Human Developmental Biology ELISA Kits
Human AGRN (Agrin) CLIA Kit (HUES00219)
- SKU:
- HUES00219
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- AGRN
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Developmental Biology
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection method: | Chemiluminescence |
| Detection range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample volume: | 100µL |
| Sample type: | Serum, plasma and other biological fluids |
| Repeatability: | CV < 15% |
| Specificity: | This kit recognizes Human AGRN in samples. No significant cross-reactivity or interference between Human AGRN 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 AGRN. 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 AGRN 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 AGRN, 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 AGRN. The concentration of Human AGRN in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | agrin: Plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ), the synapse between motor neuron and skeletal muscle. Ligand of the MUSK signaling complex that directly binds LRP4 in this complex and induces the phosphorylation of MUSK, the kinase of the complex. The activation of MUSK in myotubes induces the formation of NMJ by regulating different processes including the transcription of specific genes and the clustering of AChR in the postsynaptic membrane. Defects in AGRN are a cause of myasthenia, limb-girdle, familial (LGM). A congenital myasthenic syndrome characterized by a typical 'limb girdle' pattern of muscle weakness with small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. |
| UniProt Protein Details: | Protein type:Cell adhesion; Cell development/differentiation; Extracellular matrix; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1p36. 33 Cellular Component: basal lamina; cell junction; cytosol; extracellular matrix; extracellular region; Golgi lumen; integral component of membrane; lysosomal lumen; plasma membrane; synapse Molecular Function:calcium ion binding; heparan sulfate proteoglycan binding; laminin binding; protein binding; sialic acid binding; structural constituent of cytoskeleton Biological Process: clustering of voltage-gated sodium channels; cytoskeleton organization; extracellular matrix organization; G-protein coupled acetylcholine receptor signaling pathway; glycosaminoglycan biosynthetic process; glycosaminoglycan catabolic process; glycosaminoglycan metabolic process; positive regulation of filopodium formation; positive regulation of GTPase activity; positive regulation of synaptic growth at neuromuscular junction; positive regulation of transcription from RNA polymerase II promoter; receptor clustering; retinoid metabolic process; signal transduction; synapse organization Disease: Myasthenic Syndrome, Congenital, With Pre- And Postsynaptic Defects |
| NCBI Summary: | This gene encodes one of several proteins that are critical in the development of the neuromuscular junction (NMJ), as identified in mouse knock-out studies. The encoded protein contains several laminin G, Kazal type serine protease inhibitor, and epidermal growth factor domains. Additional post-translational modifications occur to add glycosaminoglycans and disulfide bonds. In one family with congenital myasthenic syndrome affecting limb-girdle muscles, a mutation in this gene was found. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Mar 2015] |
| UniProt Code: | O00468 |
| NCBI GenInfo Identifier: | 54873613 |
| NCBI Gene ID: | 375790 |
| NCBI Accession: | NP_940978. 2 |
| UniProt Secondary Accession: | O00468,Q5SVA1, Q5SVA2, Q60FE1, Q7KYS8, Q8N4J5, Q96IC1 Q9BTD4, |
| UniProt Related Accession: | O00468 |
| Molecular Weight: | |
| NCBI Full Name: | agrin isoform 2 |
| NCBI Synonym Full Names: | agrin |
| NCBI Official Symbol: | AGRN |
| NCBI Official Synonym Symbols: | CMS8; AGRIN; CMSPPD |
| NCBI Protein Information: | agrin |
| UniProt Protein Name: | Agrin |
| Protein Family: | Agrin |
| UniProt Gene Name: | AGRN |
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 |
| 2000 | 51554 59888 | 55721 | 55693 |
| 1000 | 22267 25681 | 23974 | 23946 |
| 500 | 11098 10966 | 11032 | 11004 |
| 250 | 4963 5625 | 5294 | 5266 |
| 125 | 2775 2441 | 2608 | 2580 |
| 62.5 | 1331 1291 | 1311 | 1283 |
| 31.25 | 629 719 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human AGRN were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AGRN 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) | 107.02 | 228.85 | 745.59 | 102.80 | 226.55 | 806.41 |
| Standard deviation | 13.58 | 22.13 | 72.62 | 11.30 | 23.56 | 52.09 |
| C V (%) | 12.69 | 9.67 | 9.74 | 10.99 | 10.40 | 6.46 |
Recovery
The recovery of Human AGRN 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) | 93-110 | 100 |
| EDTA plasma (n=5) | 88-104 | 96 |
| Cell culture media (n=5) | 92-108 | 99 |
Linearity
Samples were spiked with high concentrations of Human AGRN 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 (%) | 86-96 | 91-107 | 92-106 |
| Average (%) | 91 | 97 | 98 | |
| 1:4 | Range (%) | 96-112 | 93-106 | 92-102 |
| Average (%) | 102 | 99 | 97 | |
| 1:8 | Range (%) | 98-114 | 99-116 | 96-112 |
| Average (%) | 105 | 107 | 104 | |
| 1:16 | Range (%) | 89-104 | 90-102 | 94-110 |
| Average (%) | 96 | 96 | 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.
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