Swine IL-10 ELISA Assay
The Swine IL-10 ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 15.625 – 1000 pg/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Porcine Interleukin 10, Porcine IL-10, Swine Interleukin 10, Cytokine Synthesis Inhibitory Factor, CSIF
Interleukin 10, also known as cytokine synthesis inhibitory factor (CSIF), is the charter member of the IL-10 cytokine family. This family currently comprises IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26/AK155. All IL-10 family members are secreted α-helical proteins. Porcine IL-10 is a secreted, possibly glycosylated, polypeptide with an 18 kDa molecular weight. Based on human studies, porcine IL-10 is likely to circulate as a nondisulfide-linked homodimer. Porcine IL-10 is synthesized as a 175 amino acid (aa) precursor with an 18 aa signal sequence and a 157 aa mature form. The mature segment has one potential N-linked glycosylation site plus four cysteines which form two intrachain disulfide bridges. Mature porcine IL-10 shows 71%, 70%, 76%, 75%, 77%, and 71% aa sequence identity to rat, mouse, human, guinea pig, canine, and cotton rat IL-10, respectively. Upon activation, mammalian cells known to secrete IL-10 include NK cells, cytotoxic CD8+ T cells secreting Th2-like cytokines, CD4+CD45RA- (memory) Th1 and Th2 cells, macrophages, monocytes, CD5+ and CD5- B cells, dendritic cells, hepatic stellate (Ito cells), keratinocytes, melanoma cells, mast cells, placental cytotrophoblasts, and fetal erythroblasts.
The functional receptor for IL-10 (IL-10 R) in pigs has not been reported. By analogy to human, it would be expected to be composed of two 110 kDa a-chains (or IL-10 R1) and two 75 kDa b-chains (or IL-10 R2). The a-chain binds IL-10 and transduces a signal in the presence of a b-chain complex. Both receptors are members of the class II cytokine receptor family (CRF2) that is characterized by the presence of type III fibronectin domains and conserved tryptophans. This class does not possess the WSXWS motif characteristic of the class I CRF. There is no significant aa sequence identity (< 30%) between human IL-10 R1 and IL-10 R2.
IL-10 has myriad effects on a variety of cell types. On activated B cells, IL-10 can induce plasma cell formation (32) and the secretion of either IgG or IgA (in the presence of TGF-b1 and/or IL-4). In the presence of IL-2, CD56+ NK cells will respond to IL-10 with increased proliferation plus IFN-g and TNF-a secretion. Conversely, on macrophages, IL-10 is known to downregulate IL-1, TNF-a, and IL-6 production . On dendritic cells, IL-10 has been shown to interfere with antigen-presenting cell function by downmodulating stimulatory and co-stimulatory molecules. On monocytes, IL-10 is reported to direct monocyte differentiation into cytotoxic CD16+ macrophages rather than antigen-presenting dendritic cells.
Swine IL-4 ELISA Assay
Swine IL-2 ELISA Assay
Mouse IL-10 ELISA Assay
The Eagle Biosciences Porcine Interleukin 10 (IL-10) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-10 has been pre-coated onto a microplate. Standard, control, or sample and the working solution of Biotin-Conjugate are pipetted into the wells. Following incubation and wash steps any IL-10 present is bound by the immobilized antibody and the detection antibody specific for IL-10 is binds to the combination of capture antibody-IL-10 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-10 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-10 standard dilutions and IL-10 sample concentration determined.
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µL of Standard, control, or sample, per well, then add 50 µL of the working solution of Biotin-Conjugate to each well. Cover with the adhesive strip provided and incubate 2 hours at RT. Adequate mixing is very important for good result. Use a mini-vortexer at the lowest frequency.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at RT Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at RT. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. (optionally 630nm as the reference wave length; 610-650nm is acceptable)
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
Ren, J;Lu, H;Wen, S;Sun, W;Yan, F;Chen, X;Jing, J;Liu, H;Liu, C;Xue, F;Xiao, P;Xin, S;Jin, N;, Enhanced immune responses in pigs by DNA vaccine coexpressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus, Journal of Virol. Methods, Volume 206C, Page 27-37, https://www.sciencedirect.com/science/article/pii/S0166093414002134
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