Optimized ELISPOT assays can reliably detect antigen-specific T cells even if they occur in frequencies of 0.0001% within PBMC; that is, a single analyte-secreting T cell can be reliably identified within one million bystander cells.
This sensitivity is orders of magnitudes higher than that of flow cytometry. All ImmunoSpot® Kits are quality-controlled to meet this exquisite sensitivity threshold. The kits come with a serum-free test medium that was specially developed to ensure that the assay performs with low background and high signal.
T cells secrete more cytokines on a per-cell level than cells of the innate immune system. Therefore, one can identify antigen-specific T cells vs. the irrelevant bystander cells based on per-cell productivity. ImmunoSpot® Kits come with a substrate that precisely resolves the quantity of analyte produced by each cell, thereby permitting accurate gating and data analysis. The CTL substrate technology prevents overdevelopment which allows adjacent spots in the most crowed wells to be resolved. Additionally, established development times can be used, avoiding the subjective judgement calls required when using common substrates.
ImmunoSpot® Kits and protocols were designed to minimize assay variables such as the substrate development time and serum. Serum is a major source for assay variability resulting from undefined suppressive or stimulatory effects. We offer ImmunoSpot® Kits with a special serum-free medium, CTL-Test™, that we developed for standardized ELISPOT work. For those relying on cryopreserved PBMC, serum-free media are available for standardized freezing and thawing of PBMC.
We also offer reference PBMC to verify that the assays are performing to expectations. Using this platform, the intra- and inter-laboratory variability can be reduced to a level that until recently had been considered “incredible” for a T cell assay (Zhang et al., J. Immunotoxicology, 2009, 6:227), and that can be readily verified today: for you, all it takes is to try the ImmunoSpot® Kit!
ELISPOT assays have the reputation of being lengthy and complicated. The reagents contained in the ImmunoSpot® Kit have been tailored to shorten and simplify the assay procedure. No prewetting of the membrane with alcohol needed! No blocking necessary! Save 14 plate-washing steps! All buffers and washing solutions are included. Performing an ELISPOT assay has never been faster or easier!
The accurate, objective evaluation of ELISPOT data requires quality instrumentation which comes at a substantial expense. Before most researchers can get started with ELISPOT, they have to wait to obtain funding for a reader. To help get you started, we offer free scanning services of the plates purchased with our kits, and a discounted rental option for the gold standard ImmunoSpot® Software or instruments. Getting started with ELISPOT has never been more simple!
NEW: 384-well MiniSpot™ Format
The standard 96-well ELISPOT format is already very economic with PBMC utilization compared to flow-based assays in which most cells are lost in the tubing. In the standard 96-well ELISPOT assay, as few as 100,000 cells are sufficient for each measurement/well. The cells even survive the assay unharmed and can be reutilized for subsequent studies. Our new 384-well MiniSpot™ format allows a four-fold decrease in the number of PBMC needed, utilizing only 25,000 PBMC per well. Thus, one can, for example, perform 40 tests with only 1 million PBMC obtained from 1ml of blood! This economic cell utilization is critical whenever the number of cells is limited, such as in pediatric studies. It allows the screening of extensive antigen/peptide libraries for T cell reactivity, to study T cell avidity by titrating the antigen, or to multiplex analyte measurements by running assays in parallel. Utilize the new possibilities that the MiniSpot™ format enables!
Validation of ELISPOT Assay in 384-well Format
There is a precise number of antigen-specific T cells in each PBMC sample. Join the community of those who pursue T cell measurements as a standardized, reproducible, and exact science.