Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	1.56-100 ng/mL
Sensitivity:	0.94 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human VWF in samples. No significant cross-reactivity or interference between Human VWF 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 Human VWF. 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 Human VWF 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 VWF, 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 Human VWF. The concentration of Human VWF in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	VWF: Important in the maintenance of hemostasis, it promotes adhesion of platelets to the sites of vascular injury by forming a molecular bridge between sub-endothelial collagen matrix and platelet-surface receptor complex GPIb-IX-V. Also acts as a chaperone for coagulation factor VIII, delivering it to the site of injury, stabilizing its heterodimeric structure and protecting it from premature clearance from plasma. Defects in VWF are the cause of von Willebrand disease type 1 (VWD1). A common hemorrhagic disorder due to defects in von Willebrand factor protein and resulting in impaired platelet aggregation. Von Willebrand disease type 1 is characterized by partial quantitative deficiency of circulating von Willebrand factor, that is otherwise structurally and functionally normal. Clinical manifestations are mucocutaneous bleeding, such as epistaxis and menorrhagia, and prolonged bleeding after surgery or trauma. Defects in VWF are the cause of von Willebrand disease type 2 (VWD2). A hemorrhagic disorder due to defects in von Willebrand factor protein and resulting in impaired platelet aggregation. Von Willebrand disease type 2 is characterized by qualitative deficiency and functional anomalies of von Willebrand factor. It is divided in different subtypes including 2A, 2B, 2M and 2N (Normandy variant). The mutant VWF protein in types 2A, 2B and 2M are defective in their platelet- dependent function, whereas the mutant protein in type 2N is defective in its ability to bind factor VIII. Clinical manifestations are mucocutaneous bleeding, such as epistaxis and menorrhagia, and prolonged bleeding after surgery or trauma. Defects in VWF are the cause of von Willebrand disease type 3 (VWD3). A severe hemorrhagic disorder due to a total or near total absence of von Willebrand factor in the plasma and cellular compartments, also leading to a profound deficiency of plasmatic factor VIII. Bleeding usually starts in infancy and can include epistaxis, recurrent mucocutaneous bleeding, excessive bleeding after minor trauma, and hemarthroses.
UniProt Protein Details:	Protein type:Cell adhesion; Secreted, signal peptide; Extracellular matrix; Motility/polarity/chemotaxis; Secreted Chromosomal Location of Human Ortholog: 12p13. 3 Cellular Component: extracellular matrix; proteinaceous extracellular matrix; endoplasmic reticulum; extracellular region; external side of plasma membrane Molecular Function:integrin binding; collagen binding; identical protein binding; protein binding; protein homodimerization activity; protease binding; chaperone binding; immunoglobulin binding; protein N-terminus binding; glycoprotein binding Biological Process: platelet activation; extracellular matrix organization and biogenesis; platelet degranulation; hemostasis; response to wounding; liver development; blood coagulation; cell adhesion; blood coagulation, intrinsic pathway; protein homooligomerization; cell-substrate adhesion; placenta development Disease: Von Willebrand Disease, Type 3; Von Willebrand Disease, Type 1; Von Willebrand Disease, Type 2
NCBI Summary:	The glycoprotein encoded by this gene functions as both an antihemophilic factor carrier and a platelet-vessel wall mediator in the blood coagulation system. It is crucial to the hemostasis process. Mutations in this gene or deficiencies in this protein result in von Willebrand's disease. An unprocessed pseudogene has been found on chromosome 22. [provided by RefSeq, Jul 2008]
UniProt Code:	P04275
NCBI GenInfo Identifier:	317373549
NCBI Gene ID:	7450
NCBI Accession:	P04275. 4
UniProt Secondary Accession:	P04275,Q8TCE8, Q99806,
UniProt Related Accession:	P04275
Molecular Weight:	Calculated MW: 38kDa/309kDaObserved MW: Refer to figures
NCBI Full Name:	von Willebrand factor
NCBI Synonym Full Names:	von Willebrand factor
NCBI Official Symbol:	VWF
NCBI Official Synonym Symbols:	VWD; F8VWF
NCBI Protein Information:	von Willebrand factor; coagulation factor VIII VWF
UniProt Protein Name:	von Willebrand factor
UniProt Synonym Protein Names:	von Willebrand antigen II
Protein Family:	von Willebrand factor
UniProt Gene Name:	VWF
UniProt Entry Name:	VWF_HUMAN

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
100	2.462 2.468	2.465	2.374
50	1.571 1.591	1.581	1.49
25	0.994 0.982	0.988	0.897
12.5	0.488 0.498	0.493	0.402
6.25	0.294 0.278	0.286	0.195
3.13	0.201 0.183	0.192	0.101
1.56	0.139 0.147	0.143	0.052
0	0.083 0.099	0.091	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human VWF 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)	4.94	13.78	40.52	4.74	13.70	42.12
Standard deviation	0.30	0.64	1.94	0.25	0.68	1.44
C V (%)	6.07	4.64	4.79	5.27	4.96	3.42

Recovery

The recovery of Human VWF 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)	90-106	97
EDTA plasma (n=5)	91-103	98
Cell culture media (n=5)	92-104	98

Linearity

Samples were spiked with high concentrations of Human VWF 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 (%)	90-102	94-109	84-97
1:2	Average (%)	96	101	91
1:4	Range (%)	94-105	80-93	89-100
1:4	Average (%)	100	86	95
1:8	Range (%)	93-106	84-96	88-101
1:8	Average (%)	98	91	94
1:16	Range (%)	87-103	81-94	83-95
1:16	Average (%)	94	88	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.