Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	1.56-100 ng/mL
Sensitivity:	0.94 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human COL5 alpha2 in samples. No significant cross-reactivity or interference between Human COL5 alpha2 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 COL5 alpha2. 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 COL5 alpha2 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 COL5 alpha2, 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 COL5 alpha2. The concentration of Human COL5 alpha2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	COL5A2: Type V collagen is a member of group I collagen (fibrillar forming collagen). It is a minor connective tissue component of nearly ubiquitous distribution. Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin. Type V collagen is a key determinant in the assembly of tissue- specific matrices. Defects in COL5A2 are a cause of Ehlers-Danlos syndrome type 1 (EDS1); also known as Ehlers-Danlos syndrome gravis or severe classic type Ehlers-Danlos syndrome. EDS is a connective tissue disorder characterized by hyperextensible skin, atrophic cutaneous scars due to tissue fragility and joint hyperlaxity. EDS1 is the severe form of classic Ehlers-Danlos syndrome. Defects in COL5A2 are a cause of Ehlers-Danlos syndrome type 2 (EDS2); also known as Ehlers-Danlos syndrome mitis or mild classic type Ehlers Danlos syndrome. Belongs to the fibrillar collagen family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 2q14-q32 Cellular Component: collagen type V; endoplasmic reticulum lumen; extracellular region Biological Process: collagen catabolic process; collagen fibril organization; extracellular matrix organization and biogenesis; eye morphogenesis; skin development Disease: Ehlers-danlos Syndrome, Type I
NCBI Summary:	This gene encodes an alpha chain for one of the low abundance fibrillar collagens. Fibrillar collagen molecules are trimers that can be composed of one or more types of alpha chains. Type V collagen is found in tissues containing type I collagen and appears to regulate the assembly of heterotypic fibers composed of both type I and type V collagen. This gene product is closely related to type XI collagen and it is possible that the collagen chains of types V and XI constitute a single collagen type with tissue-specific chain combinations. Mutations in this gene are associated with Ehlers-Danlos syndrome, types I and II. [provided by RefSeq, Jul 2008]
UniProt Code:	P05997
NCBI GenInfo Identifier:	143811378
NCBI Gene ID:	1290
NCBI Accession:	P05997. 3
UniProt Secondary Accession:	P05997,P78440, Q13908, Q53WR4, Q59GR4, Q6LDJ5, Q7KZ55 Q86XF6, Q96QB0, Q96QB3,
UniProt Related Accession:	P05997
Molecular Weight:	144,910 Da
NCBI Full Name:	Collagen alpha-2(V) chain
NCBI Synonym Full Names:	collagen type V alpha 2 chain
NCBI Official Symbol:	COL5A2
NCBI Official Synonym Symbols:	EDSC
NCBI Protein Information:	collagen alpha-2(V) chain
UniProt Protein Name:	Collagen alpha-2(V) chain
Protein Family:	Collagen
UniProt Gene Name:	COL5A2
UniProt Entry Name:	CO5A2_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
100	35756 39352	37554	37521
50	15535 16947	16241	16208
25	7587 7497	7542	7509
12.5	3457 3907	3682	3649
6.25	1939 1811	1875	1842
3.13	1057 945	1001	968
1.56	539 605	572	539
0	33 33	33	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human COL5 alpha2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human COL5 alpha2 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)	5.55	15.16	45.91	5.30	15.65	47.40
Standard deviation	0.67	1.42	3.27	0.61	1.61	4.35
C V (%)	12.07	9.37	7.12	11.51	10.29	9.18

Recovery

The recovery of Human COL5 alpha2 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)	84-96	90
EDTA plasma (n=5)	94-111	102
Cell culture media (n=5)	93-109	101

Linearity

Samples were spiked with high concentrations of Human COL5 alpha2 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 (%)	94-106	94-106	95-111
1:2	Average (%)	99	100	102
1:4	Range (%)	90-105	88-103	91-102
1:4	Average (%)	98	94	97
1:8	Range (%)	95-111	90-105	91-104
1:8	Average (%)	101	96	97
1:16	Range (%)	89-102	91-105	99-115
1:16	Average (%)	94	97	105

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.