Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Plasma
Repeatability:	CV < 15%
Specificity:	This kit recognizes Rat VWF in samples. No significant cross-reactivity or interference between Rat VWF 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 Rat VWF. 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 Rat 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 Rat VWF, 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 Rat VWF. The concentration of Rat VWF in the samples can be calculated by comparing the RLU 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:Extracellular matrix; Secreted; Secreted, signal peptide; Cell adhesion; Motility/polarity/chemotaxis 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 homodimerization activity; protease binding; chaperone binding; protein N-terminus binding; immunoglobulin binding; glycoprotein binding Biological Process: platelet activation; tissue regeneration; hemostasis; liver development; cell adhesion; blood coagulation; cell-substrate adhesion; protein homooligomerization; placenta development
NCBI Summary:	human homolog is a plasma glycoprotein that mediates platelet adhesion to damaged blood vessels and stabilizes blood coagulation factor VIII [RGD, Feb 2006]
UniProt Code:	Q62935
NCBI GenInfo Identifier:	56404678
NCBI Gene ID:	116669
NCBI Accession:	Q62935. 2
UniProt Secondary Accession:	Q62935,Q78E31, Q9Z0P2,
UniProt Related Accession:	Q62935
Molecular Weight:	Predicted Molecular Mass: 22. 3kDaAccurate Molecular Mass: 22kDa as determined by SDS-PAGE reducing conditions.
NCBI Full Name:	von Willebrand factor
NCBI Synonym Full Names:	von Willebrand factor
NCBI Official Symbol:	Vwf
NCBI Protein Information:	von Willebrand factor
UniProt Protein Name:	von Willebrand factor
Protein Family:	von Willebrand factor
UniProt Gene Name:	Vwf
UniProt Entry Name:	VWF_RAT

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	55595 55847	55721	55693
1000	23565 24383	23974	23946
500	11748 10316	11032	11004
250	5196 5392	5294	5266
125	2804 2412	2608	2580
62.5	1385 1237	1311	1283
31.25	655 693	674	646
0	27 29	28	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat 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 (pg/mL)	106.77	206.02	743.87	115.47	190.30	718.62
Standard deviation	10.84	15.39	51.48	10.38	17.20	81.13
C V (%)	10.15	7.47	6.92	8.99	9.04	11.29

Recovery

The recovery of Rat 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)	94-109	101
EDTA plasma (n=5)	95-109	100
Cell culture media (n=5)	88-102	95

Linearity

Samples were spiked with high concentrations of Rat 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 (%)	86-96	97-112	101-115
1:2	Average (%)	91	103	108
1:4	Range (%)	92-105	89-103	101-115
1:4	Average (%)	97	96	109
1:8	Range (%)	103-116	91-106	94-109
1:8	Average (%)	109	97	101
1:16	Range (%)	98-112	89-104	88-100
1:16	Average (%)	104	96	93

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.