Human Cell Biology ELISA Kits 3
Human NEFL (Neurofilament, Light Polypeptide) CLIA Kit (HUES00467)
- SKU:
- HUES00467
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 7.5pg/mL
- Range:
- 12.5-800pg/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: | 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 NEFL in samples. No significant cross-reactivity or interference between Human NEFL 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 NEFL. 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 NEFL 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 NEFL, 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 NEFL. The concentration of Human NEFL in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | NFL: one of the three (L, M, and H) intermediate filament proteins that form neurofilaments. Neurofilaments are involved in the maintenance of neuronal caliber. NF-L is the most abundant of the three neurofilament proteins. Defects cause Charcot-Marie-Tooth disease type 1F (CMT1F). |
| UniProt Protein Details: | Protein type:Cytoskeletal Chromosomal Location of Human Ortholog: 8p21 Cellular Component: TSC1-TSC2 complex; growth cone; axon; cytoplasm; neurofilament; cytosol Molecular Function:protein C-terminus binding; protein binding, bridging; identical protein binding; protein domain specific binding; protein binding; phospholipase binding; structural constituent of cytoskeleton Biological Process: protein polymerization; response to peptide hormone stimulus; neurofilament bundle assembly; response to toxin; intermediate filament organization; microtubule cytoskeleton organization and biogenesis; retrograde axon cargo transport; axon regeneration in the peripheral nervous system; synaptic transmission; axon transport of mitochondrion; regulation of axon diameter; positive regulation of axonogenesis; negative regulation of neuron apoptosis; anterograde axon cargo transport; neuromuscular process controlling balance; locomotion; neurite morphogenesis; response to corticosterone stimulus Disease: Charcot-marie-tooth Disease, Axonal, Type 2e; Charcot-marie-tooth Disease, Demyelinating, Type 1f |
| NCBI Summary: | Neurofilaments are type IV intermediate filament heteropolymers composed of light, medium, and heavy chains. Neurofilaments comprise the axoskeleton and they functionally maintain the neuronal caliber. They may also play a role in intracellular transport to axons and dendrites. This gene encodes the light chain neurofilament protein. Mutations in this gene cause Charcot-Marie-Tooth disease types 1F (CMT1F) and 2E (CMT2E), disorders of the peripheral nervous system that are characterized by distinct neuropathies. A pseudogene has been identified on chromosome Y. [provided by RefSeq, Oct 2008] |
| UniProt Code: | P07196 |
| NCBI GenInfo Identifier: | 62511894 |
| NCBI Gene ID: | 4747 |
| NCBI Accession: | P07196. 3 |
| UniProt Related Accession: | P07196 |
| Molecular Weight: | |
| NCBI Full Name: | Neurofilament light polypeptide |
| NCBI Synonym Full Names: | neurofilament light |
| NCBI Official Symbol: | NEFL |
| NCBI Official Synonym Symbols: | NFL; NF-L; NF68; CMT1F; CMT2E; CMTDIG; PPP1R110 |
| NCBI Protein Information: | neurofilament light polypeptide |
| UniProt Protein Name: | Neurofilament light polypeptide |
| UniProt Synonym Protein Names: | 68 kDa neurofilament protein; Neurofilament triplet L protein |
| Protein Family: | Neurofilament heavy polypeptide |
| UniProt Gene Name: | NEFL |
| UniProt Entry Name: | NFL_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 | 48841 56443 | 52642 | 52616 |
| 400 | 20800 22668 | 21734 | 21708 |
| 200 | 10262 9154 | 9708 | 9682 |
| 100 | 4445 4659 | 4552 | 4526 |
| 50 | 2196 2180 | 2188 | 2162 |
| 25 | 1139 981 | 1060 | 1034 |
| 12.50 | 505 513 | 509 | 483 |
| 0 | 25 27 | 26 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NEFL were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NEFL 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) | 42.81 | 110.42 | 359.68 | 45.57 | 109.06 | 388.00 |
| Standard deviation | 4.76 | 8.26 | 23.49 | 5.50 | 13.02 | 28.75 |
| C V (%) | 11.12 | 7.48 | 6.53 | 12.07 | 11.94 | 7.41 |
Recovery
The recovery of Human NEFL 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) | 95-109 | 100 |
| EDTA plasma (n=5) | 96-111 | 104 |
| Cell culture media (n=5) | 97-108 | 103 |
Linearity
Samples were spiked with high concentrations of Human NEFL 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 (%) | 88-102 | 95-108 | 94-109 |
| Average (%) | 93 | 101 | 102 | |
| 1:4 | Range (%) | 89-101 | 101-116 | 89-100 |
| Average (%) | 96 | 107 | 95 | |
| 1:8 | Range (%) | 96-113 | 90-105 | 89-102 |
| Average (%) | 104 | 96 | 95 | |
| 1:16 | Range (%) | 85-98 | 87-99 | 101-114 |
| Average (%) | 92 | 94 | 107 |
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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