Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	23.44-1500 pg/mL
Sensitivity:	14.06 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human FXN in samples. No significant cross-reactivity or interference between Human FXN and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human FXN. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human FXN 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 FXN, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human FXN. The concentration of Human FXN in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	FXN: Promotes the biosynthesis of heme and assembly and repair of iron-sulfur clusters by delivering Fe(2+) to proteins involved in these pathways. May play a role in the protection against iron-catalyzed oxidative stress through its ability to catalyze the oxidation of Fe(2+) to Fe(3+); the oligomeric form but not the monomeric form has in vitro ferroxidase activity. May be able to store large amounts of iron in the form of a ferrihydrite mineral by oligomerization; however, the physiological relevance is unsure as reports are conflicting and the function has only been shown using heterologous overexpression systems. Modulates the RNA-binding activity of ACO1. Defects in FXN are the cause of Friedreich ataxia (FRDA). FRDA is an autosomal recessive, progressive degenerative disease characterized by neurodegeneration and cardiomyopathy it is the most common inherited ataxia. The disorder is usually manifest before adolescence and is generally characterized by incoordination of limb movements, dysarthria, nystagmus, diminished or absent tendon reflexes, Babinski sign, impairment of position and vibratory senses, scoliosis, pes cavus, and hammer toe. In most patients, FRDA is due to GAA triplet repeat expansions in the first intron of the frataxin gene. But in some cases the disease is due to mutations in the coding region. Belongs to the frataxin family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Mitochondrial; EC 1. 16. 3. 1 Chromosomal Location of Human Ortholog: 9q21. 11 Cellular Component: mitochondrion; mitochondrial matrix; cytosol Molecular Function:2 iron, 2 sulfur cluster binding; ferroxidase activity; protein binding; ferric iron binding; ferrous iron binding; iron-sulfur cluster binding Biological Process: mitochondrion organization and biogenesis; negative regulation of multicellular organism growth; cellular iron ion homeostasis; positive regulation of metalloenzyme activity; positive regulation of transferase activity; proprioception; negative regulation of organ growth; positive regulation of cell growth; adult walking behavior; protein autoprocessing; embryonic development ending in birth or egg hatching; iron incorporation into metallo-sulfur cluster; positive regulation of lyase activity; positive regulation of cell proliferation; aerobic respiration; ion transport; response to iron ion; negative regulation of apoptosis; positive regulation of oxidoreductase activity; oxidative phosphorylation; heme biosynthetic process Disease: Friedreich Ataxia 1
NCBI Summary:	This nuclear gene encodes a mitochondrial protein which belongs to the FRATAXIN family. The protein functions in regulating mitochondrial iron transport and respiration. The expansion of intronic trinucleotide repeat GAA from 8-33 repeats to >90 repeats results in Friedreich ataxia. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2016]
UniProt Code:	Q16595
NCBI GenInfo Identifier:	6166193
NCBI Gene ID:	2395
NCBI Accession:	Q16595. 2
UniProt Related Accession:	Q16595
Molecular Weight:	23kDa
NCBI Full Name:	Frataxin, mitochondrial
NCBI Synonym Full Names:	frataxin
NCBI Official Symbol:	FXN
NCBI Official Synonym Symbols:	FA; X25; CyaY; FARR; FRDA
NCBI Protein Information:	frataxin, mitochondrial
UniProt Protein Name:	Frataxin, mitochondrial
UniProt Synonym Protein Names:	Friedreich ataxia protein; Fxn
UniProt Gene Name:	FXN
UniProt Entry Name:	FRDA_HUMAN

As the OD 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)	O.D	Average	Corrected
1500	2.542 2.554	2.548	2.46
750	1.734 1.77	1.752	1.664
375	1.021 1.013	1.017	0.929
187.5	0.488 0.502	0.495	0.407
93.75	0.284 0.262	0.273	0.185
46.88	0.199 0.185	0.192	0.104
23.44	0.14 0.142	0.141	0.053
0	0.087 0.089	0.088	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FXN 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)	69.06	138.67	606.92	75.37	148.26	635.12
Standard deviation	4.03	5.67	31.92	3.98	6.75	32.58
C V (%)	5.84	4.09	5.26	5.28	4.55	5.13

Recovery

The recovery of Human FXN 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)	92-105	98
EDTA plasma (n=5)	93-107	98
Cell culture media (n=5)	85-100	92

Linearity

Samples were spiked with high concentrations of Human FXN 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 (%)	89-101	93-109	99-109
1:2	Average (%)	94	99	104
1:4	Range (%)	88-100	85-98	87-101
1:4	Average (%)	95	90	94
1:8	Range (%)	90-100	83-97	86-98
1:8	Average (%)	95	90	92
1:16	Range (%)	87-101	83-94	81-94
1:16	Average (%)	93	88	88

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.