Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human SCF in samples. No significant cross-reactivity or interference between Human SCF 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 SCF. 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 SCF 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 SCF, 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 SCF. The concentration of Human SCF in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	SCF: Ligand for the receptor-type protein-tyrosine kinase KIT. Plays an essential role in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and in melanogenesis. KITLG/SCF binding can activate several signaling pathways. Promotes phosphorylation of PIK3R1, the regulatory subunit of phosphatidylinositol 3-kinase, and subsequent activation of the kinase AKT1. KITLG/SCF and KIT also transmit signals via GRB2 and activation of RAS, RAF1 and the MAP kinases MAPK1/ERK2 and/or MAPK3/ERK1. KITLG/SCF and KIT promote activation of STAT family members STAT1, STAT3 and STAT5. KITLG/SCF and KIT promote activation of PLCG1, leading to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5- trisphosphate. KITLG/SCF acts synergistically with other cytokines, probably interleukins. Homodimer, non-covalently linked (Probable). Heterotetramer with KIT, binding two KIT molecules; thereby mediates KIT dimerization and subsequent activation by autophosphorylation. Belongs to the SCF family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral Chromosomal Location of Human Ortholog: 12q22 Cellular Component: extracellular space; cytoskeleton; cytoplasm; integral to membrane; extracellular region; plasma membrane Molecular Function:protein binding; growth factor activity; cytokine activity; stem cell factor receptor binding Biological Process: epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; phosphoinositide-mediated signaling; nerve growth factor receptor signaling pathway; embryonic hemopoiesis; male gonad development; germ cell programmed cell death; signal transduction; positive regulation of MAP kinase activity; cell proliferation; positive regulation of peptidyl-tyrosine phosphorylation; negative regulation of mast cell apoptosis; positive regulation of melanocyte differentiation; innate immune response; positive regulation of Ras protein signal transduction; positive regulation of myeloid leukocyte differentiation; cell adhesion; neural crest cell migration; positive regulation of DNA replication Disease: Hyperpigmentation, Familial Progressive, 2; Skin/hair/eye Pigmentation, Variation In, 7
NCBI Summary:	This gene encodes the ligand of the tyrosine-kinase receptor encoded by the KIT locus. This ligand is a pleiotropic factor that acts in utero in germ cell and neural cell development, and hematopoiesis, all believed to reflect a role in cell migration. In adults, it functions pleiotropically, while mostly noted for its continued requirement in hematopoiesis. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P21583
NCBI GenInfo Identifier:	134289
NCBI Gene ID:	4254
NCBI Accession:	P21583. 1
UniProt Secondary Accession:	P21583,Q16487, Q68DZ2, Q7M4N8, Q9UQK7, A0AV09, A8K2Q4 B7ZLM4,
UniProt Related Accession:	P21583
Molecular Weight:	Approximately 150 kDa
NCBI Full Name:	Kit ligand
NCBI Synonym Full Names:	KIT ligand
NCBI Official Symbol:	KITLG
NCBI Official Synonym Symbols:	SF; MGF; SCF; FPH2; KL-1; Kitl; SHEP7
NCBI Protein Information:	kit ligand; c-Kit ligand; steel factor; stem cell factor; mast cell growth factor; familial progressive hyperpigmentation 2
UniProt Protein Name:	Kit ligand
UniProt Synonym Protein Names:	Mast cell growth factor; MGF; Stem cell factor; SCF; c-Kit ligandCleaved into the following chain:Soluble KIT ligand; sKITLG
Protein Family:	SCF-associated factor
UniProt Gene Name:	KITLG
UniProt Entry Name:	SCF_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
1000	50489 56861	53675	53650
500	20317 24661	22489	22464
250	10344 10078	10211	10186
125	4678 5124	4901	4876
62.5	2546 2360	2453	2428
31.25	1349 1213	1281	1256
15.63	687 729	708	683
0	24 26	25	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SCF 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)	52.31	133.07	424.59	52.42	140.94	385.49
Standard deviation	5.26	10.22	42.59	5.23	12.15	25.44
C V (%)	10.06	7.68	10.03	9.98	8.62	6.60

Recovery

The recovery of Human SCF 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)	91-105	98
EDTA plasma (n=5)	93-105	100
Cell culture media (n=5)	88-102	93

Linearity

Samples were spiked with high concentrations of Human SCF 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 (%)	92-108	83-97	101-116
1:2	Average (%)	100	90	109
1:4	Range (%)	102-119	91-105	96-109
1:4	Average (%)	109	98	102
1:8	Range (%)	97-109	102-117	90-105
1:8	Average (%)	102	109	96
1:16	Range (%)	100-114	101-118	101-116
1:16	Average (%)	107	108	109

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.