Leptin (LEP) ELISA Kit

Details for Product No. ABIN612800
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Target Name (Antigen)
Synonyms ob, obese, LEPD, OB, OBS
Reactivity
Alternatives Mouse (Murine)
Kits with alternative reactivity to:
(1), (6), (5), (3), (2), (2), (3), (20), (3), (19), (6), (6), (12), (1), (2), (1)
Methode Type Sandwich ELISA
Minimum Detection Limit 300 pg/mL
Application
ELISA
Pubmed 6 references available
Quantity 96 tests
Options
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Availability Will be delivered in 2 to 3 Business Days
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Catalog No. ABIN612800
429.00 $
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Purpose The AssayMax Mouse Leptin ELISA kit is designed for detection of mouse leptin in plasma, and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Cross-Reactivity (Details) No significant cross-reactivity or interference was observed. No significant cross-reactivity or interference was observed.
Components µleptin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse Leptin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. µleptin Standard: Mouse Leptin in a buffered protein base (96 ng, lyophilized). 1 Biotinylated Leptin Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against mouse leptin (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.
Alternative Name Leptin
Background Leptin, a 16-kDa protein secreted from white adipocytes, has been implicated in the regulation of food intake, energy expenditure, and whole-body energy balance in rodents and humans. Leptin has been a potential target for treating obesity. The plasma insulin response appears more closely associated with the plasma leptin concentration. Neonatal leptin levels are strongly associated with female gender, birth length, and formula feeding. Leptin concentrations were higher in women than in men. In women, serum leptin was the most important predictor of myocardial infarction (MI). In patients with angiographically confirmed coronary atherosclerosis, leptin is a novel predictor of future cardiovascular events independent of other risk factors, including lipid status and CRP. Leptin may also play an important role in the pathophysiology of osteoarthritis (OA).
Research Area Cardiovascular, Atherosclerosis, Hormones
Sample Volume 50 µL
Assay Time < 5 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures leptin in less than 5 hours. A polyclonal antibody specific for leptin has been pre-coated onto a microplate. Leptin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for leptin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Reagent Preparation Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 96 ng of Mouse Leptin Standard with 4 ml of MIx Diluent to generate a stock solution of 24 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the leptin standard solution (24 ng/ml) 1:2 with MIx Diluent to produce 12, 6, 3, 1.5, 0.75, and 0.325 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Leptin] (ng/ml) P1 1 part Standard (24 ng/ml) 24.000 P2 1 part P1 + 1 part MIx Diluent 12.000 P3 1 part P2 + 1 part MIx Diluent 6.000 P4 1 part P3 + 1 part MIx Diluent 3.000 P5 1 part P4 + 1 part MIx Diluent 1.500 P6 1 part P5 + 1 part MIx Diluent 0.750 P7 1 part P6 + 1 part MIx Diluent 0.325 P8 MIx Diluent 0.000 Biotinylated Leptin Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.
Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:4 with MIx Diluent. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:4 into MIx Diluent. Store serum at -20°C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Assay Procedure Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated Leptin Antibody to each well and incubate for two hours. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for about 8 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.
Calculation of Results Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or semi-log curve fit. Determine the unknown sample concentration from the Standard Curve and multiply the urine value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.
Assay Precision Intra-assay and inter-assay coefficients of variation were 4.5 % and 7.7% respectively.
Restrictions For Research Use only
Handling Advice The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Product cited in: Houseknecht, Baile, Matteri et al.: "The biology of leptin: a review." in: Journal of animal science, Vol. 76, Issue 5, pp. 1405-20, 1998 (PubMed).

Abbasi, Carantoni, McLaughlin et al.: "Plasma insulin concentration is more tightly linked to plasma leptin concentration than is the body mass index." in: Metabolism: clinical and experimental, Vol. 49, Issue 4, pp. 544-7, 2000 (PubMed).

Dumond, Presle, Terlain et al.: "Evidence for a key role of leptin in osteoarthritis." in: Arthritis and rheumatism, Vol. 48, Issue 11, pp. 3118-29, 2003 (PubMed).

Wolk, Berger, Lennon et al.: "Plasma leptin and prognosis in patients with established coronary atherosclerosis." in: Journal of the American College of Cardiology, Vol. 44, Issue 9, pp. 1819-24, 2004 (PubMed).

Wallerstedt, Eriksson, Niklason et al.: "Serum leptin and myocardial infarction in hypertension." in: Blood pressure, Vol. 13, Issue 4, pp. 243-6, 2004 (PubMed).

Petridou, Mantzoros, Belechri et al.: "Neonatal leptin levels are strongly associated with female gender, birth length, IGF-I levels and formula feeding." in: Clinical endocrinology, Vol. 62, Issue 3, pp. 366-71, 2005 (PubMed).

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