Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.63-40 ng/mL
Sensitivity:	0.38 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human FBLN5 in samples. No significant cross-reactivity or interference between Human FBLN5 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 FBLN5. 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 FBLN5 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 FBLN5, 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 FBLN5. The concentration of Human FBLN5 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	FBLN5: Promotes adhesion of endothelial cells through interaction of integrins and the RGD motif. Could be a vascular ligand for integrin receptors and may play a role in vascular development and remodeling. Defects in FBLN5 are the cause of cutis laxa, autosomal dominant, type 2 (ADCL2). 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 FBLN5 are a cause of cutis laxa, autosomal recessive, type 1A (ARCL1A). 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. The clinical spectrum of autosomal recessive cutis laxa is highly heterogeneous with respect to organ involvement and severity. Type I autosomal recessive cutis laxa is a specific, life-threatening disorder with organ involvement, lung atelectasis and emphysema, diverticula of the gastrointestinal and genitourinary systems, and vascular anomalies. Associated cranial anomalies, late closure of the fontanel, joint laxity, hip dislocation, and inguinal hernia have been observed but are uncommon. Defects in FBLN5 are the cause of age-related macular degeneration type 3 (ARMD3). ARMD is a multifactorial disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid (known as drusen) that lie beneath the retinal pigment epithelium and within an elastin-containing structure known as Bruch membrane. Belongs to the fibulin family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 14q32. 1 Cellular Component: extracellular matrix; extracellular region; extracellular space; proteinaceous extracellular matrix Molecular Function:calcium ion binding; integrin binding; protein binding; protein C-terminus binding; protein homodimerization activity Biological Process: cell-matrix adhesion; elastic fiber assembly; extracellular matrix organization and biogenesis; regulation of cell growth; secretion Disease: Cutis Laxa, Autosomal Dominant 2; Cutis Laxa, Autosomal Recessive, Type Ia; Macular Degeneration, Age-related, 3
NCBI Summary:	The protein encoded by this gene is a secreted, extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif and calcium-binding EGF-like domains. It promotes adhesion of endothelial cells through interaction of integrins and the RGD motif. It is prominently expressed in developing arteries but less so in adult vessels. However, its expression is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells. Therefore, the protein encoded by this gene may play a role in vascular development and remodeling. Defects in this gene are a cause of autosomal dominant cutis laxa, autosomal recessive cutis laxa type I (CL type I), and age-related macular degeneration type 3 (ARMD3). [provided by RefSeq, Jul 2008]
UniProt Code:	Q9UBX5
NCBI GenInfo Identifier:	12643876
NCBI Gene ID:	10516
NCBI Accession:	Q9UBX5. 1
UniProt Secondary Accession:	Q9UBX5,O75966, Q6IAL4, Q6UWA3,
UniProt Related Accession:	Q9UBX5
Molecular Weight:	50,180 Da
NCBI Full Name:	Fibulin-5
NCBI Synonym Full Names:	fibulin 5
NCBI Official Symbol:	FBLN5
NCBI Official Synonym Symbols:	EVEC; UP50; ADCL2; ARMD3; DANCE; ARCL1A; FIBL-5; HNARMD
NCBI Protein Information:	fibulin-5
UniProt Protein Name:	Fibulin-5
UniProt Synonym Protein Names:	Developmental arteries and neural crest EGF-like protein; Dance; Urine p50 protein; UP50
Protein Family:	Fibulin
UniProt Gene Name:	FBLN5
UniProt Entry Name:	FBLN5_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 (ng/mL)	RLU	Average	Corrected
40	36001 37075	36538	36508
20	13411 15221	14316	14286
10	6277 6269	6273	6243
5	2920 3116	3018	2988
2.5	1648 1516	1582	1552
1.25	928 896	912	882
0.63	566 612	589	559
0	29 31	30	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human FBLN5 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human FBLN5 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)	2.01	4.64	16.36	2.03	5.09	17.42
Standard deviation	0.19	0.41	1.66	0.22	0.52	1.56
C V (%)	9.45	8.84	10.15	10.84	10.22	8.96

Recovery

The recovery of Human FBLN5 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)	91-103	98
EDTA plasma (n=5)	90-105	96
Cell culture media (n=5)	89-104	95

Linearity

Samples were spiked with high concentrations of Human FBLN5 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 (%)	88-102	84-98	86-101
1:2	Average (%)	95	90	92
1:4	Range (%)	100-114	86-102	90-105
1:4	Average (%)	106	93	96
1:8	Range (%)	87-100	103-120	98-110
1:8	Average (%)	93	110	104
1:16	Range (%)	98-113	87-100	104-119
1:16	Average (%)	104	92	110

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.