Contents of Kit
1. Porcine IL-10 Microplate, 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody speciffc for porcine IL-10. Return unused wells to the foil pouch containing the desiccant pack. Reseal along entire edge of the zip-seal. May be stored for up to 1 month at 2-8°C.** Provided this is within the expiration date of the kit.
2. Porcine IL-10 Standard, 2 vials of recombinant porcine IL-10 in a buffered protein base with preservatives; lyophilized. Refer to the vial label for reconstitution volume. Use a new standard and control for each assay. Discard after use.
3. Porcine IL-10 Control, 2 vials of recombinant porcine IL-10 in a buffered protein base with preservatives; lyophilized. The assay value of the control should be within the range speciffed on the label. Use a new standard and control for each assay. Discard after use.
4. Porcine IL-10 Conjugate, 23 mL of a polyclonal antibody speciffc for porcine IL-10 conjugated to horseradish peroxidase with preservatives.May be stored for up to 1 month at 2-8°C.*
5. Assay Diluent CD1W, 12 mL of a buffered protein base with preservatives. May be stored for up to 1 month at 2-8 °C.*
6. Calibrator Diluent CD6-33, 21 mL of a buffered protein base with preservatives. May be stored for up to 1 month at 2-8 °C.*
7. Wash Buffer Concentrate, 21 mL of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time. May be stored for up to 1 month at 2-8°C.*
8. Color Reagent A, 12 mL of stabilized hydrogen peroxide. May be stored for up to 1 month at 2-8°C.*
9. Color Reagent B, 12 mL of stabilized chromogen (tetramethylbenzidine). May be stored for up to 1 month at 2-8°C.*
10. Stop Solution, 23 mL of diluted hydrochloric acid. May be stored for up to 1 month at 2-8 °C.*
11. Plate Sealers, 4 adhesive strips.
General Description
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 nondisulffde-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 disulffde 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 α-chains (or IL-10 R1) and two 75 kDa β-chains (or IL-10 R2). The α-chain binds IL-10 and transduces a signal in the presence of a β-chain complex. Both receptors are members of the class II cytokine receptor family (CRF2) that is characterized by the presence of type III ffbronectin domains and conserved tryptophans. This class does not possess the WSXWS motif characteristic of the class I CRF. There is no signiffcant 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 and the secretion of either IgG or IgA (in the presence of TGF-β1 and/or IL-4). In the presence of IL-2, CD56+ NK cells will respond to IL-10 with increased proliferation plus IFN-γ and TNF-α secretion. Conversely, on macrophages, IL-10 is known to downregulate IL-1, TNF-α, 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.
The Porcine IL-10 Immunoassay is a 4.5 hour solid phase ELISA designed to measure porcine IL-10 levels in cell culture supernates, serum, and EDTA plasma. It contains E. coli-expressed recombinant porcine IL-10 and antibodies raised against the recombinant protein. Results obtained for naturally occurring porcine IL-10 showed linear curves that were parallel to the standard curves obtained using the kit standards. These results indicate that this kit can be used to determine relative mass values of natural porcine IL-10.
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