Human Cell Biology ELISA Kits 1
Human ADORA2a (Adenosine A2a Receptor) ELISA Kit (HUES03587)
- SKU:
- HUES03587
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P29274
- Sensitivity:
- 0.19ng/mL
- Range:
- 0.31-20ng/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: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human ADORA2a in samples. No significant cross-reactivity or interference between Human ADORA2a 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 ADORA2a. 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 ADORA2a 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 ADORA2a, 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 ADORA2a. The concentration of Human ADORA2a in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | ADORA2A: one of several receptor subtypes for adenosine. A G-protein coupled receptor. Activation is mediated by G proteins which activate adenylyl cyclase. Abundant in basal ganglia, vasculature and platelets and it is a major target of caffeine. |
| UniProt Protein Details: | Protein type:Receptor, GPCR; Membrane protein, integral; GPCR, family 1; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 22q11. 23 Cellular Component: integral to plasma membrane; membrane; plasma membrane Molecular Function:adenosine receptor activity, G-protein coupled; enzyme binding; identical protein binding; protein binding Biological Process: adenosine receptor signaling pathway; adenylate cyclase activation; apoptosis; blood circulation; blood coagulation; cAMP biosynthetic process; cell-cell signaling; cellular defense response; cellular protein metabolic process; central nervous system development; G-protein signaling, coupled to cAMP nucleotide second messenger; inflammatory response; nerve growth factor receptor signaling pathway; phagocytosis; sensory perception; transmembrane receptor protein tyrosine kinase signaling pathway |
| NCBI Summary: | This gene encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily, which is subdivided into classes and subtypes. The receptors are seven-pass transmembrane proteins that respond to extracellular cues and activate intracellular signal transduction pathways. This protein, an adenosine receptor of A2A subtype, uses adenosine as the preferred endogenous agonist and preferentially interacts with the G(s) and G(olf) family of G proteins to increase intracellular cAMP levels. It plays an important role in many biological functions, such as cardiac rhythm and circulation, cerebral and renal blood flow, immune function, pain regulation, and sleep. It has been implicated in pathophysiological conditions such as inflammatory diseases and neurodegenerative disorders. Alternative splicing results in multiple transcript variants. A read-through transcript composed of the upstream SPECC1L (sperm antigen with calponin homology and coiled-coil domains 1-like) and ADORA2A (adenosine A2a receptor) gene sequence has been identified, but it is thought to be non-coding. [provided by RefSeq, Jun 2013] |
| UniProt Code: | P29274 |
| NCBI GenInfo Identifier: | 543740 |
| NCBI Gene ID: | 135 |
| NCBI Accession: | P29274. 2 |
| UniProt Secondary Accession: | P29274,B2R7E0, |
| UniProt Related Accession: | P29274 |
| Molecular Weight: | |
| NCBI Full Name: | Adenosine receptor A2a |
| NCBI Synonym Full Names: | adenosine A2a receptor |
| NCBI Official Symbol: | ADORA2A |
| NCBI Official Synonym Symbols: | A2aR; RDC8; ADORA2 |
| NCBI Protein Information: | adenosine receptor A2a |
| UniProt Protein Name: | Adenosine receptor A2a |
| Protein Family: | Adenosine receptor |
| UniProt Gene Name: | ADORA2A |
| UniProt Entry Name: | AA2AR_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 (ng/mL) | O.D | Average | Corrected |
| 20 | 2.325 2.385 | 2.355 | 2.283 |
| 10 | 1.43 1.464 | 1.447 | 1.375 |
| 5 | 0.843 0.829 | 0.836 | 0.764 |
| 2.5 | 0.389 0.401 | 0.395 | 0.323 |
| 1.25 | 0.242 0.218 | 0.23 | 0.158 |
| 0.63 | 0.177 0.147 | 0.162 | 0.09 |
| 0.31 | 0.119 0.119 | 0.119 | 0.047 |
| 0 | 0.066 0.078 | 0.072 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ADORA2a were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ADORA2a 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 (ng/mL) | 0.91 | 1.67 | 9.91 | 0.84 | 1.77 | 9.94 |
| Standard deviation | 0.06 | 0.10 | 0.35 | 0.05 | 0.08 | 0.41 |
| C V (%) | 6.59 | 5.99 | 3.53 | 5.95 | 4.52 | 4.12 |
Recovery
The recovery of Human ADORA2a 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) | 88-103 | 95 |
| EDTA plasma (n=5) | 93-106 | 98 |
| Cell culture media (n=5) | 90-101 | 96 |
Linearity
Samples were spiked with high concentrations of Human ADORA2a 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 (%) | 89-102 | 86-99 | 91-105 |
| Average (%) | 94 | 91 | 99 | |
| 1:4 | Range (%) | 90-103 | 79-93 | 87-101 |
| Average (%) | 96 | 85 | 94 | |
| 1:8 | Range (%) | 86-98 | 86-102 | 88-99 |
| Average (%) | 92 | 93 | 94 | |
| 1:16 | Range (%) | 90-108 | 86-97 | 88-98 |
| Average (%) | 98 | 91 | 93 |
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.
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