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

UniProt Protein Function:	elastin: Major structural protein of tissues such as aorta and nuchal ligament, which must expand rapidly and recover completely. Molecular determinant of the late arterial morphogenesis, stabilizing arterial structure by regulating proliferation and organization of vascular smooth muscle. Defects in ELN are the cause of cutis laxa, autosomal dominant, type 1 (ADCL1). A connective tissue disorder characterized by loose, hyperextensible skin with decreased resilience and elasticity leading to a premature aged appearance. Face, hands, feet, joints, and torso may be differentially affected. Additional variable clinical features are gastrointestinal diverticula, hernia, and genital prolapse. Rare manifestations are pulmonary artery stenosis, aortic aneurysm, bronchiectasis, and emphysema. Defects in ELN are the cause of supravalvular aortic stenosis (SVAS). SVAS is a congenital narrowing of the ascending aorta which can occur sporadically, as an autosomal dominant condition, or as one component of Williams-Beuren syndrome. ELN is located in the Williams-Beuren syndrome (WBS) critical region. WBS results from a hemizygous deletion of several genes on chromosome 7q11. 23, thought to arise as a consequence of unequal crossing over between highly homologous low-copy repeat sequences flanking the deleted region. Haploinsufficiency of ELN may be the cause of certain cardiovascular and musculo-skeletal abnormalities observed in the disease. Belongs to the elastin family. 13 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide; Extracellular matrix Cellular Component: proteinaceous extracellular matrix; mitochondrion; extracellular region Molecular Function:protein binding; extracellular matrix binding; extracellular matrix structural constituent Biological Process: regulation of actin filament polymerization; extracellular matrix organization and biogenesis; skeletal muscle development; stress fiber formation
NCBI Summary:	This gene encodes elastin, the extracellular matrix protein that forms a major structural component of several tissues including lungs and arterial walls. Cleavage of the signal peptide from the encoded precursor generates soluble tropoelastin which undergoes lysine-derived crosslinking to form elastin polymers. Mice lacking the encoded protein exhibit defective lung development, and die of an obstructive arterial disease resulting from subendothelial cell proliferation and reorganization of smooth muscle. [provided by RefSeq, Aug 2015]
UniProt Code:	P54320
NCBI GenInfo Identifier:	31542606
NCBI Gene ID:	13717
NCBI Accession:	NP_031951. 2
UniProt Secondary Accession:	P54320,Q8C9L8,
UniProt Related Accession:	P54320
Molecular Weight:	71,938 Da
NCBI Full Name:	elastin
NCBI Synonym Full Names:	elastin
NCBI Official Symbol:	Eln
NCBI Official Synonym Symbols:	AI385707; AI480567; E030024M20Rik
NCBI Protein Information:	elastin; tropoelastin
UniProt Protein Name:	Elastin
UniProt Synonym Protein Names:	Tropoelastin
Protein Family:	Elastin
UniProt Gene Name:	Eln
UniProt Entry Name:	ELN_MOUSE

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	2.488 2.518	2.503	2.424
5	1.576 1.624	1.6	1.521
2.5	0.946 0.916	0.931	0.852
1.25	0.513 0.541	0.527	0.448
0.63	0.276 0.264	0.27	0.191
0.32	0.182 0.18	0.181	0.102
0.16	0.126 0.136	0.131	0.052
0	0.077 0.081	0.079	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse ELN 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)	0.50	0.94	3.67	0.52	1.02	3.89
Standard deviation	0.03	0.04	0.11	0.03	0.05	0.18
C V (%)	6.00	4.26	3.00	5.77	4.90	4.63

Recovery

The recovery of Mouse ELN 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)	94-107	101
EDTA plasma (n=5)	90-106	98
Cell culture media (n=5)	94-105	99

Linearity

Samples were spiked with high concentrations of Mouse ELN 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 (%)	99-110	96-112	89-105
1:2	Average (%)	105	104	96
1:4	Range (%)	91-105	80-93	84-95
1:4	Average (%)	98	86	88
1:8	Range (%)	91-103	82-95	88-101
1:8	Average (%)	97	86	95
1:16	Range (%)	91-103	84-99	85-99
1:16	Average (%)	96	90	90

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.