Rat Signaling ELISA Kits 2
Rat OPG (Osteoprotegerin) CLIA Kit (RTES00030)
- SKU:
- RTES00030
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 9.38pg/mL
- Range:
- 15.63-1000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- TNFRSF11B , OCIF, TR1
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cell Death
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| 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 Rat OPG in samples. No significant cross-reactivity or interference between Rat OPG 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 Rat OPG. 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 Rat OPG 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 Rat OPG, 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 Rat OPG. The concentration of Rat OPG in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | TNFRSF11B: Acts as decoy receptor for RANKL and thereby neutralizes its function in osteoclastogenesis. Inhibits the activation of osteoclasts and promotes osteoclast apoptosis in vitro. Bone homeostasis seems to depend on the local RANKL/OPG ratio. May also play a role in preventing arterial calcification. May act as decoy receptor for TRAIL and protect against apoptosis. TRAIL binding blocks the inhibition of osteoclastogenesis. Defects in TNFRSF11B are the cause of juvenile Paget disease (JPD); also known as hyperostosis corticalis deformans juvenilis or hereditary hyperphosphatasia or chronic congenital idiopathic hyperphosphatasia. JPD is a rare autosomal recessive osteopathy that presents in infancy or early childhood. The disorder is characterized by rapidly remodeling woven bone, osteopenia, debilitating fractures, and deformities due to a markedly accelerated rate of bone remodeling throughout the skeleton. Approximately 40 cases of JPD have been reported worldwide. Unless it is treated with drugs that block osteoclast- mediated skeletal resorption, the disease can be fatal. |
| UniProt Protein Details: | Protein type:Secreted; Inhibitor; Secreted, signal peptide Chromosomal Location of Human Ortholog: 8q24 Cellular Component: extracellular space; proteinaceous extracellular matrix; extracellular region Molecular Function:cytokine activity; receptor activity Biological Process: response to drug; response to magnesium ion; extracellular matrix organization and biogenesis; negative regulation of odontogenesis of dentine-containing teeth; response to estrogen stimulus; apoptosis; response to arsenic; signal transduction; negative regulation of bone resorption; skeletal development; response to nutrient Disease: Paget Disease, Juvenile |
| NCBI Summary: | The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein is an osteoblast-secreted decoy receptor that functions as a negative regulator of bone resorption. This protein specifically binds to its ligand, osteoprotegerin ligand, both of which are key extracellular regulators of osteoclast development. Studies of the mouse counterpart also suggest that this protein and its ligand play a role in lymph-node organogenesis and vascular calcification. Alternatively spliced transcript variants of this gene have been reported, but their full length nature has not been determined. [provided by RefSeq, Jul 2008] |
| UniProt Code: | O08727 |
| NCBI GenInfo Identifier: | 148743793 |
| NCBI Gene ID: | 4982 |
| NCBI Accession: | NP_002537. 3 |
| UniProt Secondary Accession: | O08727,O08712, O08727, |
| UniProt Related Accession: | O00300 |
| Molecular Weight: | |
| NCBI Full Name: | tumor necrosis factor receptor superfamily member 11B |
| NCBI Synonym Full Names: | TNF receptor superfamily member 11b |
| NCBI Official Symbol: | TNFRSF11B |
| NCBI Official Synonym Symbols: | OPG; TR1; OCIF; PDB5 |
| NCBI Protein Information: | tumor necrosis factor receptor superfamily member 11B |
| UniProt Protein Name: | Tumor necrosis factor receptor superfamily member 11B |
| UniProt Synonym Protein Names: | Osteoclastogenesis inhibitory factor; Osteoprotegerin |
| UniProt Gene Name: | TNFRSF11B |
| UniProt Entry Name: | TR11B_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 | 49398 57952 | 53675 | 53650 |
| 500 | 21392 23586 | 22489 | 22464 |
| 250 | 10302 10120 | 10211 | 10186 |
| 125 | 4518 5284 | 4901 | 4876 |
| 62.5 | 2552 2354 | 2453 | 2428 |
| 31.25 | 1335 1227 | 1281 | 1256 |
| 15.63 | 689 727 | 708 | 683 |
| 0 | 24 26 | 25 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat OPG were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat OPG 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) | 51.77 | 148.83 | 434.52 | 53.52 | 151.66 | 447.81 |
| Standard deviation | 4.82 | 15.70 | 39.06 | 6.10 | 11.24 | 46.80 |
| C V (%) | 9.31 | 10.55 | 8.99 | 11.40 | 7.41 | 10.45 |
Recovery
The recovery of Rat OPG 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) | 96-109 | 103 |
| EDTA plasma (n=5) | 96-111 | 103 |
| Cell culture media (n=5) | 88-101 | 96 |
Linearity
Samples were spiked with high concentrations of Rat OPG 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 (%) | 95-108 | 96-107 | 89-103 |
| Average (%) | 103 | 101 | 94 | |
| 1:4 | Range (%) | 95-109 | 88-102 | 101-118 |
| Average (%) | 102 | 94 | 107 | |
| 1:8 | Range (%) | 86-101 | 99-112 | 99-113 |
| Average (%) | 93 | 107 | 106 | |
| 1:16 | Range (%) | 100-118 | 101-116 | 100-112 |
| Average (%) | 108 | 107 | 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.
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