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Leptin ELISA Kit (LEP)

Details for Product LEP ELISA Kit No. ABIN625156, Supplier: Log in to see
Antigen
  • ob
  • obese
  • LEPD
  • OB
  • OBS
  • leptin
  • Lep
  • LEP
  • lep
Alternatives
Mouse (Murine) Leptin ELISA Kit
Reactivity
Mouse (Murine)
Alternatives
Kits with alternative reactivity to:
56
33
25
16
12
9
9
7
6
5
4
4
3
2
1
1
1
Methode Type
Sandwich ELISA
Detection Range
4-1000 pg/mL
Minimum Detection Limit
4 pg/mL
Application
ELISA
Options
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Purpose Mouse Leptin ELISA Kit for cell culture supernatants, plasma, and serum samples.
Sample Type Serum, Plasma, Cell Culture Supernatant
Analytical Method Quantitative
Specificity This ELISA kit shows no cross-reactivity with any of the cytokines tested: Mouse CD30, L CD30, T CD40, CRG-2, CTACK, CXCL16, Eotaxin , Eotaxin-2, Fas Ligand, Fractalkine, GCSF, GM-CFS, IGFBP-3, IGFBP-5, IGFBP-6, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-3 Rb, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17, KC, Leptin R, LIX, L-Selectin, Lymphotactin, MCP-1, MCP-5, M-CSF, MIG, MIP-1 alpha, MIP-1 gamma, MIP-2, MIP-3 beta, MIP-3 alpha, PF-4, P-Selectin, RANTES, SCF, SDF- 1alpha, TARC, TCA-3, TECK, TIMP-1, TNF-alpha, TNF RI, TNF RII, TPO, VCAM-1, VEGF.
Sensitivity < 4 pg/mL
Characteristics
  • Strip plates and additional reagents allow for use in multiple experiments
  • Quantitative protein detection
  • Establishes normal range
  • The best products for confirmation of antibody array data
Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Stop Solution
  • Assay Diluent(s)
  • Lyophilized Standard
  • Biotinylated Detection Antibody
  • Streptavidin-Conjugated HRP
  • TMB One-Step Substrate
Material not included
  • Distilled or deionized water
  • Precision pipettes to deliver 2 μL to 1 μL volumes
  • Adjustable 1-25 μL pipettes for reagent preparation
  • 100 μL and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • Log-log graph paper or computer and software for ELISA data analysis
Alternative Name Leptin (LEP ELISA Kit Abstract)
Background Leptin is a secreted protein of 16 kDa. Human and murine leptin show approximately 84 percent identity at the protein level. Leptin inhibits food intake and stimulates energy expenditure. Leptin also has thermogenic actions and regulates enzymes of fatty acid oxidation. Studies indicate that human obesity may be associated with leptin receptor deficiencies rather than constituting a problem with leptin itself. The Mouse Leptin ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of mouse Leptin in serum, plasma and cell culture supernatants. This assay employs an antibody specific for mouse Leptin coated on a 96-well plate. Standards and samples are pipetted into the wells and Leptin present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-mouse Leptin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Leptin bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.
Gene ID 16846
UniProt P41160
Research Area Cardiovascular, Atherosclerosis, Hormones
Pathways JAK-STAT Signaling, AMPK Signaling, Hormone Transport, Peptide Hormone Metabolism, Hormone Activity, Negative Regulation of Hormone Secretion
Application Notes Recommended Dilution for serum and plasma samples10 - 40 fold
Sample Volume 100 μL
Plate Pre-coated,Strips (12 x 8)
Protocol
  1. Prepare all reagents, samples and standards as instructed in the manual.
  2. Add 100 μL of standard or sample to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 μL of prepared biotin antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 μL of prepared Streptavidin solution to each well.
  7. Incubate 45 min at RT.
