Description
Rat VF (Visfatin) ELISA Kit
The Rat VF (Visfatin) ELISA Kit is a powerful tool for researchers looking to accurately measure visfatin levels in rat serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring precise and consistent results for a variety of research applications.Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), is a key protein involved in regulating metabolism, inflammation, and insulin sensitivity.
Elevated visfatin levels have been linked to various metabolic disorders, including obesity, diabetes, and cardiovascular diseases, making it a valuable biomarker for understanding and potentially treating these conditions.With its innovative design and reliable performance, the Rat VF ELISA Kit from Assay Genie is an essential tool for researchers studying visfatin and its role in metabolic health and disease.
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| Detection Method: | Colormetric |
| Detection Range: | 0.16-10 ng/mL |
| Sensitivity: | 0.10 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Rat VF in samples. No significant cross-reactivity or interference between Rat VF 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 Rat VF. 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 Rat VF 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 Rat VF, 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 Rat VF. The concentration of Rat VF in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | Catalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide, an intermediate in the biosynthesis of NAD. It is the rate limiting component in the mammalian NAD biosynthesis pathway. The secreted form behaves both as a cytokine with immunomodulating properties and an adipokine with anti-diabetic properties, it has no enzymatic activity, partly because of lack of activation by ATP, which has a low level in extracellular space and plasma. Plays a role in the modulation of circadian clock function. NAMPT-dependent oscillatory production of NAD regulates oscillation of clock target gene expression by releasing the core clock component: CLOCK-ARNTL/BMAL1 heterodimer from NAD-dependent SIRT1-mediated suppression. |
| NCBI Summary: | has similarity to cytokines but is not secreted; localized to the nucleus and cytoplasm, changes in subcellular localization may be associated with the cell cycle [RGD, Feb 2006] |
| UniProt Code: | Q80Z29 |
| NCBI GenInfo Identifier: | 29293813 |
| NCBI Gene ID: | 297508 |
| NCBI Accession: | NP_808789. 1 |
| UniProt Secondary Accession: | Q80Z29,Q2VT47, |
| UniProt Related Accession: | Q80Z29 |
| Molecular Weight: | 55,438 Da |
| NCBI Full Name: | nicotinamide phosphoribosyltransferase |
| NCBI Synonym Full Names: | nicotinamide phosphoribosyltransferase |
| NCBI Official Symbol: | Nampt |
| NCBI Official Synonym Symbols: | Pbef; Pbef1 |
| NCBI Protein Information: | nicotinamide phosphoribosyltransferase |
| UniProt Protein Name: | Nicotinamide phosphoribosyltransferase |
| UniProt Synonym Protein Names: | Pre-B-cell colony-enhancing factor 1 homolog; PBEF; Visfatin |
| UniProt Gene Name: | Nampt |
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 |
| 10 | 2.25 2.254 | 2.252 | 2.194 |
| 5 | 1.478 1.52 | 1.499 | 1.441 |
| 2.5 | 0.86 0.846 | 0.853 | 0.795 |
| 1.25 | 0.442 0.476 | 0.459 | 0.401 |
| 0.63 | 0.245 0.227 | 0.236 | 0.178 |
| 0.32 | 0.155 0.153 | 0.154 | 0.096 |
| 0.16 | 0.106 0.11 | 0.108 | 0.05 |
| 0 | 0.057 0.059 | 0.058 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat VF were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat VF 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) | 0.55 | 1.30 | 4.79 | 0.54 | 1.33 | 4.80 |
| Standard deviation | 0.03 | 0.07 | 0.18 | 0.03 | 0.07 | 0.23 |
| C V (%) | 5.45 | 5.38 | 3.76 | 5.56 | 5.26 | 4.79 |
Recovery
The recovery of Rat VF 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) | 87-100 | 93 |
| EDTA plasma (n=5) | 94-108 | 101 |
| Cell culture media (n=5) | 94-110 | 101 |
Linearity
Samples were spiked with high concentrations of Rat VF 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 (%) | 94-107 | 98-113 | 94-109 |
| Average (%) | 101 | 105 | 101 | |
| 1:4 | Range (%) | 89-105 | 80-93 | 86-99 |
| Average (%) | 96 | 87 | 91 | |
| 1:8 | Range (%) | 85-101 | 81-96 | 87-100 |
| Average (%) | 92 | 88 | 94 | |
| 1:16 | Range (%) | 92-105 | 85-98 | 86-99 |
| Average (%) | 98 | 91 | 91 |
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.
