Interferon-gamma (IFN-, also known as type II interferon) is an important immunoregulatory cytokine that was originally identified because of its anti-viral activity (1). It plays key roles in host defense by exerting antiviral, antiproliferative and immunoregulatory activities (2 - 5). On many cell types, IFN- induces the production of cytokines and upregulates the expression of various membrane proteins including class I and II MHC antigens, Fc receptors, leukocyte adhesion molecules and B7 family antigens. IFN- is a potent activator of macrophage effector functions. It potentiates the secretion of immunoglobulins by B cells, and directs the synthesis of IgG. IFN- also influences T-helper cell phenotype development by inhibiting Th2 differentiation and stimulating Th1 development (2 - 5). Finally, IFN- promotes mononuclear cell chemotaxis by inducing the synthesis of CXCL9, CXCL10, CCL2, CCL3, CCL4, and CCL5(3). IFN- is produced by a number of cell types, including dendritic epidermal/ T cells (6), keratinocytes (7), peripheral blood T cells (8), mast cells (9), neurons (10), CD8 T cells (11), macrophages (12), B cells (13), neutrophils (14), NK cells (15), CD4 T cells (16) and testicular spermatids (17). The production of IFN- is upregulated synergistically by IL-12, IL-18, IL-23 and IL-27 (18 - 21). Porcine IFN- cDNA encodes a 166 amino acid (aa) residue precursor protein with a 20 aa signal sequence that is cleaved to generate a 146 aa residue mature IFN- (22, 23). Porcine IFN- is presumably a noncovalently linked homodimer (3). In the mature segment, porcine IFN- shares 60%, 55%, 41%, 42%, 72%, and 72% aa sequence identity with human (24), guinea pig (25), mouse (26), rat (27), feline (28), and canine (29) IFN-, respectively.

The functional IFN- receptor complex consists of two distinct subunits (30). The alpha-subunit(IFN-R1) binds IFN- with high affinity and species specificity. The beta-subunit [IFN-R2, also known as accessory factor-1 (AF-1)] interacts with the IFN- occupied-subunit in a species-specific manner and participates in JAK-STAT mediated signal transduction. Although the functional receptor is suggested to consist of homodimeric IFN- in combination with two-chains, and two-chains (30, 31), it has been suggested that additional subunits may be involved (32, 33). Whereas the-chain is expressed constitutively on many cell types, the cellular regulation of the-chain correlates with an IFN- responsive state and is tightly regulated (30).

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IFN-γ 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 IFN-γ present is bound by the immobilized antibody and the detection antibody specific for IFN-γ is binds to the combination of capture antibody- IFN-γ 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 coloured product is formed in proportion to the amount of IFN-γ 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 IFN-γ standard dilutions and IFN-γ sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to swine IFN-γ (812)

2. 2 vials swine IFN-γ Standard lyophilized, 4000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-swine IFN-γ antibody

4. 2 vials Streptavidin-HRP solution,

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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