* Sold separately (see Other Reagents Required, below)
** Following an overnight incubation with capture antibody, 2 hour blocking step, and cell stimulation.
*** When using the protocol provided.

---

PRODUCT SUMMARY
Complete are highly sensitive, microplate-based assays for the detection of cytokine secreting cells. Kits are available for detection and enumeration of a single analyte or two analytes simultaneously. Complete ELISpot kits are ready-to-run and require no assay development or refinement. contain the basic components required to develop an ELISpot assay. They offer an economical alternative to buying separate antibodies.

ELISpot development modules are an alternative to ELISpot kits. A basic understanding of ELISpot assay development is required for the successful use of these reagents. Each investigator should optimize the coating conditions, the assay sensitivity, the type of enzyme and substrate, as well as the concentrations of the capture and detection antibodies to achieve desired results. The analyte-specific ELISpot Development Module and the contain the necessary components for analyte detection and visualization, respectively. These modules can be used together but are sold separately. Each module contains enough reagents for at least five 96-well microplates.

---

PRODUCT FEATURES

• An economical alternative to ELISpot Kits
• Optimized capture and detection antibody pairings and recommended concentrations save lengthy development time
• Generic development protocols provide direction to start an optimization protocol
• Customize the assay to your specific needs

---

MODULE CONTENTS

• Capture Antibody
• Biotinylated-conjugated Detection Antibody

---

OTHER REAGENTS REQUIRED

• ELISpot Blue Color Module or equivalent (R&D Systems, Catalog # )
• PBS - 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2 - 7.4, 0.2 µm filtered
• Wash Buffer - 0.05% Tween^® 20 in PBS
• Blocking Buffer - 1% BSA, 5% Sucrose in PBS
• Reagent Diluent - 1% BSA in PBS, pH 7.2 - 7.4, 0.2 µm filtered
• 2 °C – 8 °C refrigerator
• 37 °C CO₂ incubator
• Positive Control - Use Recombinant or cells known to secrete
• 96-well plates - Nitrocellulose-bottom plates, PVDF-bottom Immunospot^® plates, or flat-bottom polystyrene Immulon^® ELISA plates
• Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer
• Dissection microscope or an automated ELISpot Reader
• Deionized H₂O

Trademarks and registered trademarks are the property of their respective companies.

Preparation and Storage

Background: IFN-gamma

IFN-gamma (Interferon-gamma) is the prototype proinflammatory cytokine and is produced by a variety of immune cells under inflammatory conditions, notably by T cells and NK cells. It plays a key role in host defense by promoting the development and activation of Th1 cells, chemoattraction and activation of monocytes and macrophages, upregulation of antigen presentation molecules, and immunoglobulin class switching in B cells. It also exhibits antiviral, antiproliferative, and apoptotic effects. In addition, IFN-gamma functions as an anti-inflammatory mediator by promoting the development of regulatory T cells and inhibiting Th17 cell differentiation. IFN-gamma dimers signal through a receptor complex of two IFN-gamma R1 and two IFN-gamma R2 subunits.