Rat Signaling ELISA Kits 3
Rat LEP (Leptin) CLIA Kit (RTES00359)
- SKU:
- RTES00359
- Product Type:
- ELISA Kit
- ELISA Type:
- CLIA Kit
- Size:
- 96 Assays
- Sensitivity:
- 18.75pg/mL
- Range:
- 31.25-2000pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- LEPD, OB, OBS, Obesity Homolog
- Reactivity:
- Rat
- Sample Type:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Rat |
| 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 Rat LEP in samples. No significant cross-reactivity or interference between Rat LEP 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 Rat LEP. 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 Rat LEP 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 LEP, 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 Rat LEP. The concentration of Rat LEP in the samples can be calculated by comparing the RLU of the samples to the standard curve.
| UniProt Protein Function: | Key player in the regulation of energy balance and body weight control. Once released into the circulation, has central and peripheral effects by binding LEPR, found in many tissues, which results in the activation of several major signaling pathways (PubMed:11677594). In the hypothalamus, acts as an appetite-regulating factor that induces a decrease in food intake and an increase in energy consumption by inducing anorexinogenic factors and suppressing orexigenic neuropeptides, also regulates bone mass and secretion of hypothalamo-pituitary-adrenal hormones. In the periphery, increases basal metabolism, influences reproductive function, regulates pancreatic beta-cell function and insulin secretion, is pro-angiogenic for endothelial cell and affects innate and adaptive immunity (PubMed:11460888, PubMed:10482243, PubMed:9003024, PubMed:11677594). In the arcuate nucleus of the hypothalamus, activates by depolarization POMC neurons inducing FOS and SOCS3 expression to release anorexigenic peptides and inhibits by hyperpolarization NPY neurons inducing SOCS3 with a consequent reduction on release of orexigenic peptides (PubMed:10482243, PubMed:9003024). In addition to its known satiety inducing effect, has a modulatory role in nutrient absorption. In the intestine, reduces glucose absorption by enterocytes by activating PKC and leading to a sequential activation of p38, PI3K and ERK signaling pathways which exerts an inhibitory effect on glucose absorption. Acts as a growth factor on certain tissues, through the activation of different signaling pathways increases expression of genes involved in cell cycle regulation such as CCND1, via JAK2-STAT3 pathway, or VEGFA, via MAPK1/3 and PI3K-AKT1 pathways. May also play an apoptotic role via JAK2-STAT3 pathway and up-regulation of BIRC5 expression. Pro-angiogenic, has mitogenic activity on vascular endothelial cells and plays a role in matrix remodeling by regulating the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) (PubMed:11460888). In innate immunity, modulates the activity and function of neutrophils by increasing chemotaxis and the secretion of oxygen radicals. Increases phagocytosis by macrophages and enhances secretion of pro-inflammatory mediators. Increases cytotoxic ability of NK cells (Probable). Plays a pro-inflammatory role, in synergy with IL1B, by inducing NOS2 wich promotes the production of IL6, IL8 and Prostaglandin E2, through a signaling pathway that involves JAK2, PI3K, MAP2K1/MEK1 and MAPK14/p38. In adaptive immunity, promotes the switch of memory T-cells towards T helper-1 cell immune responses. Increases CD4+CD25- T-cell proliferation and reduces autophagy during TCR (T-cell receptor) stimulation, through MTOR signaling pathway activation and BCL2 up-regulation. |
| NCBI Summary: | involved in regulation of body weight, and may have a role in angiogenesis [RGD, Feb 2006] |
| UniProt Code: | P50596 |
| NCBI GenInfo Identifier: | 1709436 |
| NCBI Gene ID: | 25608 |
| NCBI Accession: | P50596. 1 |
| UniProt Related Accession: | P50596 |
| Molecular Weight: | 18,866 Da |
| NCBI Full Name: | Leptin |
| NCBI Synonym Full Names: | leptin |
| NCBI Official Symbol: | Lep |
| NCBI Official Synonym Symbols: | OB; obese |
| NCBI Protein Information: | leptin; obesity factor |
| UniProt Protein Name: | Leptin |
| UniProt Synonym Protein Names: | Obesity factor |
| Protein Family: | Leptin |
| UniProt Gene Name: | Lep |
| UniProt Entry Name: | LEP_RAT |
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 | 53727 57715 | 55721 | 55693 |
| 1000 | 23526 24422 | 23974 | 23946 |
| 500 | 11303 10761 | 11032 | 11004 |
| 250 | 5217 5371 | 5294 | 5266 |
| 125 | 2700 2516 | 2608 | 2580 |
| 62.5 | 1320 1302 | 1311 | 1283 |
| 31.25 | 627 721 | 674 | 646 |
| 0 | 27 29 | 28 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat LEP were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat LEP 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) | 99.97 | 293.39 | 865.85 | 101.83 | 275.26 | 800.74 |
| Standard deviation | 9.56 | 31.57 | 65.89 | 10.44 | 27.17 | 89.52 |
| C V (%) | 9.56 | 10.76 | 7.61 | 10.25 | 9.87 | 11.18 |
Recovery
The recovery of Rat LEP 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) | 96-107 | 102 |
| EDTA plasma (n=5) | 94-107 | 100 |
| Cell culture media (n=5) | 92-108 | 99 |
Linearity
Samples were spiked with high concentrations of Rat LEP 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 (%) | 89-104 | 95-108 | 85-98 |
| Average (%) | 96 | 101 | 90 | |
| 1:4 | Range (%) | 88-99 | 88-100 | 88-101 |
| Average (%) | 94 | 93 | 93 | |
| 1:8 | Range (%) | 86-98 | 90-103 | 87-100 |
| Average (%) | 93 | 97 | 92 | |
| 1:16 | Range (%) | 101-116 | 86-101 | 101-116 |
| Average (%) | 107 | 93 | 107 |
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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