Leptin (LEP) ELISA Kit

Details for Product No. ABIN625156
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Target Name (Antigen)
Synonyms LEPD, OB, OBS, ob, obese
Alternatives Mouse (Murine)
Kits with alternative reactivity to:
(1), (1), (6), (5), (3), (3), (2), (1), (3), (18), (3), (17), (6), (5), (10), (1), (3), (1)
Pubmed 2 references available
Catalog no. ABIN625156
Quantity 96 tests
416.90 $   Plus shipping costs $45.00
Shipping to United States (Change)
Availability Will be delivered in 5 to 7 Business Days

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Sample Type Serum, Plasma, Cell Culture Supernatant
Specificity This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., 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-1beta, 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-1alpha, MIP-1gamma, MIP-2, MIP-3beta, MIP-3alpha, 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 The minimum detectable dose of Leptin is typically less than 1.5 pg/ml.
Characteristics Mouse Leptin (OB) ELISA Kit For Serum, Plasma, Cell Culture Supernatant
Components 1. Leptin Microplate (Item A): 96 wells (12 strips x 8 wells) coated with anti-mouse Leptin. 2. Wash Buffer Concentrate (20x) (Item B): 25 ml of 20x concentrated solution 3. Standards (Item C): 2 vials, recombinant mouse Leptin. 4. Assay Diluent A (Item D): 30 ml, 0.09% sodium azide as preservative. For Standard/Sample (serum/plasma) diluent. 5. Assay Diluent B (Item E): 15 ml of 5x concentrated buffer. For Standard/Sample (cell culture medium/urine) diluent. 6. Detection Antibody Leptin (Item F): 2 vial of biotinylated anti-mouse Leptin (each vial is enough to assay half microplate). 7. HRP-Streptavidin Concentrate (Item G): 8 µl of 6,000x concentrated HRP-conjugated streptavidin. 8. TMB One-Step Substrate Reagent (Item H): 12 ml of 3,3™,5,5™-tetramethylbenzidine (TMB) in buffered solution. 9. Stop Solution (Item I): 8 ml of 2 M sulfuric acid.
Material not included 1. Microplate reader capable of measuring absorbance at 450 nm. 2. Precision pipettes to deliver 2 µl to 1 ml volumes. 3. Adjustable 1-25 ml pipettes for reagent preparation. 4. 100 ml and 1 liter graduated cylinders. 5. Absorbent paper. 6. Distilled or deionized water. 7. Log-log graph paper or computer and software for ELISA data analysis. 8. Tubes to prepare standard or sample dilutions.
Alternative Name Leptin
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%.
Plate 96 wells
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) is used for dilution of serum/plasma samples, and Assay Diluent B (Item E) is used for dilution of culture supernatants and urine. 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 4 µl Leptin standard from the vial of Item C, into a tube with 596 µl Assay Diluent A or 1x Assay Diluent B to prepare 200 pg/ml stock standard solution. Pipette 300 µl Assay Diluent A or 1x Assay Diluent B into each tube. Use the stock standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Gently vortex to mix. Assay Diluent A or 1x Assay Diluent B serves as the zero standard (0 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 6,000-fold with 1x Assay Diluent B. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 2 µl of HRP-Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent B to prepare 6,000 fold diluted HRP- Streptavidin solution.
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 µl) 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 3. 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 3. 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.
Restrictions For Research Use only
Storage 4 °C
Product cited in: Jiao, Feng, Ma et al.: "Constitutive Activation of IKKβ in Adipose Tissue Prevents Diet-Induced Obesity in Mice." in: Endocrinology, 2011 (PubMed).

Martín, Hernández, Córdova et al.: "Natural Triterpenes Modulate Immune-Inflammatory Hallmarks to Protect against Experimental Autoimmune Encephalomyelitis. Therapeutic Implications for Multiple Sclerosis." in: British journal of pharmacology, 2012 (PubMed).

Reactivities (1), (1), (6), (5), (3), (3), (2), (1), (3), (18), (3), (17), (6), (5), (10), (1), (3), (1)
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