A successful development of immune checkpoint inhibitors cannot be achieved overnight. To test the immunomodulatory activities of checkpoint inhibitors, cytokine production/release of the T-cells assay is also necessary. As an industry-leading CRO company, Creative Biolabs has focused on immune checkpoint assay for more than ten years. We have a comprehensive technology platform to satisfy customers' different requirements. With advanced foundations and an excellent expert team, we can provide high-quality cytokine production/release assay to global clients.
Brief introduction to Cytokine
Cytokines are small protein, glycoprotein, or peptide molecules that, through cell signaling, allow intricate cellular communication. These molecules are produced by cells of various embryological origins and are classified according to structure or function. Upon binding with their cell surface receptor, these molecules initiate an intracellular signaling cascade that may result in the up-regulation and/or down-regulation of gene expression or take on transcription factor activity, subsequently inducing further cytokine production or curbing their activity via feedback inhibition.
Cytokine Production/Release Assay Platform
Creative Biolabs is an indubitable leader in drug development and manufacture. Equipped with the most advanced platforms, scientists at Creative Biolabs can help customers design and perform suitable cytokine production/release assays to meet each customer's individual requirements.
The ELISPOT assay is based on the principle of the ELISA detecting antigen-induced secretion of cytokines trapped by an immobilized antibody and visualized by an enzyme-coupled second antibody. The good reproducibility of the ELISPOT assay for the determination of antigen-specific T-cells in different laboratories. Frequencies of influenza-reactive T-cells determined with the ELISPOT assay are correlated with frequencies derived from the limiting dilution assay (LDA).
Fig.1 The principle of the ELISPOT assay for detecting cytokine-secreting T-cells. (Letsch, 2003)
Intracellular cytokine flow cytometry (ICC)
Another elegant, functional approach to detect cytokine production in T-cells is by intracellular cytokine flow cytometry. Following brief antigen exposure, cytokines are trapped intracellularly by the addition of brefeldin A to block the secretory pathways. After permeabilization, specific anti-cytokine fluorescent antibody conjugates can pass into the cells.
Fig.2 The principle of the ICC for detecting cytokine-producing T-cells. (Letsch, 2003)
Cytokine bead array
Cytokine bead array is another efficient method for testing cytokine production. Beads of different sizes or colors are used for multiplexed immunoassays. The beads were differentiated by light scatter characteristics, and the immunoassay signal was generated through the binding of fluorescent conjugates. Larger sets of different bead populations can be prepared by staining the beads with two or more fluorescent dyes at various ratios. The immunoassay signal is generated by detection reagents coupled to the third type of fluorescent dye.
Having focused on immune checkpoint drug development for years, Creative Biolabs has accumulated extensive cytokine production/release assay experience from practice. We are confident in bringing our clients with top-rated customer experience. If you are interested in our services or have any other questions, please contact us for more information.
Letsch, A.; Scheibenbogen, C. Quantification and characterization of specific t-cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods. 2003, 31(2): 143-9.
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