Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection method:	Chemiluminescence
Detection range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Mouse Slit2 in samples. No significant cross-reactivity or interference between Mouse Slit2 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 Mouse Slit2. 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 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 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 Mouse Slit2. The concentration of Mouse Slit2 in the samples can be calculated by comparing the RLU 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 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
4000	56603 61559	59081	59046
2000	26594 29124	27859	27824
1000	14353 12615	13484	13449
500	6111 7099	6605	6570
250	3260 3226	3243	3208
125	1643 1519	1581	1546
62.50	713 797	755	720
0	35 35	35	--

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 (pg/mL)	197.81	505.80	1654.53	193.60	479.88	1568.12
Standard deviation	25.42	41.43	132.20	15.64	52.79	126.70
C V (%)	12.85	8.19	7.99	8.08	11.00	8.08

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)	97-112	104
EDTA plasma (n=5)	94-109	101
Cell culture media (n=5)	87-101	94

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 (%)	89-99	85-98	94-108
1:2	Average (%)	94	91	101
1:4	Range (%)	88-101	84-100	92-105
1:4	Average (%)	94	91	99
1:8	Range (%)	100-115	88-102	99-112
1:8	Average (%)	107	94	105
1:16	Range (%)	93-111	100-115	97-114
1:16	Average (%)	101	105	104

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.