Mouse FV ELISA Kit (MOES00872)- High Sensitivity

Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	3.13-200 ng/mL
Sensitivity:	1.88 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse FV in samples. No significant cross-reactivity or interference between Mouse FV 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 Mouse FV. 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 Mouse FV 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 FV, 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 Mouse FV. The concentration of Mouse FV in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	factor V: Central regulator of hemostasis. It serves as a critical cofactor for the prothrombinase activity of factor Xa that results in the activation of prothrombin to thrombin. Defects in F5 are the cause of factor V deficiency (FA5D); also known as Owren parahemophilia. It is an hemorrhagic diastesis. Defects in F5 are the cause of thrombophilia due to activated protein C resistance (THPH2). THPH2 is a hemostatic disorder due to defective degradation of factor Va by activated protein C. It is characterized by a poor anticoagulant response to activated protein C resulting in tendency to thrombosis. Defects in F5 are a cause of susceptibility to Budd- Chiari syndrome (BDCHS). A syndrome caused by obstruction of hepatic venous outflow involving either the hepatic veins or the terminal segment of the inferior vena cava. Obstructions are generally caused by thrombosis and lead to hepatic congestion and ischemic necrosis. Clinical manifestations observed in the majority of patients include hepatomegaly, right upper quadrant pain and abdominal ascites. Budd-Chiari syndrome is associated with a combination of disease states including primary myeloproliferative syndromes and thrombophilia due to factor V Leiden, protein C deficiency and antithrombin III deficiency. Budd-Chiari syndrome is a rare but typical complication in patients with polycythemia vera. Defects in F5 may be a cause of susceptibility to ischemic stroke (ISCHSTR); also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. Defects in F5 are associated with susceptibility to pregnancy loss, recurrent, type 1 (RPRGL1). RPRGL1 is a common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions. Belongs to the multicopper oxidase family.
UniProt Protein Details:	Protein type:Protease; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1 H2. 2\|1 71. 46 cM Cellular Component: endoplasmic reticulum; extracellular region; extracellular space; Golgi apparatus; membrane Molecular Function:copper ion binding; metal ion binding Biological Process: blood circulation; blood coagulation; hemostasis
NCBI Summary:	This gene encodes a glycoprotein coagulation factor that plays a critical role in the process of blood coagulation and hemostasis. The encoded protein is activated by thrombin, to generate a heterodimer containing heavy and light chains held together by calcium ions. About half of the mice lacking the encoded protein die at an embryonic stage possible due to abnormal yolk-sac vasculature while the remaining animals succumbed to massive hemorrhage immediately after birth. A point mutation in this gene has been shown to cause disseminated intravascular thrombosis in the perinatal period, resulting in frequent deaths of newborn mice. [provided by RefSeq, Apr 2015]
UniProt Code:	O88783
NCBI GenInfo Identifier:	6679731
NCBI Gene ID:	14067
NCBI Accession:	NP_032002. 1
UniProt Secondary Accession:	O88783,Q3UTQ2, Q3UV80,
UniProt Related Accession:	O88783
Molecular Weight:
NCBI Full Name:	coagulation factor V preproprotein
NCBI Synonym Full Names:	coagulation factor V
NCBI Official Symbol:	F5
NCBI Official Synonym Symbols:	Cf5; Cf-5; AI173222
NCBI Protein Information:	coagulation factor V
UniProt Protein Name:	Coagulation factor V
UniProt Synonym Protein Names:	Activated protein C cofactor
Protein Family:	Coagulation factor
UniProt Gene Name:	F5

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
200	2.305 2.315	2.31	2.244
100	1.485 1.527	1.506	1.44
50	0.847 0.835	0.841	0.775
25	0.404 0.444	0.424	0.358
12.5	0.214 0.208	0.211	0.145
6.25	0.165 0.141	0.153	0.087
3.13	0.108 0.116	0.112	0.046
0	0.064 0.068	0.066	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse FV 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)	9.14	23.00	73.02	8.76	23.03	67.34
Standard deviation	0.48	1.36	3.61	0.52	1.08	2.30
C V (%)	5.25	5.91	4.94	5.94	4.69	3.42

Recovery

The recovery of Mouse FV 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)	87-101	93
EDTA plasma (n=5)	93-107	99
Cell culture media (n=5)	86-98	92

Linearity

Samples were spiked with high concentrations of Mouse FV 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 (%)	86-101	97-111	86-100
1:2	Average (%)	92	103	93
1:4	Range (%)	90-104	86-96	85-95
1:4	Average (%)	96	91	90
1:8	Range (%)	89-102	83-94	84-97
1:8	Average (%)	95	88	89
1:16	Range (%)	94-105	82-93	82-95
1:16	Average (%)	99	87	87

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.