Human Metabolism ELISA Kits
Human ACO1 (Aconitase 1) CLIA Kit (HUES00249)
- SKU:
- HUES00249
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- IREB1, IRP1, IREBP
- Reactivity:
- Human
- Sample Type:
- Serum, plasma and other biological fluids
- Research Area:
- Metabolism
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 ACO1 in samples. No significant cross-reactivity or interference between Human ACO1 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 ACO1. 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 ACO1 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 ACO1, 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 ACO1. The concentration of Human ACO1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | IREB1: Iron sensor. Binds a 4Fe-4S cluster and functions as aconitase when cellular iron levels are high. Functions as mRNA binding protein that regulates uptake, sequestration and utilization of iron when cellular iron levels are low. Binds to iron-responsive elements (IRES) in target mRNA species when iron levels are low. Binding of a 4Fe-4S cluster precludes RNA binding. Belongs to the aconitase/IPM isomerase family. |
| UniProt Protein Details: | Protein type:EC 4. 2. 1. 3; Carbohydrate Metabolism - citrate (TCA) cycle; Carbohydrate Metabolism - glyoxylate and dicarboxylate; RNA-binding; Lyase; Endoplasmic reticulum; Translation Chromosomal Location of Human Ortholog: 9p21. 1 Cellular Component: cytoplasm; cytosol; endoplasmic reticulum; Golgi apparatus; mitochondrion Molecular Function:4 iron, 4 sulfur cluster binding; aconitate hydratase activity; iron-responsive element binding; protein binding; RNA binding Biological Process: citrate metabolic process; response to iron(II) ion |
| NCBI Summary: | The protein encoded by this gene is a bifunctional, cytosolic protein that functions as an essential enzyme in the TCA cycle and interacts with mRNA to control the levels of iron inside cells. When cellular iron levels are high, this protein binds to a 4Fe-4S cluster and functions as an aconitase. Aconitases are iron-sulfur proteins that function to catalyze the conversion of citrate to isocitrate. When cellular iron levels are low, the protein binds to iron-responsive elements (IREs), which are stem-loop structures found in the 5' UTR of ferritin mRNA, and in the 3' UTR of transferrin receptor mRNA. When the protein binds to IRE, it results in repression of translation of ferritin mRNA, and inhibition of degradation of the otherwise rapidly degraded transferrin receptor mRNA. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. Alternative splicing results in multiple transcript variants [provided by RefSeq, Jan 2014] |
| UniProt Code: | P21399 |
| NCBI GenInfo Identifier: | 3123225 |
| NCBI Gene ID: | 48 |
| NCBI Accession: | P21399. 3 |
| UniProt Secondary Accession: | P21399,Q14652, Q5VZA7, D3DRK7, |
| UniProt Related Accession: | P21399 |
| Molecular Weight: | 98,399 Da |
| NCBI Full Name: | Cytoplasmic aconitate hydratase |
| NCBI Synonym Full Names: | aconitase 1 |
| NCBI Official Symbol: | ACO1 |
| NCBI Official Synonym Symbols: | IRP1; ACONS; HEL60; IREB1; IREBP; IREBP1 |
| NCBI Protein Information: | cytoplasmic aconitate hydratase |
| UniProt Protein Name: | Cytoplasmic aconitate hydratase |
| UniProt Synonym Protein Names: | Citrate hydro-lyase; Ferritin repressor protein; Iron regulatory protein 1; IRP1; Iron-responsive element-binding protein 1; IRE-BP 1 |
| Protein Family: | Aconitate hydratase |
| UniProt Gene Name: | ACO1 |
| UniProt Entry Name: | ACOC_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 |
| 2000 | 53153 58289 | 55721 | 55693 |
| 1000 | 22710 25238 | 23974 | 23946 |
| 500 | 11284 10780 | 11032 | 11004 |
| 250 | 5108 5480 | 5294 | 5266 |
| 125 | 2773 2443 | 2608 | 2580 |
| 62.5 | 1403 1219 | 1311 | 1283 |
| 31.25 | 657 691 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human ACO1 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ACO1 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) | 106.87 | 286.48 | 886.35 | 115.85 | 276.99 | 826.38 |
| Standard deviation | 11.42 | 28.39 | 70.64 | 12.91 | 25.46 | 50.24 |
| C V (%) | 10.69 | 9.91 | 7.97 | 11.14 | 9.19 | 6.08 |
Recovery
The recovery of Human ACO1 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) | 92-106 | 100 |
| EDTA plasma (n=5) | 98-110 | 104 |
| Cell culture media (n=5) | 92-105 | 97 |
Linearity
Samples were spiked with high concentrations of Human ACO1 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 (%) | 89-101 | 100-118 | 89-101 |
| Average (%) | 96 | 107 | 96 | |
| 1:4 | Range (%) | 99-116 | 86-99 | 103-118 |
| Average (%) | 106 | 91 | 109 | |
| 1:8 | Range (%) | 86-99 | 87-100 | 86-96 |
| Average (%) | 92 | 93 | 91 | |
| 1:16 | Range (%) | 103-117 | 99-116 | 97-113 |
| Average (%) | 109 | 106 | 104 |
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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