Human TNF-alpha ELISPOT Kit

Materials Provided

Product Overview

Tumor necrosis factor-alpha (TNF-α) is a multifunctional Th1 cytokine and one of the most important inflammatory cytokines. It is produced by macrophages during inflammation and can also be activated by the endotoxin lipopolysaccharide (LPS). The Human TNF-α ELISpot assay is designed for the detection of human TNF-α secreting cells at the single cell level and can be used to quantitate the frequency of human TNF-α secreting cells. ELISpot assays are highly reproducible and sensitive, and can be used to measure responses with frequencies well below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and they are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in vaccine development, cytokine secretion and the monitoring of various clinical trials.

Application

This kit is used to detect TNF-alpha at the cellular level.

It is for research use only.

Reconstitution

Please see Certificate of Analysis for details of reconstitution instruction and specific concentration.

Storage

1. Unopened kit should be stored at 2℃-8℃ upon receiving.

2. Find the expiration date on the outside packaging and do not use reagents past their expiration date.

3. The opened kit should be stored per components table. The shelf life is 30 days from the date of opening.

Assay Principles

ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. The Kit consists of Pre-coated Anti-TNF-α Antibody Microplate, Positive Stimulus, Biotin-Anti-TNF-α Antibody, Streptavidin-HRP, AEC and buffers.

Your experiment will include 6 simple steps:

a) A monoclonal antibody specific for human TNF-α has been pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate.

b) Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time.During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted TNF-α.

c) After washing away any cells and unbound substances, a biotinylated antibody specific for human TNF-α is added to the wells. Following a wash to remove any unbound biotinylated antibody, HRP conjugated to streptavidin is added.

d) Wash the plate and add the Streptavidin-HRP diluted by Dilution Buffer to the plate.

e) Wash the plate and add AEC. A reddish-brown colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual TNF-α secreting cell.

f) SThe spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.

ACRO Quality Management System

1. QMS(ISO, GMP)
2. Quality Advantages
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