Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 12.50-800 pg/mL |
| Sensitivity: | 7.50 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human F13A1 in samples. No significant cross-reactivity or interference between Human F13A1 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 F13A1. 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 F13A1 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 F13A1, 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 F13A1. The concentration of Human F13A1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | F13A1: Factor XIII is activated by thrombin and calcium ion to a transglutaminase that catalyzes the formation of gamma-glutamyl- epsilon-lysine cross-links between fibrin chains, thus stabilizing the fibrin clot. Also cross-link alpha-2-plasmin inhibitor, or fibronectin, to the alpha chains of fibrin. Defects in F13A1 are the cause of factor XIII subunit A deficiency (FA13AD). FA13AD is an autosomal recessive disorder characterized by a life-long bleeding tendency, impaired wound healing and spontaneous abortion in affected women. Belongs to the transglutaminase superfamily. Transglutaminase family. |
| UniProt Protein Details: | Protein type:Transferase; EC 2. 3. 2. 13 Chromosomal Location of Human Ortholog: 6p25. 3-p24. 3 Cellular Component: extracellular region Molecular Function:protein-glutamine gamma-glutamyltransferase activity; metal ion binding Biological Process: platelet activation; platelet degranulation; peptide cross-linking; blood coagulation Disease: Thrombophilia Due To Thrombin Defect; Myocardial Infarction, Susceptibility To; Factor Xiii, A Subunit, Deficiency Of |
| NCBI Summary: | This gene encodes the coagulation factor XIII A subunit. Coagulation factor XIII is the last zymogen to become activated in the blood coagulation cascade. Plasma factor XIII is a heterotetramer composed of 2 A subunits and 2 B subunits. The A subunits have catalytic function, and the B subunits do not have enzymatic activity and may serve as plasma carrier molecules. Platelet factor XIII is comprised only of 2 A subunits, which are identical to those of plasma origin. Upon cleavage of the activation peptide by thrombin and in the presence of calcium ion, the plasma factor XIII dissociates its B subunits and yields the same active enzyme, factor XIIIa, as platelet factor XIII. This enzyme acts as a transglutaminase to catalyze the formation of gamma-glutamyl-epsilon-lysine crosslinking between fibrin molecules, thus stabilizing the fibrin clot. It also crosslinks alpha-2-plasmin inhibitor, or fibronectin, to the alpha chains of fibrin. Factor XIII deficiency is classified into two categories: type I deficiency, characterized by the lack of both the A and B subunits; and type II deficiency, characterized by the lack of the A subunit alone. These defects can result in a lifelong bleeding tendency, defective wound healing, and habitual abortion. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P00488 |
| NCBI GenInfo Identifier: | 119395709 |
| NCBI Gene ID: | 2162 |
| NCBI Accession: | NP_000120. 2 |
| UniProt Secondary Accession: | P00488,Q59HA7, Q8N6X2, Q96P24, Q9BX29, |
| UniProt Related Accession: | P00488 |
| Molecular Weight: | 312,000 |
| NCBI Full Name: | coagulation factor XIII A chain |
| NCBI Synonym Full Names: | coagulation factor XIII, A1 polypeptide |
| NCBI Official Symbol: | F13A1 |
| NCBI Official Synonym Symbols: | F13A |
| NCBI Protein Information: | coagulation factor XIII A chain; TGase; factor XIIIa; fibrinoligase; FSF, A subunit; coagulation factor XIIIa; transglutaminase A chain; transglutaminase. plasma; fibrin stabilizing factor, A subunit; coagulation factor XIII, A polypeptide; protein-glutamine gamma-glutamyltransferase A chain; bA525O21. 1 (coagulation factor XIII, A1 polypeptide) |
| UniProt Protein Name: | Coagulation factor XIII A chain |
| UniProt Synonym Protein Names: | Protein-glutamine gamma-glutamyltransferase A chain; Transglutaminase A chain |
| Protein Family: | 36 kDa major membrane protein |
| UniProt Gene Name: | F13A1 |
| UniProt Entry Name: | F13A_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 |
| 800 | 50662 54622 | 52642 | 52616 |
| 400 | 20267 23201 | 21734 | 21708 |
| 200 | 10518 8898 | 9708 | 9682 |
| 100 | 4357 4747 | 4552 | 4526 |
| 50 | 2359 2017 | 2188 | 2162 |
| 25 | 1090 1030 | 1060 | 1034 |
| 12.50 | 506 512 | 509 | 483 |
| 0 | 25 27 | 26 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human F13A1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human F13A1 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) | 37.66 | 72.96 | 305.26 | 40.70 | 73.29 | 277.75 |
| Standard deviation | 4.56 | 7.30 | 34.40 | 3.51 | 6.63 | 23.64 |
| C V (%) | 12.11 | 10.01 | 11.27 | 8.62 | 9.05 | 8.51 |
Recovery
The recovery of Human F13A1 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) | 86-99 | 92 |
| EDTA plasma (n=5) | 91-103 | 98 |
| Cell culture media (n=5) | 85-97 | 91 |
Linearity
Samples were spiked with high concentrations of Human F13A1 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 (%) | 98-110 | 96-110 | 98-113 |
| Average (%) | 104 | 101 | 103 | |
| 1:4 | Range (%) | 98-115 | 100-115 | 85-99 |
| Average (%) | 106 | 106 | 91 | |
| 1:8 | Range (%) | 102-119 | 100-116 | 102-118 |
| Average (%) | 109 | 106 | 108 | |
| 1:16 | Range (%) | 85-95 | 100-116 | 99-112 |
| Average (%) | 90 | 109 | 106 |
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.
