital ddPCR Gene Expression Service can help identify and quantify cytokines using ddPCR readout in proximity ligation assay protocol. This platform can analyze low-abundant cytokines by amplifying signals from the protein-antibody interactions. With the availability of a diverse array of cytokine assays, it is crucial to make a rational choice based on the research objectives and the advantages and limitations of the assays.
