Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 31.25-2000 pg/mL |
| Sensitivity: | 18.75 pg/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human REN in samples. No significant cross-reactivity or interference between Human REN 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 REN. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human REN 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 REN, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of 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 REN. You can calculate the concentration of Human REN in the samples by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | REN: Renin is a highly specific endopeptidase, whose only known function is to generate angiotensin I from angiotensinogen in the plasma, initiating a cascade of reactions that produce an elevation of blood pressure and increased sodium retention by the kidney. Interacts with ATP6AP2. Interaction with ATP6AP2 results in a 5-fold increased efficiency in angiotensinogen processing. Belongs to the peptidase A1 family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; EC 3. 4. 23. 15; Protease; Secreted Chromosomal Location of Human Ortholog: 1q32 Cellular Component: extracellular space; cytoplasm; plasma membrane; extracellular region Molecular Function:peptidase activity; insulin-like growth factor receptor binding; aspartic-type endopeptidase activity; receptor binding Biological Process: response to cAMP; hormone-mediated signaling; renin-angiotensin regulation of aldosterone production; male gonad development; cell maturation; drinking behavior; response to lipopolysaccharide; angiotensin maturation; proteolysis; cellular protein metabolic process; beta-amyloid metabolic process; regulation of MAPKKK cascade; regulation of blood pressure; mesonephros development; kidney development Disease: Renal Tubular Dysgenesis; Hyperuricemic Nephropathy, Familial Juvenile, 2 |
| NCBI Summary: | Renin catalyzes the first step in the activation pathway of angiotensinogen--a cascade that can result in aldosterone release,vasoconstriction, and increase in blood pressure. Renin, an aspartyl protease, cleaves angiotensinogen to form angiotensin I, which is converted to angiotensin II by angiotensin I converting enzyme, an important regulator of blood pressure and electrolyte balance. Transcript variants that encode different protein isoforms and that arise from alternative splicing and the use of alternative promoters have been described, but their full-length nature has not been determined. Mutations in this gene have been shown to cause familial hyperproreninemia. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P00797 |
| NCBI GenInfo Identifier: | 132326 |
| NCBI Gene ID: | 5972 |
| NCBI Accession: | P00797. 1 |
| UniProt Secondary Accession: | P00797,Q6FI38, Q6T5C2, |
| UniProt Related Accession: | P00797 |
| Molecular Weight: | 44,726 Da |
| NCBI Full Name: | Renin |
| NCBI Synonym Full Names: | renin |
| NCBI Official Symbol: | REN |
| NCBI Official Synonym Symbols: | HNFJ2 |
| NCBI Protein Information: | renin; angiotensinogenase; renin precursor, renal; angiotensin-forming enzyme |
| UniProt Protein Name: | Renin |
| UniProt Synonym Protein Names: | Angiotensinogenase |
| Protein Family: | Renin |
| UniProt Gene Name: | REN |
| UniProt Entry Name: | RENI_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 (pg/mL) | O.D | Average | Corrected |
| 2000 | 2.29 2.34 | 2.315 | 2.262 |
| 1000 | 1.544 1.56 | 1.552 | 1.499 |
| 500 | 0.973 0.959 | 0.966 | 0.913 |
| 250 | 0.464 0.482 | 0.473 | 0.42 |
| 125 | 0.249 0.235 | 0.242 | 0.189 |
| 62.5 | 0.166 0.142 | 0.154 | 0.101 |
| 31.25 | 0.099 0.111 | 0.105 | 0.052 |
| 0 | 0.051 0.055 | 0.053 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human REN were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human REN 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) | 108.90 | 258.70 | 836.60 | 98.90 | 234.70 | 771.40 |
| Standard deviation | 5.90 | 11.90 | 36.80 | 6.40 | 10.80 | 28.50 |
| C V (%) | 5.42 | 4.60 | 4.40 | 6.47 | 4.60 | 3.69 |
Recovery
The recovery of Human REN 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) | 86-98 | 92 |
| EDTA plasma (n=5) | 84-98 | 90 |
| Cell culture media (n=5) | 83-96 | 90 |
Linearity
Samples were spiked with high concentrations of Human REN 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 (%) | 98-111 | 96-107 | 98-110 |
| Average (%) | 104 | 101 | 104 | |
| 1:4 | Range (%) | 94-106 | 86-98 | 97-114 |
| Average (%) | 100 | 93 | 104 | |
| 1:8 | Range (%) | 99-112 | 82-95 | 93-107 |
| Average (%) | 106 | 88 | 100 | |
| 1:16 | Range (%) | 98-113 | 86-101 | 93-106 |
| Average (%) | 106 | 93 | 98 |
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 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.
