IFN GAMMA HUMAN ELISA

BRAND:                 DEMEDITEC DIAGNOSTICS GMBH

PRODUCT CODE: DE4434

AVAILABILITY:     IN STOCK

DESCRIPTION

• CE certifed for IVD use

• 96 well ELISA microplate

• Incubation time: 2 h 15 min

• Range: 0.51 - 21.2 IU/ml

• Sensitivity: 0.03 IU/ml

• Sample size: 50 µl

• Sample type: serum, plasma

• Substrate: TMB 450nm

1. INTENDED USE

Immunoenzymetric assay for the in vitro quantitative measurement of human interferon gamma (IFN-gamma) in serum and plasma.

2. CLINICAL BACKGROUND

A. Biological activities

IFN-γ (type 2, immune IFN) is structurally and functionally distinct from type 1 (alpha/beta) interferon and acts on a separate receptor. Only one IFN-γ gene has been identified, coding for a 146 AA protein that is post-translationaly processed into two glycosylated species of 20 and 25 Kd. Native IFN-γ is pH2-labile, highly basic, and can aggregate to form dimers that are biologically active. IFN-γ is a real lymphokine produced by activated T (and NK) cells. Despite its clear antiviral and cellular growth regulating activities, its immunomodulatory properties are believed to be the most important. IFN-γ is the principal activator of macrophage function (Macrophage Activating Factor, MAF), and it also regulates the pathway of differentiation of myeloid cells. It plays an important role in the growth and differentiation of cytotoxic (and possibly suppressor) T cells, activates NK cells and acts as a B cell maturation factor. It regulates Ig isotype production and inhibits IgE responses. One of the modes of action of IFN-γ is to induce the expression of membrane proteins, such as class 1 and class 2 MHC antigens and adhesion molecules on various cell types, high affinity Fc receptors for IgG on myelomonocytic cells, etc. Integrated in the cytokine network, IFN-γ interacts with other cytokines, in either a synergistic (e.g. TNF) or antagonistic (e.g. IL-4) way.

B. Clinical application

The precise role of IFN-γ in human diseases and therapy is still poorly defined. Clearly, it is involved in the defense against parasites, intercellular pathogens and possibly tumour cells. Its therapeutic administration partially corrects the deficient immune response observed in lepromatous leprosy and the phagocyte defect of patients with X-Linked chronic granulomatous disease. A deficiency in IFN-γ production has been related to persistent (e.g. EBV) viral infections, and a correlation could be established between the secretion of IFN-γ by peripheral blood mononuclear cells during an herpetic infection and the time of a next recurrence. A defect in IFN-γ production has also been recorded in several primary or secondary immunodeficiency states. IFN-γ is seldom detected in the serum of healthy persons. Its production may be demonstrated "in situ" in several inflammatory disorders (Sarcoidosis, rheumatoid-arthritis, subacute thyroiditis, polymyositis, multiple sclerosis). Higher levels of serum IFN-γ are measured during severe parasitic diseases (e.g. Plasmodium Falciparium malaria); during cytokine (IL-2) therapy; and after the first injections of OKT3.

3. PRINCIPLES OF THE METHOD

The IFN-γ-ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on microtiterplate. The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of IFN-γ. Calibrators and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP). After an incubation period allowing the formation of a sandwich: coated MAb 1 – human IFN-γ – MAb 2 – HRP, the microtiterplate is washed to remove unbound enzyme labelled antibody. Bound enzyme- labelled antibody is measured through a chromogenic reaction. Chromogenic solution (TMB) is added and incubated. The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength. The amount of substrate turnover is determined colourimetrically by measuring the absorbance, which is proportional to the IFN-γ concentration.

A calibration curve is plotted and IFN-γ concentration in samples is determined by interpolation from the calibration curve. The use of the ELISA reader (linearity up to 3 OD units) and a sophisticated data reduction method (polychromatic data reduction) result in a high sensitivity in the low range and in an extended calibration range.