Human Signal Transduction ELISA Kits
Human OC/BGP (Osteocalcin) ELISA Kit (HUES02389)
- SKU:
- HUES02389
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P02818
- Sensitivity:
- 0.75ng/mL
- Range:
- 1.25-80ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- BGLAP, OT, Bone Gamma-carboxyglutamate Protein, Bone Gla Protein
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Signal Transduction
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 1.25-80 ng/mL |
| Sensitivity: | 0.75 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human OC/BGP in samples. No significant cross-reactivity or interference between Human OC/BGP 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 OC/BGP. 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 OC/BGP 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 OC/BGP, 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 OC/BGP. The concentration of Human OC/BGP in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | osteocalcin: Constitutes 1-2% of the total bone protein. It binds strongly to apatite and calcium. Belongs to the osteocalcin/matrix Gla protein family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted; Cell development/differentiation Chromosomal Location of Human Ortholog: 1q22 Cellular Component: Golgi apparatus; extracellular space; rough endoplasmic reticulum; dendrite; cytoplasm; perikaryon Molecular Function:structural constituent of bone; hydroxyapatite binding; calcium ion binding; structural molecule activity Biological Process: response to drug; response to gravity; regulation of bone resorption; response to glucocorticoid stimulus; cell aging; response to testosterone stimulus; bone mineralization; osteoblast development; osteoblast differentiation; odontogenesis; response to vitamin D; regulation of osteoclast differentiation; response to ethanol; response to vitamin K; response to zinc ion; response to mechanical stimulus; response to estrogen stimulus; response to hydroxyisoflavone; response to activity; cell adhesion; skeletal development; regulation of bone mineralization |
| NCBI Summary: | This gene encodes a highly abundant bone protein secreted by osteoblasts that regulates bone remodeling and energy metabolism. The encoded protein contains a Gla (gamma carboxyglutamate) domain, which functions in binding to calcium and hydroxyapatite, the mineral component of bone. Serum osteocalcin levels may be negatively correlated with metabolic syndrome. Read-through transcription exists between this gene and the neighboring upstream gene, PMF1 (polyamine-modulated factor 1), but the encoded protein only shows sequence identity with the upstream gene product. [provided by RefSeq, Jun 2015] |
| UniProt Code: | P02818 |
| NCBI GenInfo Identifier: | 129253 |
| NCBI Gene ID: | 632 |
| NCBI Accession: | P02818. 2 |
| UniProt Secondary Accession: | P02818,Q5TCK6, |
| UniProt Related Accession: | P02818 |
| Molecular Weight: | 10,963 Da |
| NCBI Full Name: | Osteocalcin |
| NCBI Synonym Full Names: | bone gamma-carboxyglutamate (gla) protein |
| NCBI Official Symbol: | BGLAP |
| NCBI Official Synonym Symbols: | OC; BGP; OCN |
| NCBI Protein Information: | osteocalcin; bone Gla protein; gamma-carboxyglutamic acid-containing protein; bone gamma-carboxyglutamate (gla) protein (osteocalcin) |
| UniProt Protein Name: | Osteocalcin |
| UniProt Synonym Protein Names: | Bone Gla protein; BGP; Gamma-carboxyglutamic acid-containing protein |
| Protein Family: | Osteocalcin |
| UniProt Gene Name: | BGLAP |
| UniProt Entry Name: | OSTCN_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 (ng/mL) | O.D | Average | Corrected |
| 80 | 2.402 2.442 | 2.422 | 2.357 |
| 40 | 1.519 1.543 | 1.531 | 1.466 |
| 20 | 0.927 0.895 | 0.911 | 0.846 |
| 10 | 0.452 0.482 | 0.467 | 0.402 |
| 5 | 0.268 0.266 | 0.267 | 0.202 |
| 2.5 | 0.175 0.149 | 0.162 | 0.097 |
| 1.25 | 0.11 0.12 | 0.115 | 0.05 |
| 0 | 0.06 0.07 | 0.065 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human OC/BGP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human OC/BGP 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) | 3.70 | 9.20 | 32.90 | 3.80 | 9.50 | 36.10 |
| Standard deviation | 0.20 | 0.40 | 1.60 | 0.30 | 0.50 | 1.30 |
| C V (%) | 5.41 | 4.35 | 4.86 | 7.89 | 5.26 | 3.60 |
Recovery
The recovery of Human OC/BGP 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) | 92-109 | 99 |
| EDTA plasma (n=5) | 93-109 | 99 |
| Cell culture media (n=5) | 85-97 | 90 |
Linearity
Samples were spiked with high concentrations of Human OC/BGP 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 (%) | 87-102 | 91-108 | 91-105 |
| Average (%) | 94 | 98 | 97 | |
| 1:4 | Range (%) | 94-109 | 80-95 | 95-108 |
| Average (%) | 101 | 87 | 102 | |
| 1:8 | Range (%) | 101-117 | 83-95 | 98-112 |
| Average (%) | 107 | 88 | 104 | |
| 1:16 | Range (%) | 99-113 | 83-97 | 90-102 |
| Average (%) | 107 | 89 | 97 |
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 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 90µL 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.
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