Human Cardiovascular ELISA Kits
Human EFNA1 (Ephrin A1) CLIA Kit (HUES00528)
- SKU:
- HUES00528
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Cardiovascular
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 EFNA1 in samples. No significant cross-reactivity or interference between Human EFNA1 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 EFNA1. 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 EFNA1 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 EFNA1, 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 EFNA1. The concentration of Human EFNA1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | Cell surface GPI-bound ligand for Eph receptors, a family of receptor tyrosine kinases which are crucial for migration, repulsion and adhesion during neuronal, vascular and epithelial development. Binds promiscuously Eph receptors residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. Plays an important role in angiogenesis and tumor neovascularization. The recruitment of VAV2, VAV3 and PI3-kinase p85 subunit by phosphorylated EPHA2 is critical for EFNA1-induced RAC1 GTPase activation and vascular endothelial cell migration and assembly. Exerts anti-oncogenic effects in tumor cells through activation and down-regulation of EPHA2. Activates EPHA2 by inducing tyrosine phosphorylation which leads to its internalization and degradation. Acts as a negative regulator in the tumorigenesis of gliomas by down-regulating EPHA2 and FAK. Can evoke collapse of embryonic neuronal growth cone and regulates dendritic spine morphogenesis. |
| NCBI Summary: | This gene encodes a member of the ephrin (EPH) family. The ephrins and EPH-related receptors comprise the largest subfamily of receptor protein-tyrosine kinases and have been implicated in mediating developmental events, especially in the nervous system and in erythropoiesis. Based on their structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. This gene encodes an EFNA class ephrin which binds to the EPHA2, EPHA4, EPHA5, EPHA6, and EPHA7 receptors. Two transcript variants that encode different isoforms were identified through sequence analysis. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P20827 |
| NCBI GenInfo Identifier: | 73920206 |
| NCBI Gene ID: | 1942 |
| NCBI Accession: | P20827. 2 |
| UniProt Secondary Accession: | P20827,Q5SR60, Q5SR61, Q6I9T9, Q8N578, D3DV86, |
| UniProt Related Accession: | P20827 |
| Molecular Weight: | 21,246 Da |
| NCBI Full Name: | Ephrin-A1 |
| NCBI Synonym Full Names: | ephrin A1 |
| NCBI Official Symbol: | EFNA1 |
| NCBI Official Synonym Symbols: | B61; EFL1; ECKLG; EPLG1; LERK1; LERK-1; TNFAIP4 |
| NCBI Protein Information: | ephrin-A1 |
| UniProt Protein Name: | Ephrin-A1 |
| UniProt Synonym Protein Names: | EPH-related receptor tyrosine kinase ligand 1; LERK-1 |
| Protein Family: | Ephrin |
| UniProt Gene Name: | EFNA1 |
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 | 50680 60762 | 55721 | 55693 |
| 1000 | 22593 25355 | 23974 | 23946 |
| 500 | 11604 10460 | 11032 | 11004 |
| 250 | 5179 5409 | 5294 | 5266 |
| 125 | 2665 2551 | 2608 | 2580 |
| 62.5 | 1334 1288 | 1311 | 1283 |
| 31.25 | 651 697 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human EFNA1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human EFNA1 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) | 100.41 | 272.79 | 849.06 | 96.49 | 267.35 | 772.86 |
| Standard deviation | 8.77 | 22.31 | 55.27 | 8.80 | 30.26 | 54.56 |
| C V (%) | 8.73 | 8.18 | 6.51 | 9.12 | 11.32 | 7.06 |
Recovery
The recovery of Human EFNA1 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) | 87-99 | 94 |
| EDTA plasma (n=5) | 90-104 | 96 |
| Cell culture media (n=5) | 90-103 | 97 |
Linearity
Samples were spiked with high concentrations of Human EFNA1 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 (%) | 101-117 | 94-108 | 87-100 |
| Average (%) | 108 | 100 | 93 | |
| 1:4 | Range (%) | 90-105 | 101-114 | 93-105 |
| Average (%) | 96 | 107 | 99 | |
| 1:8 | Range (%) | 102-115 | 85-99 | 102-116 |
| Average (%) | 108 | 92 | 110 | |
| 1:16 | Range (%) | 86-99 | 87-99 | 86-98 |
| Average (%) | 92 | 92 | 92 |
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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