Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human IL-4.
Principle:Add stimulated cells or cells and stimulant to the wells and incubate at 37°C in CO2 incubator for a specified period. Secreted human IL-4 binds to antibody coated microtiter plate
microtiter plate coated with human IL-4 Capture Antibody. Cells and unbounded proteins are washed away. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. Unbounded proteins are washed away. Biotinylated detection antibody is added and binds to the secreted human IL-4. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human IL-4 secreting location. Each individual spot representing an individual human IL-4 secreting cell.
Background:Interleukin 4 (IL-4) was initially characterized as a B cell stimulatory factor (BSF-1) (1) and B-cell differentiation factor (BCDFγ) (2). It was subsequently revealed that IL-4 is a pleiotropic cytokine with multiple immune response modulating functions on diverse cell types including T cells, monocytes, macrophages, mast cells, fibroblasts, endothelial cells, osteoblasts, keratinocytes, hepatocytes and astrocytes (3-8). IL- 4 is produced by CD4+ TH0 and TH2 cells (9,10), fetal thymocytes (11), CD8+ T cells (12), mast cells (13) and basophils (14).
Human and mouse cDNAs for IL-4 encode precursor proteins containing 153 and 140 amino acid residues, respectively. The signal peptides from the precursors are cleaved to yield mature proteins of 129 amino acid residues (human) and 120 amino acid residues (mouse) (17, 18). Both human and mouse proteins have multiple potential glycosylation sites and six cysteines that are all involved in intro-molecular disulfide bridges.
IL- 4 exhibits approximately 25% amino acid sequence homology to IL-13 that shares a number of biological functions with IL-4 (19). The human and mouse IL-4 gene, each composed of four exons and three introns, have been localized to human chromosome 5q23-31 and mouse chromosome 11 in tandem with genes for IL-3, IL-5, IL-9, IL-13 and GM-CSF (20, 21). IL-4 is an anti-inflammatory cytokine that exhibits multiple immuno-modulation functions on a variety of cell types, including T cells, B cells, monocytes, neutrophils, hematopoietic progenitors, fibroblasts, endothelial cells, and epithelial cells (15, 16).
The diverse effects exhibited by IL-4 in vitro suggest that it may play a central role in the modulation of immune and inflammatory responses in vivo. It was reported that introduction of malignant tumor cells transfected with the gene for IL-4 and producing IL-4 in athymic mice can block tumor formation by other transplantable tumor lines in vivo. Since the anti-tumor activity of IL-4 is evident in these athymic mice, IL-4-mediated host-defense responses other than T cell immunity must be involved (22). It was reported that IL-4 is important for protective immunity in parasitic nematode-infected mice, since IL-4 or IL-4R antibodies can blocked the polyclonal IgE response and abrogated protective immunity to the infection (23). In contrast to results obtained with the nematode-infected mice, endogenous IL-4 was reported to inhibit protective immunity in mice infected with the protozoan Leishmania major (24, 25). Clearly, much more work is required to unravel the complex network of IL-4-dependent processes in order to utilize IL-4 successfully in immunotherapy (16).
I8426-01A2: Positive Control 1x1vial. Supplied as a lyophilized recombinant human IL-4. Reconstitute with 250ul Cell Culture Media before use. Use in 1 hour. The final concentration is 8ng/ml
I8426-01A3: Wash Buffer (20X) 1x60ml. Add 1 volume of Wash Buffer (20X) to 19 volume of ddH2O. Use in 1 week. If the concentrate is stored ar 4ºC, crystals may form. Allow to reach RT before reconstitution
I8426-01A4: Mab Detection Antibody (Biotin) 1x11ml. Biotinylated mouse anti-human IL-4 monoclonal antibody. Ready to use. I8426-01A5: Streptavidin (AP) (100X) 1x120ul. Add 1 volume of I8426-01A5 Streptavidin (AP)(100X) to 100 volumes of I8426-01A6 Streptavidin (AP) Diluent before use. Use in 1 month
I8426-01A6: Streptavidin (AP) Diluent 1x11ml. Ready to use. I8426-01A7: BCIP/NBT Substrate Solution 1x11ml. Ready to use.
Storage and Stability:Store all components at 4ºC. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
