Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human GDNF in samples. No significant cross-reactivity or interference between Human GDNF 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 GDNF. 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 GDNF 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 GDNF, 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 GDNF. The concentration of Human GDNF in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	GDNF: Neurotrophic factor that enhances survival and morphological differentiation of dopaminergic neurons and increases their high-affinity dopamine uptake. Defects in GDNF may be a cause of Hirschsprung disease type 3 (HSCR3). In association with mutations of RET gene, defects in GDNF may be involved in Hirschsprung disease. This genetic disorder of neural crest development is characterized by the absence of intramural ganglion cells in the hindgut, often resulting in intestinal obstruction. Defects in GDNF are a cause of congenital central hypoventilation syndrome (CCHS); also known as congenital failure of autonomic control or Ondine curse. CCHS is a rare disorder characterized by abnormal control of respiration in the absence of neuromuscular or lung disease, or an identifiable brain stem lesion. A deficiency in autonomic control of respiration results in inadequate or negligible ventilatory and arousal responses to hypercapnia and hypoxemia. Belongs to the TGF-beta family. GDNF subfamily. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 5p13. 1-p12 Cellular Component: extracellular region Molecular Function:protein homodimerization activity; growth factor activity; receptor binding Biological Process: positive regulation of dopamine secretion; axon guidance; nervous system development; peristalsis; adult locomotory behavior; mRNA stabilization; regulation of dopamine uptake; positive regulation of monooxygenase activity; signal transduction; enteric nervous system development; sympathetic nervous system development; ureteric bud branching; regulation of gene expression; induction of an organ; positive regulation of cell proliferation; postganglionic parasympathetic nervous system development; positive regulation of transcription from RNA polymerase II promoter; negative regulation of neuron apoptosis; positive regulation of cell differentiation; postsynaptic membrane organization; metanephros development; neural crest cell migration; neurite development; negative regulation of apoptosis Disease: Hirschsprung Disease, Susceptibility To, 3; Central Hypoventilation Syndrome, Congenital; Pheochromocytoma
NCBI Summary:	This gene encodes a highly conserved neurotrophic factor. The recombinant form of this protein was shown to promote the survival and differentiation of dopaminergic neurons in culture, and was able to prevent apoptosis of motor neurons induced by axotomy. The encoded protein is processed to a mature secreted form that exists as a homodimer. The mature form of the protein is a ligand for the product of the RET (rearranged during transfection) protooncogene. Multiple transcript variants encoding different isoforms have been found for this gene. Mutations in this gene may be associated with Hirschsprung disease. [provided by RefSeq, Jun 2010]
UniProt Code:	P39905
NCBI GenInfo Identifier:	729567
NCBI Gene ID:	2668
NCBI Accession:	P39905. 1
UniProt Secondary Accession:	P39905,O95448, O95449, O95986, Q6FH33, Q96L44, Q9UD32 Q9UD33, Q9UMV2, Q9UP67, Q9UP97, B7WPK7,
UniProt Related Accession:	P39905,AAB33493,AAB33494
Molecular Weight:	18,123 Da
NCBI Full Name:	Glial cell line-derived neurotrophic factor
NCBI Synonym Full Names:	glial cell derived neurotrophic factor
NCBI Official Symbol:	GDNF
NCBI Official Synonym Symbols:	ATF1; ATF2; HSCR3; HFB1-GDNF
NCBI Protein Information:	glial cell line-derived neurotrophic factor; ATF; astrocyte-derived trophic factor
UniProt Protein Name:	Glial cell line-derived neurotrophic factor
UniProt Synonym Protein Names:	Astrocyte-derived trophic factor; ATF
Protein Family:	GDNF family receptor
UniProt Gene Name:	GDNF
UniProt Entry Name:	GDNF_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 (ng/mL)	O.D	Average	Corrected
20	2.501 2.515	2.508	2.438
10	1.64 1.7	1.67	1.6
5	1.008 0.984	0.996	0.926
2.5	0.443 0.465	0.454	0.384
1.25	0.27 0.252	0.261	0.191
0.63	0.183 0.163	0.173	0.103
0.31	0.112 0.132	0.122	0.052
0	0.061 0.079	0.07	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GDNF 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 (ng/mL)	1.06	2.18	9.18	1.06	2.09	9.23
Standard deviation	0.06	0.11	0.35	0.07	0.11	0.40
C V (%)	5.66	5.05	3.81	6.60	5.26	4.33

Recovery

The recovery of Human GDNF 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)	92-104	98
EDTA plasma (n=5)	87-99	92
Cell culture media (n=5)	86-98	92

Linearity

Samples were spiked with high concentrations of Human GDNF 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 (%)	95-112	86-96	99-113
1:2	Average (%)	103	91	105
1:4	Range (%)	89-106	82-97	83-96
1:4	Average (%)	97	88	90
1:8	Range (%)	90-102	81-93	87-102
1:8	Average (%)	96	86	93
1:16	Range (%)	88-104	86-98	87-99
1:16	Average (%)	95	91	93

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 Ã—12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100Ã—)	1 vial, 120 µL
Concentrated HRP Conjugate (100Ã—)	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 (25Ã—)	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.