Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Mouse COL1 alpha2 in samples. No significant cross-reactivity or interference between Mouse COL1 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 Mouse COL1 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 Mouse COL1 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 Mouse COL1 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 Mouse COL1 alpha2. The concentration of Mouse COL1 alpha2 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	COL1A2: Type I collagen is a member of group I collagen (fibrillar forming collagen). Defects in COL1A2 are the cause of Ehlers-Danlos syndrome type 7B (EDS7B). EDS is a connective tissue disorder characterized by hyperextensible skin, atrophic cutaneous scars due to tissue fragility and joint hyperlaxity. EDS7B is marked by bilateral congenital hip dislocation, hyperlaxity of the joints, and recurrent partial dislocations. Defects in COL1A2 are a cause of osteogenesis imperfecta type 1 (OI1). A dominantly inherited connective tissue disorder characterized by bone fragility and blue sclerae. Osteogenesis imperfecta type 1 is non-deforming with normal height or mild short stature, and no dentinogenesis imperfecta. Defects in COL1A2 are a cause of osteogenesis imperfecta type 2 (OI2); also known as osteogenesis imperfecta congenita (OIC) or lethal perinatal. A connective tissue disorder characterized by bone fragility, with many perinatal fractures, severe bowing of long bones, undermineralization, and death in the perinatal period due to respiratory insufficiency. Defects in COL1A2 are the cause of Ehlers-Danlos syndrome autosomal recessive cardiac valvular form (EDSCV). A connective tissue disorder characterized by hyperextensible skin, atrophic cutaneous scars due to tissue fragility and joint hyperlaxity. In addition to joint laxity, skin hyperextensibility and friability, and abnormal scar formation, patients have mitral valve prolapse and insufficiency, mitral regurgitation, and aortic insufficiency. Defects in COL1A2 are a cause of osteogenesis imperfecta type 3 (OI3). A connective tissue disorder characterized by progressively deforming bones, very short stature, a triangular face, severe scoliosis, grayish sclera, and dentinogenesis imperfecta. Defects in COL1A2 are a cause of osteogenesis imperfecta type 4 (OI4); also known as osteogenesis imperfecta with normal sclerae. A connective tissue disorder characterized by moderately short stature, mild to moderate scoliosis, grayish or white sclera and dentinogenesis imperfecta. A chromosomal aberration involving COL1A2 may be a cause of lipoblastomas, which are benign tumors resulting from transformation of adipocytes, usually diagnosed in children. Translocation t(7;8)(p22;q13) with PLAG1. Belongs to the fibrillar collagen family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Cellular Component: extracellular matrix; proteinaceous extracellular matrix; extracellular space; collagen; extracellular region; collagen type I; intracellular Molecular Function:protein binding, bridging; identical protein binding; protein binding; platelet-derived growth factor binding; extracellular matrix structural constituent; metal ion binding; SMAD binding Biological Process: blood vessel development; collagen fibril organization; skin morphogenesis; transforming growth factor beta receptor signaling pathway; regulation of blood pressure; skeletal development; Rho protein signal transduction
UniProt Code:	Q01149
NCBI GenInfo Identifier:	111120329
NCBI Gene ID:	12843
NCBI Accession:	NP_031769. 2
UniProt Secondary Accession:	Q01149,Q8CGA5,
UniProt Related Accession:	Q01149
Molecular Weight:	129,557 Da
NCBI Full Name:	collagen alpha-2(I) chain
NCBI Synonym Full Names:	collagen, type I, alpha 2
NCBI Official Symbol:	Col1a2
NCBI Official Synonym Symbols:	oim; Cola2; Cola-2; Col1a-2; AA960264; AI325291
NCBI Protein Information:	collagen alpha-2(I) chain; collagen alpha-2(I) chain; alpha-2 type I collagen; collagen COL1A2; osteogenesis imperfecta; procollagen, type I, alpha 2
UniProt Protein Name:	Collagen alpha-2(I) chain
UniProt Synonym Protein Names:	Alpha-2 type I collagen
Protein Family:	Collagen
UniProt Gene Name:	Col1a2
UniProt Entry Name:	CO1A2_MOUSE

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
2000	52132 59310	55721	55693
1000	22361 25587	23974	23946
500	11292 10772	11032	11004
250	5079 5509	5294	5266
125	2675 2541	2608	2580
62.5	1353 1269	1311	1283
31.25	668 680	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse COL1 alpha2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse COL1 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 (pg/mL)	98.23	260.00	678.63	90.99	248.33	730.76
Standard deviation	11.79	18.49	76.07	11.05	20.49	54.22
C V (%)	12.00	7.11	11.21	12.14	8.25	7.42

Recovery

The recovery of Mouse COL1 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)	94-108	101
EDTA plasma (n=5)	99-113	106
Cell culture media (n=5)	99-111	105

Linearity

Samples were spiked with high concentrations of Mouse COL1 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 (%)	96-110	97-111	88-100
1:2	Average (%)	102	104	95
1:4	Range (%)	91-103	100-116	87-101
1:4	Average (%)	97	108	93
1:8	Range (%)	96-108	86-99	94-107
1:8	Average (%)	102	92	102
1:16	Range (%)	90-104	93-107	101-114
1:16	Average (%)	97	98	107

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.