Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	93.75-6000 ng/mL
Sensitivity:	56.25 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human ELN in samples. No significant cross-reactivity or interference between Human ELN 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 ELN. 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 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 Human ELN, 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 ELN. The concentration of Human ELN in the samples can be calculated by comparing the RLU 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:Extracellular matrix; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 7q11. 23 Cellular Component: extracellular region Molecular Function:protein binding Biological Process: blood circulation; cell proliferation; extracellular matrix disassembly; extracellular matrix organization and biogenesis; organ morphogenesis; respiratory gaseous exchange Disease: Cutis Laxa, Autosomal Dominant 1; Supravalvular Aortic Stenosis; Williams-beuren Syndrome
NCBI Summary:	This gene encodes a protein that is one of the two components of elastic fibers. The encoded protein is rich in hydrophobic amino acids such as glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. Deletions and mutations in this gene are associated with supravalvular aortic stenosis (SVAS) and autosomal dominant cutis laxa. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P15502
NCBI GenInfo Identifier:	306526276
NCBI Gene ID:	2006
NCBI Accession:	P15502. 3
UniProt Secondary Accession:	P15502,O15336, O15337, Q14233, Q14234, Q14235, Q14238 Q6P0L4, Q6ZWJ6, Q75MU5, Q7Z316, B3KTS6,
UniProt Related Accession:	P15502
Molecular Weight:	68kDa
NCBI Full Name:	Elastin
NCBI Synonym Full Names:	elastin
NCBI Official Symbol:	ELN
NCBI Official Synonym Symbols:	WS; WBS; SVAS
NCBI Protein Information:	elastin
UniProt Protein Name:	Elastin
UniProt Synonym Protein Names:	Tropoelastin
Protein Family:	Elastin
UniProt Gene Name:	ELN

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 (ng/mL)	RLU	Average	Corrected
6000	62806 68460	65633	65614
3000	33578 36400	34989	34970
1500	19184 16324	17754	17735
750	8251 9063	8657	8638
375	4009 3969	3989	3970
187.5	1749 1501	1625	1606
93.75	432 440	436	417
0	19 19	19	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human 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)	321.00	543.85	2645.01	339.13	561.97	2411.78
Standard deviation	34.44	43.29	204.46	36.56	47.99	252.03
C V (%)	10.73	7.96	7.73	10.78	8.54	10.45

Recovery

The recovery of Human 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)	89-104	96
EDTA plasma (n=5)	97-112	102
Cell culture media (n=5)	102-115	108

Linearity

Samples were spiked with high concentrations of Human 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 (%)	91-106	87-103	86-98
1:2	Average (%)	99	94	92
1:4	Range (%)	91-105	88-104	100-118
1:4	Average (%)	98	95	108
1:8	Range (%)	88-103	91-105	88-100
1:8	Average (%)	95	99	93
1:16	Range (%)	92-107	91-107	96-107
1:16	Average (%)	100	99	101

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.