Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human GREM1 in samples. No significant cross-reactivity or interference between Human GREM1 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 GREM1. 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 GREM1 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 GREM1, 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 GREM1. The concentration of Human GREM1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	GREM1: Cytokine that may play an important role during carcinogenesis and metanephric kidney organogenesis, as a BMP antagonist required for early limb outgrowth and patterning in maintaining the FGF4-SHH feedback loop. Down-regulates the BMP4 signaling in a dose-dependent manner. Acts as inhibitor of monocyte chemotaxis. Interacts with SLIT1 and SLIT2 in a glycosylation- dependent manner. By high glucose through TGFB1-mediated pathways in mesangial cell. Down-regulated in tumor cell lines. Highly expressed in small intestine, fetal brain and colon. Weakly expressed in brain, ovary, prostate, pancreas and skeletal muscle. In brain found in the region localized around the internal capsule in the large subcortical nuclei, including caudate, putamen, substantia nigra, thalamus and subthalamus. Predominantly expressed in normal cells including neurons, astrocytes and fibroblasts. Belongs to the DAN family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 15q13. 3 Molecular Function:morphogen activity; protein binding; receptor agonist activity; transmembrane receptor protein tyrosine kinase activator activity; vascular endothelial growth factor receptor 2 binding Biological Process: cell migration during sprouting angiogenesis; cell morphogenesis; collagen fibril organization; determination of dorsal identity; limb development; negative regulation of apoptosis; negative regulation of BMP signaling pathway; negative regulation of bone mineralization; negative regulation of bone remodeling; negative regulation of chondrocyte differentiation; negative regulation of osteoblast proliferation; positive regulation of cell proliferation; positive regulation of receptor internalization; positive regulation of telomerase activity; positive regulation of transcription from RNA polymerase II promoter; regulation of stress-activated MAPK cascade; signal transduction
NCBI Summary:	This gene encodes a member of the BMP (bone morphogenic protein) antagonist family. Like BMPs, BMP antagonists contain cystine knots and typically form homo- and heterodimers. The CAN (cerberus and dan) subfamily of BMP antagonists, to which this gene belongs, is characterized by a C-terminal cystine knot with an eight-membered ring. The antagonistic effect of the secreted glycosylated protein encoded by this gene is likely due to its direct binding to BMP proteins. As an antagonist of BMP, this gene may play a role in regulating organogenesis, body patterning, and tissue differentiation. In mouse, this protein has been shown to relay the sonic hedgehog (SHH) signal from the polarizing region to the apical ectodermal ridge during limb bud outgrowth. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2010]
UniProt Code:	O60565
NCBI GenInfo Identifier:	62510668
NCBI Gene ID:	26585
NCBI Accession:	O60565. 1
UniProt Secondary Accession:	O60565,Q52LV3, Q8N914, Q8N936,
UniProt Related Accession:	O60565
Molecular Weight:
NCBI Full Name:	Gremlin-1
NCBI Synonym Full Names:	gremlin 1, DAN family BMP antagonist
NCBI Official Symbol:	GREM1
NCBI Official Synonym Symbols:	DRM; HMPS; MPSH; PIG2; CRAC1; CRCS4; DAND2; HMPS1; IHG-2; DUP15q; C15DUPq; GREMLIN; CKTSF1B1
NCBI Protein Information:	gremlin-1
UniProt Protein Name:	Gremlin-1
UniProt Synonym Protein Names:	Cell proliferation-inducing gene 2 protein; Cysteine knot superfamily 1, BMP antagonist 1; DAN domain family member 2; Down-regulated in Mos-transformed cells protein; Increased in high glucose protein 2; IHG-2
Protein Family:	Gremlin
UniProt Gene Name:	GREM1
UniProt Entry Name:	GREM1_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
10	2.451 2.461	2.456	2.365
5	1.728 1.768	1.748	1.657
2.5	0.956 0.946	0.951	0.86
1.25	0.534 0.542	0.538	0.447
0.63	0.273 0.261	0.267	0.176
0.32	0.203 0.193	0.198	0.107
0.16	0.145 0.147	0.146	0.055
0	0.089 0.093	0.091	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human GREM1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human GREM1 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)	0.47	1.43	4.34	0.45	1.38	4.48
Standard deviation	0.03	0.06	0.13	0.03	0.08	0.24
C V (%)	6.38	4.20	3.00	6.67	5.80	5.36

Recovery

The recovery of Human GREM1 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)	84-97	90
EDTA plasma (n=5)	89-102	97
Cell culture media (n=5)	88-101	95

Linearity

Samples were spiked with high concentrations of Human GREM1 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 (%)	86-101	85-97	87-97
1:2	Average (%)	92	91	92
1:4	Range (%)	88-100	84-97	82-97
1:4	Average (%)	95	90	89
1:8	Range (%)	89-102	87-98	80-94
1:8	Average (%)	94	93	87
1:16	Range (%)	88-103	80-93	87-100
1:16	Average (%)	94	86	92

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.