  8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 μL of Stop Solution to each well.
  11. Read at 450 nm immediately.
Reagent Preparation
  1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
    2. Sample dilution: If your samples need to be diluted, Assay Diluent A (Item D) should be used for dilution of serum/plasma samples. 1x Assay Diluent B (Item E) should be used for dilution of culture supernatants. Suggested dilution for normal serum/plasma: 10-40 fold. Please note that levels of the target protein may vary between different specimens. Optimal dilution factors for each sample must be determined by the investigator.
    3. Assay Diluent B should be diluted 5-fold with deionized or distilled water.
    4. Preparation of standard: Briefly spin the vial of Item C and then add 400 µL Assay Diluent A (for serum/plasma samples) or 1x Assay Diluent B (for cell culture medium) into Item C vial to prepare a 30 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 20 µL Leptin standard from the vial of Item C, into a tube with 580 µL Assay Diluent A or 1x Assay Diluent B to prepare 1,000 pg/mL standard solution. Pipette 300 µL Assay Diluent A or 1x Assay Diluent B into each tube. Use the standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 pg/mL). 200 µL 200 µL 200 µL 200 µL 200 µL 200myl 20 µL standard + 580 µL 1,000 400 160 64 25.6 10.24 4.1 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
    5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
    6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 120-fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure.
    7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 120-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 100 µL of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare 120 fold diluted HRP- Streptavidin solution. Mix well.
Assay Procedure
  1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
    2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.
    3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
    5. Discard the solution. Repeat the wash as in step
    6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
    7. Discard the solution. Repeat the wash as in step
    8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
    9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Assay Diluent A Mouse Leptin concentrations (pg/mL) O D =4 50 n m 0.01 0.1 1 10 1 10 100 1,000 Assay Diluent B Mouse Leptin concentrations (pg/mL) O D =4 50 n m 0.01 0.1 1 10 1 10 100 1,000
Sensitivity: The minimum detectable dose of Leptin is typically less than 4 pg/mL.
Recovery: Recovery was determined by spiking various levels of mouse Leptin into mouse serum, plasma and cell culture media. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Serum 93.29 82-102 Plasma 95.38 84-104 Cell culture media 94.47 83-103
Linearity: Sample Type Serum Plasma Cell Culture Media 1:2 Average % of Expected 93 94 95 Range ( %) 83-103 83-103 84-103 1:4 Average % of Expected 95 93 96 Range ( %) 84-104 82-102 83-103
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Assay Precision Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %
Restrictions For Research Use only
Handling Advice Avoid repeated freeze-thaw cycles.
Storage -20 °C
Storage Comment The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.
Expiry Date 6 months
Supplier Images
ELISA image for Leptin ELISA Kit (LEP) (ABIN625156) Leptin (LEP) ELISA Kit
Product cited in: Eo, Jeon, Lee, Lim: "Brown Alga Ecklonia cava polyphenol extract ameliorates hepatic lipogenesis, oxidative stress, and inflammation by activation of AMPK and SIRT1 in high-fat diet-induced obese mice." in: Journal of agricultural and food chemistry, Vol. 63, Issue 1, pp. 349-59, 2015 (PubMed).

