Human Autoimmune Regulator (AIRE) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to AIRE. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to AIRE. 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 AIRE, 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 AIRE in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For use with: Tissue homogenates and Other biological fluids.

For Research Use Only. Not for use in diagnostic procedures.

Target Information

Transcription factor playing an essential role to promote self-tolerance in the thymus by regulating the expression of a wide array of self-antigens that have the commonality of being tissue-restricted in their expression pattern in the periphery, called tissue restricted antigens (TRA) (PubMed:26084028). Binds to G-doublets in an A/T-rich environment; the preferred motif is a tandem repeat of 5'-ATTGGTTA-3' combined with a 5'-TTATTA-3' box. Binds to nucleosomes (By similarity). Binds to chromatin and interacts selectively with histone H3 that is not methylated at 'Lys-4', not phosphorylated at 'Thr-3' and not methylated at 'Arg-2'. Functions as a sensor of histone H3 modifications that are important for the epigenetic regulation of gene expression. Mainly expressed by medullary thymic epithelial cells (mTECs), induces the expression of thousands of tissue-restricted proteins, which are presented on major histocompatibility complex class I (MHC-I) and MHC-II molecules to developing T-cells percolating through the thymic medulla (PubMed:26084028). Also induces self-tolerance through other mechanisms such as the regulation of the mTEC differentiation program. Controls the medullary accumulation of thymic dendritic cells and the development of regulatory T-cell through the regulation of XCL1 expression. Regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. In thimic B-cells, allows the presentation of licensing-dependent endogenous self-anitgen for negative selection. In secondary lymphoid organs, induces functional inactivation of CD4(+) T-cells. Expressed by a distinct bone marrow-derived population, induces self-tolerance through a mechanism that does not require regulatory T-cells and is resitant to innate inflammatory stimuli (By similarity).

GENE ID	326
SWISS PROT	O43918
SYNONYMS	AIRE1; APECED; APS1; APSI; PGA1; Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy

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 (30X concentrate)	1 tube
Product data sheet	1 copy

Storage

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 AIRE were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level AIRE 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 0.053ng/mL.
ASSAY RANGE	0.156–10ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of AIRE. No significant cross-reactivity or interference between AIRE and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between AIRE and all analogs, therefore, cross reactivity may still exist.