Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human AIRE in samples. No significant cross-reactivity or interference between Human AIRE and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human AIRE. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human AIRE and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human AIRE, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human AIRE. The concentration of Human AIRE in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	AIRE: Transcriptional regulator that binds to DNA as a dimer or as a tetramer, but not as a monomer. 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. 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. Functions as a transcriptional activator and promotes the expression of otherwise tissue-specific self-antigens in the thymus, which is important for self tolerance and the avoidance of autoimmune reactions. Defects in AIRE are a cause of autoimmune poly- endocrinopathy candidiasis ectodermal dystrophy (APS1). An autosomal recessive disease characterized by the combination of chronic mucocutaneous candidiasis, hypoparathyroidism and Addison disease. Symptoms of mucocutaneous candidiasis manifest first, followed by hypotension or fatigue occurring as a result of Addison disease. APS1 is associated with other autoimmune disorders including diabetes mellitus, vitiligo, alopecia, hepatitis, pernicious anemia and primary hypothyroidism. Most of the mutations alter the nucleus-cytoplasm distribution of AIRE and disturb its association with nuclear dots and cytoplasmic filaments. Most of the mutations also decrease transactivation of the protein. The HSR domain is responsible for the homomultimerization activity of AIRE. All the missense mutations of the HSR and the SAND domains decrease this activity, but those in other domains do not. The AIRE protein is present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturb the formation of these complexes. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Transcription factor Chromosomal Location of Human Ortholog: 21q22. 3 Molecular Function:chromatin binding; histone binding; protein binding; transcription cofactor activity; zinc ion binding Biological Process: immune response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of transcription, DNA-dependent Disease: Autoimmune Polyendocrine Syndrome, Type I, With Or Without Reversible Metaphyseal Dysplasia
NCBI Summary:	This gene encodes a transcriptional regulator that forms nuclear bodies and interacts with the transcriptional coactivator CREB binding protein. The encoded protein plays an important role in immunity by regulating the expression of autoantigens and negative selection of autoreactive T-cells in the thymus. Mutations in this gene cause the rare autosomal-recessive systemic autoimmune disease termed autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy (APECED). [provided by RefSeq, Jun 2012]
UniProt Code:	O43918
NCBI GenInfo Identifier:	3334119
NCBI Gene ID:	326
NCBI Accession:	O43918. 1
UniProt Secondary Accession:	O43918,O43922, O43932, O75745, B2RP50,
UniProt Related Accession:	O43918
Molecular Weight:	36,501 Da
NCBI Full Name:	Autoimmune regulator
NCBI Synonym Full Names:	autoimmune regulator
NCBI Official Symbol:	AIRE
NCBI Official Synonym Symbols:	APS1; APSI; PGA1; AIRE1; APECED
NCBI Protein Information:	autoimmune regulator
UniProt Protein Name:	Autoimmune regulator
UniProt Synonym Protein Names:	Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy protein; APECED protein
Protein Family:	Autoimmune regulator
UniProt Gene Name:	AIRE
UniProt Entry Name:	AIRE_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
2000	55299 56143	55721	55693
1000	21709 26239	23974	23946
500	11151 10913	11032	11004
250	5167 5421	5294	5266
125	2697 2519	2608	2580
62.5	1396 1226	1311	1283
31.25	646 702	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AIRE were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AIRE were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	100.52	262.77	966.93	100.87	281.12	972.00
Standard deviation	9.20	24.49	101.62	8.43	27.83	107.31
C V (%)	9.15	9.32	10.51	8.36	9.90	11.04

Recovery

The recovery of Human AIRE spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	94-106	100
EDTA plasma (n=5)	92-103	97
Cell culture media (n=5)	91-104	96

Linearity

Samples were spiked with high concentrations of Human AIRE and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	88-102	100-115	101-117
1:2	Average (%)	93	106	109
1:4	Range (%)	100-119	84-97	102-120
1:4	Average (%)	109	91	110
1:8	Range (%)	91-104	98-111	85-97
1:8	Average (%)	98	106	90
1:16	Range (%)	99-115	85-99	84-99
1:16	Average (%)	105	91	91

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.