Human Cell Biology ELISA Kits 4
Human EXT1 (Exostoses 1) CLIA Kit (HUES00800)
- SKU:
- HUES00800
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 0.09ng/mL
- Range:
- 0.16-10ng/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 0.16-10 ng/mL |
| Sensitivity: | 0.09 ng/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human EXT1 in samples. No significant cross-reactivity or interference between Human EXT1 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 EXT1. 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 EXT1 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 EXT1, 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 EXT1. The concentration of Human EXT1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | EXT1: Glycosyltransferase required for the biosynthesis of heparan-sulfate. The EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone. Appears to be a tumor suppressor. Defects in EXT1 are a cause of hereditary multiple exostoses type 1 (EXT1). EXT is a genetically heterogeneous bone disorder caused by genes segregating on human chromosomes 8, 11, and 19 and designated EXT1, EXT2 and EXT3 respectively. EXT is a dominantly inherited skeletal disorder primarily affecting endochondral bone during growth. The disease is characterized by formation of numerous cartilage-capped, benign bone tumors (osteocartilaginous exostoses or osteochondromas) that are often accompanied by skeletal deformities and short stature. In a small percentage of cases exostoses have exhibited malignant transformation resulting in an osteosarcoma or chondrosarcoma. Osteochondromas development can also occur as a sporadic event. Defects in EXT1 are a cause of tricho-rhino-phalangeal syndrome type 2 (TRPS2). A syndrome that combines the clinical features of trichorhinophalangeal syndrome type 1 and multiple exostoses type 1. Affected individuals manifest multiple dysmorphic facial features including large, laterally protruding ears, a bulbous nose, an elongated upper lip, as well as sparse scalp hair, winged scapulae, multiple cartilaginous exostoses, redundant skin, and mental retardation. A chromosomal aberration resulting in the loss of functional copies of TRPS1 and EXT1 has been found in TRPS2 patients. Defects in EXT1 are a cause of chondrosarcoma (CHDSA). It is a malignant neoplasm derived from cartilage cells. Chondrosarcomas range from slow-growing non-metastasizing lesions to highly aggressive metastasizing sarcomas. Belongs to the glycosyltransferase 47 family. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Glycan Metabolism - heparan sulfate biosynthesis; Tumor suppressor; Motility/polarity/chemotaxis; EC 2. 4. 1. 225; EC 2. 4. 1. 224; Transferase Chromosomal Location of Human Ortholog: 8q24. 11 Cellular Component: Golgi membrane; Golgi apparatus; endoplasmic reticulum membrane; endoplasmic reticulum; integral to membrane; integral to endoplasmic reticulum membrane Molecular Function:acetylglucosaminyltransferase activity; transferase activity, transferring glycosyl groups; protein homodimerization activity; glucuronosyl-N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase activity; protein heterodimerization activity; glucuronosyltransferase activity; metal ion binding; heparan sulfate N-acetylglucosaminyltransferase activity; N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase activity Biological Process: glycosaminoglycan biosynthetic process; axon guidance; ossification; cellular polysaccharide biosynthetic process; heparan sulfate proteoglycan biosynthetic process, polysaccharide chain biosynthetic process; glycosaminoglycan metabolic process; olfactory bulb development; carbohydrate metabolic process; heparan sulfate proteoglycan biosynthetic process; protein amino acid glycosylation; gastrulation; pathogenesis; mesoderm development; signal transduction; skeletal development; endoderm development Disease: Chondrosarcoma; Exostoses, Multiple, Type I |
| NCBI Summary: | This gene encodes an endoplasmic reticulum-resident type II transmembrane glycosyltransferase involved in the chain elongation step of heparan sulfate biosynthesis. Mutations in this gene cause the type I form of multiple exostoses. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q16394 |
| NCBI GenInfo Identifier: | 20141422 |
| NCBI Gene ID: | 2131 |
| NCBI Accession: | Q16394. 2 |
| UniProt Related Accession: | Q16394 |
| Molecular Weight: | ~ 86kDa |
| NCBI Full Name: | Exostosin-1 |
| NCBI Synonym Full Names: | exostosin glycosyltransferase 1 |
| NCBI Official Symbol: | EXT1 |
| NCBI Official Synonym Symbols: | EXT; LGS; TTV; LGCR; TRPS2 |
| NCBI Protein Information: | exostosin-1 |
| UniProt Protein Name: | Exostosin-1 |
| UniProt Synonym Protein Names: | Glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase; Multiple exostoses protein 1; Putative tumor suppressor protein EXT1 |
| Protein Family: | Extensin |
| UniProt Gene Name: | EXT1 |
| UniProt Entry Name: | EXT1_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 (ng/mL) | RLU | Average | Corrected |
| 10 | 29739 35203 | 32471 | 32447 |
| 5 | 13925 17015 | 15470 | 15446 |
| 2.5 | 7686 7320 | 7503 | 7479 |
| 1.25 | 3546 3760 | 3653 | 3629 |
| 0.63 | 1843 1679 | 1761 | 1737 |
| 0.31 | 860 788 | 824 | 800 |
| 0.16 | 338 376 | 357 | 333 |
| 0 | 23 25 | 24 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human EXT1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human EXT1 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) | 0.53 | 1.42 | 4.28 | 0.49 | 1.38 | 4.10 |
| Standard deviation | 0.06 | 0.14 | 0.44 | 0.05 | 0.10 | 0.47 |
| C V (%) | 11.32 | 9.86 | 10.28 | 10.20 | 7.25 | 11.46 |
Recovery
The recovery of Human EXT1 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) | 99-112 | 104 |
| EDTA plasma (n=5) | 96-106 | 101 |
| Cell culture media (n=5) | 96-110 | 101 |
Linearity
Samples were spiked with high concentrations of Human EXT1 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 (%) | 102-119 | 100-115 | 94-108 |
| Average (%) | 109 | 106 | 100 | |
| 1:4 | Range (%) | 91-102 | 94-106 | 92-106 |
| Average (%) | 96 | 101 | 99 | |
| 1:8 | Range (%) | 88-104 | 84-96 | 92-105 |
| Average (%) | 95 | 91 | 99 | |
| 1:16 | Range (%) | 96-107 | 95-107 | 86-102 |
| Average (%) | 102 | 102 | 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.
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