Description
Human F5 (Coagulation Factor V/Plasma Factor V) ELISA Kit
The Human F5 Coagulation Factor V Plasma Factor V ELISA Kit is a powerful tool for measuring levels of Factor V in human plasma samples. This kit offers high sensitivity and specificity, providing reliable and consistent results for a variety of research applications.Factor V is a key player in the blood coagulation process, essential for the formation of blood clots and maintaining hemostasis. Dysregulation of Factor V can lead to conditions such as thrombosis or bleeding disorders, making it a valuable biomarker for monitoring and studying these health conditions.
Whether investigating hemostasis, thrombosis, or other related disorders, the Human F5 Coagulation Factor V Plasma Factor V ELISA Kit provides accurate and precise measurement of Factor V levels in human plasma samples, aiding in the advancement of research and potential therapeutic interventions.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 62.50-4000 pg/mL |
| Sensitivity: | 37.50 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human F5 in samples. No significant cross-reactivity or interference between Human F5 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 F5. 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 F5 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 F5, 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 F5. The concentration of Human F5 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:Secreted; Protease; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1q23 Cellular Component: endoplasmic reticulum lumen; ER to Golgi transport vesicle; ER-Golgi intermediate compartment membrane; extracellular region; extracellular space; Golgi membrane; membrane; plasma membrane Molecular Function:copper ion binding; protein binding Biological Process: blood circulation; blood coagulation; cellular protein metabolic process; COPII coating of Golgi vesicle; ER to Golgi vesicle-mediated transport; platelet activation; platelet degranulation; post-translational protein modification; protein amino acid N-linked glycosylation via asparagine Disease: Budd-chiari Syndrome; Factor V Deficiency; Pregnancy Loss, Recurrent, Susceptibility To, 1; Stroke, Ischemic; Thrombophilia Due To Activated Protein C Resistance |
| NCBI Summary: | This gene encodes an essential cofactor of the blood coagulation cascade. This factor circulates in plasma, and is converted to the active form by the release of the activation peptide by thrombin during coagulation. This generates a heavy chain and a light chain which are held together by calcium ions. The activated protein is a cofactor that participates with activated coagulation factor X to activate prothrombin to thrombin. Defects in this gene result in either an autosomal recessive hemorrhagic diathesis or an autosomal dominant form of thrombophilia, which is known as activated protein C resistance. [provided by RefSeq, Oct 2008] |
| UniProt Code: | P12259 |
| NCBI GenInfo Identifier: | 105990535 |
| NCBI Gene ID: | 2153 |
| NCBI Accession: | NP_000121. 2 |
| UniProt Secondary Accession: | P12259,Q14285, Q2EHR5, Q5R346, Q5R347, Q6UPU6, Q8WWQ6 A8K6E8, |
| UniProt Related Accession: | P12259 |
| Molecular Weight: | 30. 4 kDa |
| NCBI Full Name: | coagulation factor V preproprotein |
| NCBI Synonym Full Names: | coagulation factor V |
| NCBI Official Symbol: | F5 |
| NCBI Official Synonym Symbols: | FVL; PCCF; THPH2; RPRGL1 |
| NCBI Protein Information: | coagulation factor V |
| UniProt Protein Name: | Coagulation factor V |
| UniProt Synonym Protein Names: | Activated protein C cofactor; Proaccelerin, labile factor |
| Protein Family: | Coagulation factor |
| UniProt Gene Name: | F5 |
| UniProt Entry Name: | FA5_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 (pg/mL) | O.D | Average | Corrected |
| 4000 | 2.352 2.358 | 2.355 | 2.286 |
| 2000 | 1.591 1.641 | 1.616 | 1.547 |
| 1000 | 0.937 0.897 | 0.917 | 0.848 |
| 500 | 0.423 0.451 | 0.437 | 0.368 |
| 250 | 0.236 0.218 | 0.227 | 0.158 |
| 125 | 0.173 0.157 | 0.165 | 0.096 |
| 62.50 | 0.115 0.123 | 0.119 | 0.05 |
| 0 | 0.06 0.078 | 0.069 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human F5 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human F5 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) | 198.40 | 567.10 | 1389.70 | 188.90 | 566.80 | 1506.00 |
| Standard deviation | 11.90 | 32.30 | 63.90 | 11.50 | 26.60 | 72.30 |
| C V (%) | 6.00 | 5.70 | 4.60 | 6.09 | 4.69 | 4.80 |
Recovery
The recovery of Human F5 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) | 93-106 | 101 |
| EDTA plasma (n=5) | 87-98 | 92 |
| Cell culture media (n=5) | 88-100 | 95 |
Linearity
Samples were spiked with high concentrations of Human F5 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 (%) | 96-111 | 98-115 | 86-99 |
| Average (%) | 103 | 105 | 91 | |
| 1:4 | Range (%) | 100-118 | 86-99 | 96-112 |
| Average (%) | 107 | 93 | 104 | |
| 1:8 | Range (%) | 95-108 | 86-99 | 97-112 |
| Average (%) | 102 | 91 | 103 | |
| 1:16 | Range (%) | 101-114 | 81-94 | 93-106 |
| Average (%) | 107 | 87 | 99 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.
