The Ideal Assay for Immune Monitoring
Using ImmunoSpot® Kits, first-time users, from various different laboratories, were able to obtain accurate data with their first-ever attempt to run an ELISPOT assay (Zhang et al., J. Immunotoxicology, 2009, 6:227).
ELISPOT assays measure the cytokine (or antibody for B-cell ELISPOT) released from a stimulated T cell in the immediate cellular environment surrounding the cell. The T cell sets upon a membrane coated with “capture Ab” during the assay. Upon stimulation via antigen or polyclonal mitogenic activation, the T cell releases cytokine, at which point it is bound by the coating Ab before diffusing into the culture medium or undergoing proteolytic degradation.
ELISPOT assays are therefore able to measure the cytokine released with a far greater sensitivity than indirect assays such as ELISA or Cytokine Bead Arrays (CBAs), which rely on testing of samples in the culture medium in which the cytokine has been diluted and/or degraded.
ELISPOT assays are orders of magnitude more sensitive than cytokine measurements using ICS/Flow Cytometry, where the cytokine must be detected within the cell and is performed using treated and fixed dead cells. The cytokine is therefore not necessarily biologically active and/or relevant.
ImmunoSpot® Assays can reliably detect antigen-specific T cells in frequencies as low as 0.0001% within PBMC; that is, a single cytokine-secreting T cell can be reliably identified among one million bystander cells.
CTL has nearly 20 years of experience in defining and establishing the criteria and software solutions for recognizing antigen-specific T cell cytokine responses. ImmunoSpot® Kits are optimized to distinguish between antigen-specific responses from “background spots” from bystander cells of the innate immune system.
Our colorimetric substrates are chosen for the precision with which they are able to resolve the cytokine signature “spot” produced by each cell, allowing for a quantitative analysis, and size-based “gating” to enumerate antigen-specific responses over bystander cytokine production.
ImmunoSpot® colorimetric substrate development is standardized and optimized for a defined incubation time, further eliminating subjective determinations regarding spot development, while enabling the resolution and discrimination of adjacent and overlapping spots from crowded assay wells.
ImmunoSpot® Kits have been standardized and optimized for the highest standards of reliability and reproducibility, which is essential for immune monitoring studies.
Serum-free CTL-Test™ Medium has been formulated to support T cell growth and performance while eliminating the variability inherent in using serum-containing media. Serum has been found to be a major source of variability resulting from uncharacterized immuno-suppressive or stimulatory effects observed in immune monitoring assays. CTL Serum-free Media products are also available for freezing and thawing of cryopreserved PBMC.
ePBMC® Reference Samples are also available for standardization of assay procedures and performance. These cryopreserved PBMC are available in large sample batches, fully HLA types and characterized for antigen responses, making them the perfect assay controls.
Using our ImmunoSpot® Platform, including ELISPOT Kits, Serum-free Media, and ePBMC®, we have demonstrated a dramatically reduced intra- and intra-laboratory variability and high degree of data conformity (Zhang et al., J. Immunotoxicology, 2009, 6:227) between and within labs performing standard ELISPOT assays.
ImmunoSpot® ELISPOT Kits have been developed and standardized to eliminate variability and subjectivity in performance and results. Our reagents and protocols eliminate the need for “prewetting” of the plate membranes with alcohol, and lengthy “blocking” and subsequent washing steps. This eliminates operator variability and error, while also eliminating 14 washing steps.
Our ImmunoSpot® ELISPOT Kits include all buffers and washing solutions, and are available with cell culture medium – further simplifying and standardizing the assay(s).
Plate Scanning and Analysis Services – To accurately image and analyze ELISPOT results requires the use of sophisticated plate scanners and analysis software. CTL has plate scanning and analysis services available and offers a trail version of ImmunoSpot® Software with the purchase of any kit.
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