I. Introduction and Protocol Overview

Obesity, and obesity-related disorders, are reaching alarming proportions in the US, and are on the
increase in Europe and Asia. A deeper understanding of the molecular and cellular dynamics of such
disorders, and their subsequent amelioration, will have a far-reaching impact on the quality of life of
millions of people worldwide.
Adipocytes (fat cells) express a variety of proteins that function in the homeostatic control of glucose
and lipid metabolism. Insulin regulates the translocation and secretion of many of these proteins in
response to changes in energy balance. Adipocyte complement-related protein of 30 kDa (Acrp30),
now known as adiponectin, is a protein whose secretion from adipocytes is enhanced by insulin
stimulation.
It has been suggested that the development of non-insulin dependent (Type II) diabetes may involve
dysregulation of adiponectin secretion. In support of the link between obesity and Type II diabetes,
it has been shown that decreased expression of adiponectin correlates with insulin resistance, and that adiponectin appears to be a potent insulin enhancer linking adipose tissue and whole-body glucose metabolism.
The Mouse/Rat Adiponectin ELISA Kit is designed to measure the concentration of
mouse/rat adiponectin from mouse/rat serum, adipocytes, or conditioned medium.
The principle of the assay is shown in Figure 1. Samples and serially diluted standard (recombinant
mouse adiponectin) solutions are added to an appropriate number of wells of the microtiter plate and
incubated. Adiponectin in the sample will be bound by the primary anti-mouse adiponectin polyclonal
antibody immobilized in the well (1st Reaction). After washing, the biotinylated secondary rabbit
anti-mouse adiponectin antibody is added to each well and allowed to incubate (2nd Reaction). The
biotinylated secondary rabbit anti-mouse adiponectin polyclonal antibody will bind to the adiponectin
trapped in the well in the 1st Reaction. After washing, a conjugate of horseradish peroxidase (HRP)
and streptavidin is added to each well and allowed to incubate (3rd Reaction). The HRP-conjugated
streptavidin will recognize and bind to biotinylated rabbit anti-adiponectin antibody trapped in the well
in the 2nd Reaction. After washing, the colorimetric substrate for the enzyme is added to all wells and
incubated. The color development is terminated by the addition of a stop solution. The intensity of the
color is measured at 450 nm. The concentrations of the samples tested are calculated using the
absorbance values of the adiponectin standard solutions assayed at the same time.

II. List of Components

·  Store all components at 2-8°C. DO NOT FREEZE.
         1 25X WASH SOLUTION                                                       1 Bottle (40mL)
         2 5X SAMPLE DILUENT                                                         1 Bottle (50mL)
         3 PRIMARY ANTIBODY-COATED PLATE                           1 Plate
One plate holds 12x8-well strips (96 wells), with adsorbed rabbit anti-mouse adiponectin polyclonal antibody. Plate is provided in a resealable foil pouch with desiccant.
         4 ADIPONECTIN STANDARD                                              1 Vial (2mL)
Recombinant mouse adiponectin (8.0 ng/mL)
         5 BIOTINYALED SECONDARY ANTIBODY SOLUTION 1 Bottle (12mL)
Biotinylated rabbit anti-mouse adiponectin polyclonal antibody
         6 HRP-CONJUGATED STREPTAVIDIN                               1 Vial (0.1mL)
Horseradish peroxidase (HRP)-conjugated streptavidin
         7 STREPTAVIDIN DILUENT                                                1 Bottle (15mL)
         8 SUBSTRATE A                                                                    1 Bottle (7.5mL)
         9 SUBSTRATE B                                                                    1 Bottle (7.5mL)
        10 STOP SOLUTION                                                              1 Bottle (15mL)
         PLATE SEALERS                                                                   1 Package
Six sealers per package

III. Additional Materials Required

The following materials are required, but not supplied:
           ·  Graduated cylinder
           ·  Micropipettor(s) and disposable pipette tips
           ·  Null strips for 96-well plate
           ·  96-well plate or manual strip washer
           ·  Paper towels or absorbent paper
           ·  Plate reader capable of measuring absorbance at a wavelength of 450nm (reference filter at
650 nm, optional)
           · Well-closed containers such as microtubes (1.5 mL or more in capacity)

IV. Reagent Preparation and Storage

            1. 1X Wash Solution
Prepare 1X Wash Solution by mixing all of the 25X Wash Solution (40mL) with 960 mL of deionized water or equivalent. If crystals are observed in the 25X Wash Solution bottle, warm the bottle in a 37°C water bath until the crystals disappear. After preparation, store 1X Wash Solution at 2-8°C.
            2. 1X Sample Diluent
Prepare 1X Sample Diluent by mixing all of the 5X Sample Diluent (50mL) with 200mL of deionized water or equivalent. After preparation, store 1X Sample Diluent at 2-8°C.
            3. Adiponectin Standard Solution
Prepare each Adiponectin Standard (4.0 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.25 ng/mL) by serially diluting the supplied adiponectin standard solution (8.0 ng/mL) with 1X Sample Diluent . Use undiluted adiponectin (8.0 ng/mL) and 1X Sample Diluent for 8.0 ng/mL and 0 ng/mL standard solutions, respectively.
           4. HRP-Conjugated Streptavidin Solution
Prepare the HRP-Conjugated Streptavidin by mixing 60 uL of HRP-Conjugated Streptavidin and 12mL of Streptavidin Diluent. Prepare only as much as needed immediately before the third reaction.
           5. Substrate Solution
Prepare the Substrate Solution by adding one part Substrate A to one part Substrate B. Prepare only as much Substrate Solution as needed. Return Substrate A to 2-8°C immediately after the necessary volume is removed.