Human Cardiovascular ELISA Kits
Human F12 (Coagulation Factor XII) CLIA Kit (HUES00479)
- SKU:
- HUES00479
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cardiovascular
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 15.63-1000 pg/mL |
| Sensitivity: | 9.38 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human F12 in samples. No significant cross-reactivity or interference between Human F12 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 Human F12. 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 Human F12 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 F12, 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 Human F12. The concentration of Human F12 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | F12: Factor XII is a serum glycoprotein that participates in the initiation of blood coagulation, fibrinolysis, and the generation of bradykinin and angiotensin. Prekallikrein is cleaved by factor XII to form kallikrein, which then cleaves factor XII first to alpha-factor XIIa and then trypsin cleaves it to beta- factor XIIa. Alpha-factor XIIa activates factor XI to factor XIa. Defects in F12 are the cause of factor XII deficiency (FA12D); also known as Hageman factor deficiency. This trait is an asymptomatic anomaly of in vitro blood coagulation. Its diagnosis is based on finding a low plasma activity of the factor in coagulating assays. It is usually only accidentally discovered through pre-operative blood tests. F12 deficiency is divided into two categories, a cross-reacting material (CRM)-negative group (negative F12 antigen detection) and a CRM-positive group (positive F12 antigen detection). Defects in F12 are the cause of hereditary angioedema type 3 (HAE3); also known as estrogen-related HAE or hereditary angioneurotic edema with normal C1 inhibitor concentration and function. HAE is characterized by episodic local subcutaneous edema, and submucosal edema involving the upper respiratory and gastrointestinal tracts. HAE3 occurs exclusively in women and is precipitated or worsened by high estrogen levels (e. g. during pregnancy or treatment with oral contraceptives). It differs from HAE types 1 and 2 in that both concentration and function of C1 inhibitor are normal. Belongs to the peptidase S1 family. |
| UniProt Protein Details: | Protein type:Protease; Secreted; Chaperone; EC 3. 4. 21. 38; Secreted, signal peptide Chromosomal Location of Human Ortholog: 5q35. 3 Cellular Component: extracellular region; extracellular space; plasma membrane Molecular Function:misfolded protein binding; protein binding; serine-type endopeptidase activity Biological Process: blood coagulation; blood coagulation, intrinsic pathway; Factor XII activation; fibrinolysis; innate immune response; plasma kallikrein-kinin cascade; positive regulation of blood coagulation; positive regulation of fibrinolysis; protein autoprocessing; protein processing; response to misfolded protein; zymogen activation Disease: Angioedema, Hereditary, Type Iii; Factor Xii Deficiency |
| NCBI Summary: | This gene encodes coagulation factor XII which circulates in blood as a zymogen. This single chain zymogen is converted to a two-chain serine protease with an heavy chain (alpha-factor XIIa) and a light chain. The heavy chain contains two fibronectin-type domains, two epidermal growth factor (EGF)-like domains, a kringle domain and a proline-rich domain, whereas the light chain contains only a catalytic domain. On activation, further cleavages takes place in the heavy chain, resulting in the production of beta-factor XIIa light chain and the alpha-factor XIIa light chain becomes beta-factor XIIa heavy chain. Prekallikrein is cleaved by factor XII to form kallikrein, which then cleaves factor XII first to alpha-factor XIIa and then to beta-factor XIIa. The active factor XIIa participates in the initiation of blood coagulation, fibrinolysis, and the generation of bradykinin and angiotensin. It activates coagulation factors VII and XI. Defects in this gene do not cause any clinical symptoms and the sole effect is that whole-blood clotting time is prolonged. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P00748 |
| NCBI GenInfo Identifier: | 317373446 |
| NCBI Gene ID: | 2161 |
| NCBI Accession: | P00748. 3 |
| UniProt Secondary Accession: | P00748,P78339, |
| UniProt Related Accession: | P00748 |
| Molecular Weight: | 67,792 Da |
| NCBI Full Name: | Coagulation factor XII |
| NCBI Synonym Full Names: | coagulation factor XII |
| NCBI Official Symbol: | F12 |
| NCBI Official Synonym Symbols: | HAF; HAE3; HAEX |
| NCBI Protein Information: | coagulation factor XII |
| UniProt Protein Name: | Coagulation factor XII |
| UniProt Synonym Protein Names: | Hageman factor; HAF |
| Protein Family: | Coagulation factor |
| UniProt Gene Name: | F12 |
| UniProt Entry Name: | FA12_HUMAN |
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 |
| 1000 | 50734 56616 | 53675 | 53650 |
| 500 | 20562 24416 | 22489 | 22464 |
| 250 | 10810 9612 | 10211 | 10186 |
| 125 | 4826 4976 | 4901 | 4876 |
| 62.5 | 2557 2349 | 2453 | 2428 |
| 31.25 | 1292 1270 | 1281 | 1256 |
| 15.63 | 682 734 | 708 | 683 |
| 0 | 25 25 | 25 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human F12 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human F12 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) | 53.84 | 103.28 | 476.02 | 52.37 | 104.88 | 506.95 |
| Standard deviation | 5.38 | 7.28 | 36.03 | 4.66 | 10.80 | 30.67 |
| C V (%) | 9.99 | 7.05 | 7.57 | 8.90 | 10.30 | 6.05 |
Recovery
The recovery of Human F12 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) | 88-104 | 95 |
| EDTA plasma (n=5) | 86-100 | 93 |
| Cell culture media (n=5) | 92-105 | 100 |
Linearity
Samples were spiked with high concentrations of Human F12 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 (%) | 90-105 | 98-112 | 93-106 |
| Average (%) | 96 | 104 | 101 | |
| 1:4 | Range (%) | 83-96 | 88-100 | 95-112 |
| Average (%) | 90 | 93 | 103 | |
| 1:8 | Range (%) | 101-118 | 97-111 | 100-113 |
| Average (%) | 109 | 104 | 106 | |
| 1:16 | Range (%) | 91-104 | 101-118 | 99-115 |
| Average (%) | 98 | 109 | 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.
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