Park, Shim, Na, Bae, Im, Song: "Orexin A regulates plasma insulin and leptin levels in a time-dependent manner following a glucose load in mice." in: Diabetologia, Vol. 58, Issue 7, pp. 1542-50, 2015 (PubMed).

Qian, Tang, Li, Liu, Zhang, Huang, Xue, Yu, Guo, Gao, Liu, Sun, Li, Jia, Tang: "BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 9, pp. E798-807, 2013

Wang, Zhang, Jiang, Deng, Zhang, Deng, Zhong, Wang, Zhu, Yang, Hong, Guo, Chen, Zhang, She, Chen, Yang, Zhang, Li: "Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice." in: Hepatology (Baltimore, Md.), Vol. 58, Issue 2, pp. 603-16, 2013 (PubMed).

Qian, Tang, Li, Liu, Zhang, Huang, Xue, Yu, Guo, Gao, Liu, Sun, Li, Jia, Tang: "BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 9, pp. E798-807, 2013

Qian, Tang, Li, Liu, Zhang, Huang, Xue, Yu, Guo, Gao, Liu, Sun, Li, Jia, Tang: "BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 9, pp. E798-807, 2013

Qian, Tang, Li, Liu, Zhang, Huang, Xue, Yu, Guo, Gao, Liu, Sun, Li, Jia, Tang: "BMP4-mediated brown fat-like changes in white adipose tissue alter glucose and energy homeostasis." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 9, pp. E798-807, 2013 (PubMed).

Nan Xia, Qin Zhang, Du, Wen: "Regulation effects of TZQ-F on adipocyte differentiation and insulin action." in: Journal of ethnopharmacology, Vol. 150, Issue 2, pp. 692-9, 2013 (PubMed).

Martín, Hernández, Córdova, Nieto: "Natural triterpenes modulate immune-inflammatory markers of experimental autoimmune encephalomyelitis: therapeutic implications for multiple sclerosis." in: British journal of pharmacology, Vol. 166, Issue 5, pp. 1708-23, 2012 (PubMed).

Uner, Sulu: "In vivo effects of leptin on lymphocyte subpopulations in mice." in: Immunobiology, Vol. 217, Issue 9, pp. 882-8, 2012 (PubMed).

Jiao, Feng, Ma, Nie, Paul, Li, Xu: "Constitutive Activation of IKKβ in Adipose Tissue Prevents Diet-Induced Obesity in Mice." in: Endocrinology, 2011 (PubMed).

Jiao, Ma, Feng, Zhang, Diehl, Chin, Yan, Xu: "FFA-induced adipocyte inflammation and insulin resistance: involvement of ER stress and IKKβ pathways." in: Obesity (Silver Spring, Md.), Vol. 19, Issue 3, pp. 483-91, 2011 (PubMed).

Kishino, Ito, Fujita, Kiuchi: "A mixture of the Salacia reticulata (Kotala himbutu) aqueous extract and cyclodextrin reduces the accumulation of visceral fat mass in mice and rats with high-fat diet-induced obesity." in: The Journal of nutrition, Vol. 136, Issue 2, pp. 433-9, 2006

Kishino, Ito, Fujita, Kiuchi: "A mixture of the Salacia reticulata (Kotala himbutu) aqueous extract and cyclodextrin reduces the accumulation of visceral fat mass in mice and rats with high-fat diet-induced obesity." in: The Journal of nutrition, Vol. 136, Issue 2, pp. 433-9, 2006

Kishino, Ito, Fujita, Kiuchi: "A mixture of the Salacia reticulata (Kotala himbutu) aqueous extract and cyclodextrin reduces the accumulation of visceral fat mass in mice and rats with high-fat diet-induced obesity." in: The Journal of nutrition, Vol. 136, Issue 2, pp. 433-9, 2006

Kishino, Ito, Fujita, Kiuchi: "A mixture of the Salacia reticulata (Kotala himbutu) aqueous extract and cyclodextrin reduces the accumulation of visceral fat mass in mice and rats with high-fat diet-induced obesity." in: The Journal of nutrition, Vol. 136, Issue 2, pp. 433-9, 2006 (PubMed).

Zhou, Shimabukuro, Koyama, Lee, Wang, Trieu, Newgard, Unger: "Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, Issue 12, pp. 6386-90, 1997 (PubMed).

Fujisawa: "[Guide for laboratory tests]." in: Nihon Ishikai zasshi. Journal of the Japan Medical Association, Vol. 61, Issue 2, pp. 196-209, 1970 (PubMed).

Background publications Maffei, Halaas, Ravussin, Pratley, Lee, Zhang, Fei, Kim, Lallone, Ranganathan: "Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects." in: Nature medicine, Vol. 1, Issue 11, pp. 1155-61, 1995 (PubMed).