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Leptin (LEP) ELISA Kit

Details for Product No. ABIN625156
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Target Name (Antigen)
Synonyms LEPD, OB, OBS, ob, obese
Reactivity
Alternatives Mouse (Murine)
Kits with alternative reactivity to:
(1), (1), (6), (5), (3), (2), (2), (1), (3), (19), (3), (18), (5), (5), (11), (1), (3), (1)
Methode Type Sandwich ELISA
Application
ELISA
Pubmed 2 references available
Quantity 96 tests
Options
Shipping to United States (Change)
Availability Will be delivered in 5 to 7 Business Days
Catalog No. ABIN625156
416.90 $
Plus shipping costs $45.00

Order hotline:

  • +1 404 474 4654
  • +1 888 205 9894 (TF)
Purpose Quantitative measurement
Sample Type Serum, Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Specificity Cross Reactivity: 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 < 4 pg/mL
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%.
Research Area Cardiovascular, Atherosclerosis, Hormones
Sample Volume 100 µL
Plate Pre-coated,96 wells
Protocol 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.
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 4 °C/-20 °C/-80 °C
Storage Comment May be stored for up to 6 months at 2 - 8 °C from the date of shipment. Standard (recombinant protein) should be stored at -20 °C or -80 °C (recommended at -80 °C) after reconstitution. Opened Microplate Wells or reagents may be store for up to 1 month at 2 - 8 °C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge. Note: the kit can be used within one year if the whole kit is stored at -20 °C.
Expiry Date 12 months
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).

Request Want additional data for this product?

The Independent Validation Initiative strives to provide you with high quality data. Find out more

Catalog No. ABIN625156
416.90 $
Plus shipping costs $45.00

Order hotline:

  • +1 404 474 4654
  • +1 888 205 9894 (TF)
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