Adiponectin Mouse ELISA

INTENDED USE

The RD293023100R Mouse Adiponectin ELISA is a sandwich enzyme immunoassay for the quantitative measurement of mouse adiponectin.

Features

• It is intended for research use only
• The total assay time is less than 3.5 hours
• The kit measures total mouse adiponectin in serum
• Assay format is 96 wells
• Quality Controls are mouse serum based
• Standard is recombinant protein based
• Components of the kit are provided ready to use, concentrated or lyophilized

STORAGE, EXPIRATION

Store the complete kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box).

Clinical Application

• Energy metabolism and weight regulation
• Coronary artery disease
• Chronic renal failure

TEST PRINCIPLE

In the BioVendor Mouse Adiponectin ELISA, standards, quality controls and samples are incubated in microplate wells pre-coated with monoclonal anti-mouse adiponectin antibody. After 60 minutes incubation and washing, biotin labelled polyclonal anti-mouse adiponectin antibody is added and incubated at RT with captured mouse adiponectin for 60 minutes. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation at RT and the last washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of mouse adiponectin. A standard curve is constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

ASSAY PROCEDURE

1. Pipet 100  μl of each individual concentration of Standards, Quality Controls, Dilution Buffer (=Blank) and diluted samples, preferably in duplicates, into the appropriate wells. See Figure 1 for example of work sheet.
2. Incubate the plate at room temperature (ca. 25°C) for 1 hour, shaking at ca. 300 rpm on an orbital microplate shaker.
3. Wash the wells 3-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel.
4. Add 100 μl of Biotin Labelled Antibody solution into each well.
5. Incubate the plate at room temperature (ca. 25°C) for 1 hour, shaking at ca. 300 rpm on an orbital microplate shaker.
6. Wash the wells 3-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel.
7. Add 100 μl of Streptavidin-HRP Conjugate into each well.
8. Incubate the plate at room temperature (ca. 25°C) for 30 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.
9. Wash the wells 3-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel.
10. Add 100 μl of Substrate Solution into each well. Avoid exposing the microtiter plate to direct sunlight. Covering the plate with e.g. aluminium foil is recommended.
11. Incubate the plate for 10 minutes at room temperature. The incubation time may be extended [up to 20 minutes] if the reaction temperature is below than 20°C. Do not shake the plate during the incubation.
12. Stop the colour development by adding 100 μl of Stop Solution.
13. Determine the absorbance of each well using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550 – 650 nm). Subtract readings at 630 nm (550 – 650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 12.

Note: If some samples and standard/s have absorbances above the upper limit of your microplate reader, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine mouse adiponectin concentration of off-scale standards and samples. The readings at 405 nm should not replace the readings for samples that were “in range” at 450 nm.

Note 2: Manual washing: Aspirate wells and pipet 0.35 ml Wash Solution into each well. Aspirate wells and repeat twice. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

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