Mouse Slit2 ELISA Kit (MOES01291)- High Sensitivity

Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
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 Mouse Slit2 in samples. No significant cross-reactivity or interference between Mouse Slit2 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 Mouse Slit2. 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 Mouse Slit2 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 Mouse Slit2, 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 Mouse Slit2. The concentration of Mouse Slit2 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	SLIT2: Thought to act as molecular guidance cue in cellular migration, and function appears to be mediated by interaction with roundabout homolog receptors. During neural development involved in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions. SLIT1 and SLIT2 seem to be essential for midline guidance in the forebrain by acting as repulsive signal preventing inappropriate midline crossing by axons projecting from the olfactory bulb. In spinal chord development may play a role in guiding commissural axons once they reached the floor plate by modulating the response to netrin. In vitro, silences the attractive effect of NTN1 but not its growth- stimulatory effect and silencing requires the formation of a ROBO1-DCC complex. May be implicated in spinal chord midline post- crossing axon repulsion. In vitro, only commissural axons that crossed the midline responded to SLIT2. In the developing visual system appears to function as repellent for retinal ganglion axons by providing a repulsion that directs these axons along their appropriate paths prior to, and after passage through, the optic chiasm. In vitro, collapses and repels retinal ganglion cell growth cones. Seems to play a role in branching and arborization of CNS sensory axons, and in neuronal cell migration. In vitro, Slit homolog 2 protein N-product, but not Slit homolog 2 protein C-product, repels olfactory bulb (OB) but not dorsal root ganglia (DRG) axons, induces OB growth cones collapse and induces branching of DRG axons. Seems to be involved in regulating leukocyte migration. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Extracellular matrix; Motility/polarity/chemotaxis; Secreted; Secreted, signal peptide Cellular Component: cytoplasm; extracellular space; plasma membrane; proteinaceous extracellular matrix Molecular Function:chemorepellent activity; GTPase inhibitor activity; heparan sulfate proteoglycan binding; heparin binding; laminin-1 binding; protein binding; protein homodimerization activity; proteoglycan binding; receptor binding; Roundabout binding Biological Process: axon extension involved in axon guidance; axon guidance; axonogenesis; branching morphogenesis of a tube; cell migration during sprouting angiogenesis; cell-cell adhesion; central nervous system projection neuron axonogenesis; chemorepulsion involved in postnatal olfactory bulb interneuron migration; corticospinal neuron axon guidance through the spinal cord; dorsal/ventral axon guidance; in utero embryonic development; induction of negative chemotaxis; metanephros development; motor axon guidance; negative chemotaxis; negative regulation of actin filament polymerization; negative regulation of axon extension; negative regulation of cell growth; negative regulation of cell migration; negative regulation of cell proliferation; negative regulation of inflammatory response; negative regulation of leukocyte chemotaxis; negative regulation of protein amino acid phosphorylation; negative regulation of small GTPase mediated signal transduction; negative regulation of smooth muscle cell migration; negative regulation of vascular permeability; neurite morphogenesis; olfactory bulb development; positive regulation of apoptosis; retinal ganglion cell axon guidance; telencephalon cell migration; ureteric bud development
UniProt Code:	Q9R1B9
NCBI GenInfo Identifier:	410304000
NCBI Gene ID:	461137
NCBI Accession:	Q9R1B9
Molecular Weight:
NCBI Full Name:	slit homolog 2
NCBI Official Symbol:	SLIT2
NCBI Protein Information:	slit homolog 2 protein
UniProt Protein Name:	Slit homolog 2
UniProt Synonym Protein Names:	Slit homolog 2
Protein Family:	Slit homolog 2 protein
UniProt Gene Name:	SLIT2
UniProt Entry Name:	K7CZV3_PANTR

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	2.327 2.387	2.357	2.276
10	1.547 1.585	1.566	1.485
5	0.929 0.905	0.917	0.836
2.5	0.43 0.448	0.439	0.358
1.25	0.281 0.275	0.278	0.197
0.63	0.185 0.169	0.177	0.096
0.31	0.122 0.138	0.13	0.049
0	0.075 0.087	0.081	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse Slit2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse Slit2 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)	0.92	2.30	9.03	0.90	2.09	8.21
Standard deviation	0.05	0.12	0.42	0.05	0.10	0.36
C V (%)	5.43	5.22	4.65	5.56	4.78	4.38

Recovery

The recovery of Mouse Slit2 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)	87-97	92
EDTA plasma (n=5)	91-105	98
Cell culture media (n=5)	92-105	98

Linearity

Samples were spiked with high concentrations of Mouse Slit2 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 (%)	88-98	89-103	94-104
1:2	Average (%)	93	96	99
1:4	Range (%)	90-105	81-90	85-97
1:4	Average (%)	97	85	90
1:8	Range (%)	90-104	81-94	82-94
1:8	Average (%)	95	86	88
1:16	Range (%)	88-103	82-95	84-98
1:16	Average (%)	94	86	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 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.