Emr1001_1.pdf

Mouse Resistin
ELISA Kit
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For any questions regarding troubleshooting or performing the assay, please contact our support team at [email protected].
Assay Summary
Add 50 μl of standard/samples per well.
Wash, then add 50 μl of SP per well.
Add 50 μl of Stop Solution per well.
Assay Template
AssayMax Mouse Resistin ELISA Kit
Introduction
Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity (1). Resistin is produced by white and brown adipose tissues but has also has been identified in several other tissues, including the hypothalamus, pituitary and adrenal glands, pancreas, gastrointestinal tract, myocytes, spleen, white blood cells, and plasma.
Resistin antagonizes insulin action, and is down regulated by rosiglitazone and peroxisome proliferator-activated receptor gamma agonists (2). Resistin is elevated in patients with type 2 diabetes and may play a role in the vascular complications of this disorder (3). Recently, resistin has been discussed controversially as a missing link between obesity and insulin resistance (4).
Principle of the Assay
The AssayMax Mouse Resistin ELISA kit is designed for detection of mouse resistin in plasma, serum, and cell culture supernatants. This assay employs a quantitative sandwich enzyme immunoassay technique which measures resistin in 5 hours. A murine monoclonal antibody specific for resistin has been pre-coated onto a microplate. Resistin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for resistin, 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.
Caution and Warning
Prepare all reagents (working diluent buffer, wash buffer, standards,
biotinylated antibody, and SP conjugate) as instructed, prior to running
the assay.
Prepare all samples prior to running the assay. The dilution factors for
the samples are suggested in this protocol. However, the user should
determine the optimal dilution factor.
Spin down the SP conjugate vial and the biotinylated antibody vial
before opening and using contents.
The kit should not be used beyond the expiration date.
The Stop Solution is an acidic solution.
Reagents
Mouse Resistin Microplate: A 96 well polystyrene microplate (12 strips
of 8 wells) coated with a monoclonal antibody against resistin.
Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing
tapes that can be cut to fit the format of the individual assay.
Mouse Resistin Standard: Mouse resistin in a buffered protein base (8
ng, lyophilized).
Biotinylated Mouse Resistin Antibody (50x): A 50-fold concentrated
biotinylated polyclonal antibody against mouse resistin (140 Pl).
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, 2 bottles).
Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold
concentrate (80 Pl).
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).
Storage Condition
Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date.
Store SP Conjugate and biotinylated antibody at -20°C.
Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C.
Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 1 month in a vacuum desiccator.
Diluent (1x) may be stored for up to 1 month at 2-8°C.
Store standard at 2-8°C before reconstituting with diluent and at -20°Cafter reconstituting with diluent.
Other Supplies Required
Microplate reader capable of measuring absorbance at 450 nm.
Pipettes (1-20 Pl, 20-200 Pl, 200-1000 Pl and multiple channel).
Deionized or distilled reagent grade water.
Sample Collection, Preparation and Storage
Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate
as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes.
Dilute samples 1:50 with MIX Diluent and assay. The undiluted samples
can be stored at -20°C or below for up to 3 months. Avoid repeated
freeze-thaw cycles. (EDTA or Heparin can also be used as an
anticoagulant.)
Serum: Samples should be collected into a serum separator tube. After
clot formation, centrifuge samples at 3000 x g for 10 minutes and remove
serum. Dilute samples 1:50 into MIX Diluent and assay. The undiluted
samples can be stored at -20°C or below for up to 3 months. Avoid
repeated freeze-thaw cycles.
Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for
10 minutes to remove debris. Collect supernatants and assay. Store
samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Reagent Preparation
Freshly dilute all reagents and bring all reagents to room temperature before use.
MIX Diluent Concentrate (10x): If crystals have formed in the
concentrate, mix gently until the crystals have completely dissolved.
Dilute MIX Diluent Concentrate 1:10 with reagent grade water. Store for
up to 1 month at 2-8°C.
Standard Curve: Reconstitute the 8 ng of Mouse Resistin Standard with 2
ml of MIX Diluent to generate a standard solution of 4 ng/ml. Allow the
standard to sit for 10 minutes with gentle agitation prior to making
dilutions. Prepare duplicate or triplicate standard points by serially
diluting the resistin standard solution (4 ng/ml) twofold with equal
volume of MIX Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.0625 ng/ml
solutions. MIX Diluent serves as the zero standard (0 ng/ml). Any
remaining solution should be frozen at -20°C and used within 30 days.
Standard
[Mouse Resistin]
Dilution
Biotinylated Mouse Resistin Antibody (50x): Spin down the antibody
briefly and dilute the desired amount of the antibody 1:50 with MIX
Diluent. Any remaining solution should be frozen at -20°C.
Wash Buffer Concentrate (20x): If crystals have formed in the
concentrate, mix gently until the crystals have completely dissolved.
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.
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 desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
Add 50 Pl of Mouse Resistin Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last sample addition.
Wash five times with 200 Pl of Wash Buffer manually. Invert the plate each time and decant the contents; hit 4-5 times on absorbent materialto completely remove the liquid. If using a machine, wash six times with 300 Pl of Wash Buffer and then invert the plate, decanting the contents; hit 4-5 times on absorbent material to completely remove the liquid.
Add 50 Pl of Biotinylated Mouse Resistin Antibody to each well and incubate for 2 hours.
Wash the microplate as described above.
Add 50 Pl of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance.
Wash the microplate as described above.
Add 50 Pl of Chromogen Substrate per well and incubate for about 30 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 Pl 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. If wavelength correction is available, subtract readings at
570 nm from those at 450 nm to correct optical imperfections.
Otherwise, read the plate at 450 nm only. 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.
Data Analysis
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 four-parameter logistic curve-fit.
Determine the unknown sample concentration from the Standard Curve and multiply the 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.
Performance Characteristics
The minimum detectable level of resistin is typically ~ 0.06 ng/ml.
Intra-assay and inter-assay coefficients of variation were 4.1 % and 7.5% respectively.
Linearity
Average Percentage of Expected Value
Sample Dilution
Recovery
Standard Added Value
Recovery %
Average Recovery %
Cross-Reactivity
No significant cross-reactivity or interference was observed.
References
Fujita H et. al. (2002) Biochem Biophys Res Commun. 298(3):345-9 Adeghate E. (2004) Cell Mol Life Sci. 61(19-20):2485-96 Calabro P et. al. (2004) Circulation 23;110(21):3335-40 Schaffler A et. al. (2004) Horm Metab Res. 36(10):702-7 Related Products
ER1001-1 AssayMax Human Resistin ELISA Kit (Plasma, Serum, and Cell Culture Supernatant samples) ǁǁǁ͘ĂƐƐĂLJƉƌŽ͘ĐŽŵͻ-mail: [email protected]

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