Mouse IL2 Inducible T-Cell Kinase (ITK) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to ITK. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to ITK. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain ITK, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of ITK in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Tyrosine kinase that plays an essential role in regulation of the adaptive immune response. Regulates the development, function and differentiation of conventional T-cells and nonconventional NKT-cells. When antigen presenting cells (APC) activate T-cell receptor (TCR), a series of phosphorylation lead to the recruitment of ITK to the cell membrane, in the vicinity of the stimulated TCR receptor, where it is phosphorylated by LCK. Phosphorylation leads to ITK autophosphorylation and full activation. Once activated, phosphorylates PLCG1, leading to the activation of this lipase and subsequent cleavage of its substrates. In turn, the endoplasmic reticulum releases calcium in the cytoplasm and the nuclear activator of activated T-cells (NFAT) translocates into the nucleus to perform its transcriptional duty. Phosphorylates 2 essential adapter proteins: the linker for activation of T-cells/LAT protein and LCP2. Then, a large number of signaling molecules such as VAV1 are recruited and ultimately lead to lymphokine production, T-cell proliferation and differentiation. Required for TCR-mediated calcium response in gamma-delta T-cells, may also be involved in the modulation of the transcriptomic signature in the Vgamma2-positive subset of immature gamma-delta T-cells (PubMed:23562159). Phosphorylates TBX21 at 'Tyr-525' and mediates its interaction with GATA3 (PubMed:15662016).

GENE ID	16428
SWISS PROT	Q03526
SYNONYMS	PSCTK2; EMT; LYK; T-cell-specific kinase; Tyrosine-protein kinase Lyk

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level ITK were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level ITK were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 0.107ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of ITK. No significant cross-reactivity or interference between ITK and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between ITK and all analogs, therefore, cross reactivity may still exist.