Human NEF3 (Neurofilament 3) CLIA Kit (HUES00466)
- SKU:
- HUES00466
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 7.5pg/mL
- Range:
- 12.5-800pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- NEFM, NF-M, NFM, Neurofilament, medium polypeptide
- 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: | 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 NEF3 in samples. No significant cross-reactivity or interference between Human NEF3 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 NEF3. 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 NEF3 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 NEF3, 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 NEF3. The concentration of Human NEF3 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | NFM: a neurofilament intermediate filament protein. Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. The levels of phosphorylation are altered developmentally and coincident with a change in the neurofilament function. |
| UniProt Protein Details: | Protein type:Cytoskeletal Chromosomal Location of Human Ortholog: 8p21 Cellular Component: axon; neurofilament; neuromuscular junction Molecular Function:protein binding; structural constituent of cytoskeleton; microtubule binding Biological Process: neurofilament bundle assembly; axon cargo transport; regulation of axon diameter; microtubule cytoskeleton organization and biogenesis |
| NCBI Summary: | Neurofilaments are type IV intermediate filament heteropolymers composed of light, medium, and heavy chains. Neurofilaments comprise the axoskeleton and functionally maintain neuronal caliber. They may also play a role in intracellular transport to axons and dendrites. This gene encodes the medium neurofilament protein. This protein is commonly used as a biomarker of neuronal damage. Alternative splicing results in multiple transcript variants encoding distinct isoforms. [provided by RefSeq, Oct 2008] |
| UniProt Code: | P07197 |
| NCBI GenInfo Identifier: | 281185500 |
| NCBI Gene ID: | 4741 |
| NCBI Accession: | P07197. 3 |
| UniProt Secondary Accession: | P07197,Q4QRK6, B4DGN2, E9PBF7, |
| UniProt Related Accession: | P07197 |
| Molecular Weight: | 59,599 Da |
| NCBI Full Name: | Neurofilament medium polypeptide |
| NCBI Synonym Full Names: | neurofilament, medium polypeptide |
| NCBI Official Symbol: | NEFM |
| NCBI Official Synonym Symbols: | NFM; NEF3; NF-M |
| NCBI Protein Information: | neurofilament medium polypeptide; neurofilament 3; 160 kDa neurofilament protein; neurofilament triplet M protein; neurofilament-3 (150 kD medium); neurofilament, medium polypeptide 150kDa |
| UniProt Protein Name: | Neurofilament medium polypeptide |
| UniProt Synonym Protein Names: | 160 kDa neurofilament protein; Neurofilament 3; Neurofilament triplet M protein |
| Protein Family: | Neurofilament medium polypeptide |
| UniProt Gene Name: | NEFM |
| UniProt Entry Name: | NFM_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 | 52341 52943 | 52642 | 52616 |
| 400 | 19632 23836 | 21734 | 21708 |
| 200 | 10089 9327 | 9708 | 9682 |
| 100 | 4187 4917 | 4552 | 4526 |
| 50 | 2327 2049 | 2188 | 2162 |
| 25 | 1071 1049 | 1060 | 1034 |
| 12.50 | 497 521 | 509 | 483 |
| 0 | 26 26 | 26 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NEF3 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NEF3 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) | 41.47 | 104.00 | 346.57 | 39.81 | 105.51 | 359.61 |
| Standard deviation | 4.76 | 9.56 | 31.16 | 4.36 | 8.40 | 36.28 |
| C V (%) | 11.48 | 9.19 | 8.99 | 10.95 | 7.96 | 10.09 |
Recovery
The recovery of Human NEF3 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) | 100-116 | 106 |
| EDTA plasma (n=5) | 97-110 | 103 |
| Cell culture media (n=5) | 96-108 | 101 |
Linearity
Samples were spiked with high concentrations of Human NEF3 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 (%) | 88-104 | 99-115 | 90-103 |
| Average (%) | 95 | 105 | 97 | |
| 1:4 | Range (%) | 85-95 | 96-109 | 97-109 |
| Average (%) | 90 | 101 | 103 | |
| 1:8 | Range (%) | 88-101 | 91-103 | 100-113 |
| Average (%) | 93 | 97 | 106 | |
| 1:16 | Range (%) | 92-106 | 95-107 | 93-108 |
| Average (%) | 99 | 101 | 100 |
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.
Related Products
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Human Neurofilament medium polypeptide (NEFM) ELISA Kit
Rat NEF3 / Neurofilament 3 ELISA Kit
