Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 125.00-8000 pg/mL |
| Sensitivity: | 75.00 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human NOG in samples. No significant cross-reactivity or interference between Human NOG 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 NOG. 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 NOG 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 NOG, 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 NOG. The concentration of Human NOG in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | NOG: Essential for cartilage morphogenesis and joint formation. Inhibitor of bone morphogenetic proteins (BMP) signaling which is required for growth and patterning of the neural tube and somite. Defects in NOG are a cause of symphalangism proximal syndrome (SYM1). SYM1 is characterized by the hereditary absence of the proximal interphalangeal (PIP) joints (Cushing symphalangism). Severity of PIP joint involvement diminishes towards the radial side. Distal interphalangeal joints are less frequently involved and metacarpophalangeal joints are rarely affected whereas carpal bone malformation and fusion are common. In the lower extremities, tarsal bone coalition is common. Conducive hearing loss is seen and is due to fusion of the stapes to the petrous part of the temporal bone. Defects in NOG are the cause of multiple synostoses syndrome type 1 (SYNS1); also known as synostoses, multiple, with brachydactyly/symphalangism-brachydactyly syndrome. SYNS1 is characterized by tubular-shaped (hemicylindrical) nose with lack of alar flare, otosclerotic deafness, and multiple progressive joint fusions commencing in the hand. The joint fusions are progressive, commencing in the fifth proximal interphalangeal joint in early childhood (or at birth in some individuals) and progressing in an ulnar-to-radial and proximal- to-distal direction. With increasing age, ankylosis of other joints, including the cervical vertebrae, hips, and humeroradial joints, develop. Defects in NOG are the cause of tarsal-carpal coalition syndrome (TCC). TCC is an autosomal dominant disorder characterized by fusion of the carpals, tarsals and phalanges, short first metacarpals causing brachydactyly, and humeroradial fusion. TCC is allelic to SYM1, and different mutations in NOG can result in either TCC or SYM1 in different families. Defects in NOG are a cause of stapes ankylosis with broad thumb and toes (SABTS); also known as Teunissen- Cremers syndrome. SABTS is a congenital autosomal dominant disorder that includes hyperopia, a hemicylindrical nose, broad thumbs, great toes, and other minor skeletal anomalies but lacked carpal and tarsal fusion and symphalangism. Defects in NOG are the cause of brachydactyly type B2 (BDB2). BDB2 is a subtype of brachydactyly characterized by hypoplasia/aplasia of distal phalanges in combination with distal symphalangism, fusion of carpal/tarsal bones, and partial cutaneous syndactyly. Belongs to the noggin family. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 17q22 Cellular Component: extracellular region; extracellular space Molecular Function:cytokine binding; protein binding; protein homodimerization activity Biological Process: BMP signaling pathway; cell differentiation in hindbrain; dorsal/ventral pattern formation; embryonic digit morphogenesis; embryonic skeletal development; epithelial to mesenchymal transition; limb development; middle ear morphogenesis; negative regulation of astrocyte differentiation; negative regulation of BMP signaling pathway; negative regulation of cardiac muscle cell proliferation; negative regulation of cell migration; negative regulation of osteoblast differentiation; nervous system development; osteoblast differentiation; skeletal development; somatic stem cell maintenance; wound healing Disease: Brachydactyly, Type B2; Multiple Synostoses Syndrome 1; Stapes Ankylosis With Broad Thumb And Toes; Symphalangism, Proximal, 1a; Tarsal-carpal Coalition Syndrome |
| NCBI Summary: | The secreted polypeptide, encoded by this gene, binds and inactivates members of the transforming growth factor-beta (TGF-beta) superfamily signaling proteins, such as bone morphogenetic protein-4 (BMP4). By diffusing through extracellular matrices more efficiently than members of the TGF-beta superfamily, this protein may have a principal role in creating morphogenic gradients. The protein appears to have pleiotropic effect, both early in development as well as in later stages. It was originally isolated from Xenopus based on its ability to restore normal dorsal-ventral body axis in embryos that had been artificially ventralized by UV treatment. The results of the mouse knockout of the ortholog suggest that it is involved in numerous developmental processes, such as neural tube fusion and joint formation. Recently, several dominant human NOG mutations in unrelated families with proximal symphalangism (SYM1) and multiple synostoses syndrome (SYNS1) were identified; both SYM1 and SYNS1 have multiple joint fusion as their principal feature, and map to the same region (17q22) as this gene. All of these mutations altered evolutionarily conserved amino acid residues. The amino acid sequence of this human gene is highly homologous to that of Xenopus, rat and mouse. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q13253 |
| NCBI GenInfo Identifier: | 15214099 |
| NCBI Gene ID: | 9241 |
| NCBI Accession: | Q13253. 1 |
| UniProt Related Accession: | Q13253 |
| Molecular Weight: | 25,774 Da |
| NCBI Full Name: | Noggin |
| NCBI Synonym Full Names: | noggin |
| NCBI Official Symbol: | NOG |
| NCBI Official Synonym Symbols: | SYM1; SYNS1; SYNS1A |
| NCBI Protein Information: | noggin |
| UniProt Protein Name: | Noggin |
| Protein Family: | Noggin |
| UniProt Gene Name: | NOG |
| UniProt Entry Name: | NOGG_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 (pg/mL) | O.D | Average | Corrected |
| 8000 | 2.504 2.55 | 2.527 | 2.452 |
| 4000 | 1.803 1.857 | 1.83 | 1.755 |
| 2000 | 1.104 1.066 | 1.085 | 1.01 |
| 1000 | 0.502 0.53 | 0.516 | 0.441 |
| 500 | 0.316 0.294 | 0.305 | 0.23 |
| 250 | 0.197 0.179 | 0.188 | 0.113 |
| 125.00 | 0.126 0.138 | 0.132 | 0.057 |
| 0 | 0.066 0.084 | 0.075 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NOG were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NOG 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) | 370.81 | 1171.82 | 3307.52 | 394.62 | 1105.13 | 3627.69 |
| Standard deviation | 22.40 | 58.24 | 168.35 | 24.82 | 59.23 | 117.17 |
| C V (%) | 6.04 | 4.97 | 5.09 | 6.29 | 5.36 | 3.23 |
Recovery
The recovery of Human NOG 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) | 90-104 | 96 |
| EDTA plasma (n=5) | 85-98 | 90 |
| Cell culture media (n=5) | 87-101 | 93 |
Linearity
Samples were spiked with high concentrations of Human NOG 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 (%) | 91-103 | 90-104 | 91-106 |
| Average (%) | 98 | 96 | 98 | |
| 1:4 | Range (%) | 90-100 | 80-95 | 87-103 |
| Average (%) | 95 | 87 | 94 | |
| 1:8 | Range (%) | 89-100 | 82-95 | 89-101 |
| Average (%) | 94 | 88 | 94 | |
| 1:16 | Range (%) | 90-105 | 86-98 | 81-94 |
| Average (%) | 97 | 91 | 87 |
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.
