Adiponectin, Mouse BioAssay™ ELISA Kit

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United States Biological

Catalog # A0725-51
Intended Use This ELISA is a sandwich enzyme immunoassay for the quantitative measurement of mouse Adiponectin/Acrp30 protein in serum. It is intended for in vitro research use only.

Features • The total assay time is less than 3 hours. • The kit measures serum Adiponectin / Acrp30. • Quality Controls are mouse serum based. • Components in the kit are provided ready-to-use, concentrated, and lyophilized.
Storage, Expiration Store the kit at 2-8C. Under these conditions, the kit is stable until the expiration date (see the label on the box).
Summary Adiponectin, also referred to as Acrp30, AdipoQ and GBP-28, is a recently discovered 244 aminoacid protein, the product of the apM1 gene, which is physiologically active and specifically and highly expressed in adipose cells (adipokine). The protein belongs to the soluble defence collagen superfamily; it has a collagen-like domain structurally homologous with collagen VIII and X and complement factor C1q-like globular domain. Adiponectin forms homotrimers, which are the building blocks for higher order complexes found circulating in serum. Adiponectin receptors AdipoR1 and AdipoR2 have been recently cloned; AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver.

Paradoxically, adipose tissue-expressed adiponectin levels are inversely related to the degree of adiposity. A reduction in adiponectin serum levels is accompanied by insulin resistance states, such as obesity and type 2 diabetes mellitus. It is also reported in patients with coronary artery disease. Increased adiponectin levels are associated with type 1 diabetes mellitus, anorexia nervosa and chronic renal failure. Adiponectin concentrations correlate negatively with glucose, insulin, triglyceride concentrations and body mass index and positively with high-density lipoprotein-cholesterol levels and insulin-stimulated glucose disposal.

Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by increasing tissue fat oxidation. It inhibits the inflammatory processes of atherosclerosis suppressing the expression of adhesion and cytokine molecules in vascular endothelial cells and macrophages, respectively. This adipokine assumes the role of a scaffold of newly formed collagen in myocardial remodeling after ischaemic injury and also stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signalling in endothelial cells.
Test Principle The surface of the wells in a microtitration plate is coated with monoclonal anti-mouse Adiponectin specific antibody. The standards, quality controls (QC), and diluted samples are pipetted into the wells. Any mouse Adiponectin/Acrp30 present is captured by immobilized antibody and unbound protein is washed away after the first incubation period. Then, a horseradish peroxidase (HRP) conjugated polyclonal anti-mouse Adiponectin antibody is added to the wells and incubated. Following another washing step, to remove unbound antibody-HRP conjugate, a substrate solution (H2O2 and TMB) is added to the wells. The enzymatic reaction yields a blue product that turns yellow when acidic stop solution is added. The intensity of the color, measured spectrophotochemically at 450nm, is directly proportional to the amount of mouse Adiponectin bound in the initial step. The concentrations of the diluted test samples are then read off the standard curve that is constructed by plotting the absorbance values against each respective mouse Adiponectin standard level using a four-parameter function. The dilution factor needs to be taken into consideration when calculating the actual concentration of Adiponectin/Acrp30 analyte in the test samples.
Precautions 1. For in vitro research use only.

2. This kit contains components of animal origin.

3. Wear gloves, eye and clothing protection when handling supplied immunodiagnostic material, particularly acidic Stop Solution, and Substrate Solution that contains hydrogen peroxide and tetramethylbenzidine (TMB). Avoid contact with these two latter reagents. The Stop and/or Substrate Solutions may cause skin/eye irritation. If this occurs, wash the skin/eyes thoroughly with water and seek medical attention if necessary.

4. Do not pipet solutions by mouth.

5. Do not drink, eat, or smoke in any area where immunodiagnostic materials are being handled.

6. Reagents with different lot numbers or sources should not be mixed.

7. Reagents should not be used beyond the expiration date marked on the kit label.
Reagents Supplied 1. Antibody Coated Microtiter Strips, 96 wells, sealed

2. Conjugate Solution, 13ml

3. Mouse Adiponectin Master Calibrator, 8ng, 2 vial

4. Quality Controls: High and Low, 2 vials each. Refer to the Certificate of Analysis for the actual Quality Control values.

5. Dilution Buffer Concentrate (10x), 22ml

6. Wash Solution Concentrate (5x), 100ml

7. Substrate Solution (TMB), 13ml

8. Stop Solution, 13ml
Reagents Required but Not Supplied 1. Test tubes for diluting samples

2. Precision pipettes to deliver 10-1000ul and disposable tips

3. Multichannel pipette 50-200ul

4. Microplate reader with 450 10nm filter

5. Orbital microplate shaker capable of agitation at approximately 300rpm

6. Software package facilitating data generation and analysis

7. Microplate washer (optional). Manual washing is possible but not preferable.

8. Glassware (graduated cylinder and bottle for Wash Solution)

9. Distilled water
Result Calculations Most microtiter plate readers perform automatic calculations of analyte concentration. The standard curve is constructed by plotting the absorbance (Y) of calibrators against the log of the known concentration (X) of calibrators using the four-parameter function. Alternatively, the logit log function can be used to linearize the calibration curve (i.e. logit of absorbance (Y) is plotted against the log of the known concentration (X) of calibrators).

The concentration of Adiponectin in diluted samples is determined using a standard curve. The actual amount of Adiponectin in the original serum is assessed by multiplying the assay result by a dilution factor of 10 000. So if, for example, the read value is 2.65ng/ml, 2.65 x 10 000 gives 26 500ng/ml which represents 26.5 ug/ml).
Limits of Assay Samples exceeding an Adiponectin level of 8ng/ml should be measured at higher dilution (e.g. 1:20 000) and the new dilution factor needs to be taken into consideration (20 000 in this case).

Performance Characteristics Typical analytical data obtained with this product are presented below. All the results in this section are valid for 10 000-fold diluted samples and laboratory conditions of 26C.

A. Sensitivity The sensitivity of the mouse Adiponectin ELISA, defined by the 99% confidence interval, is 0.104ng/ml. Sensitivity or limit of detection (LOD) is determined as a mean absorbance value plus three standard deviations of thirty Blank* readings (ABlank + 3x SDBlank). Then the corresponding concentration is calculated. *The dilution buffer is pipetted into Blank wells

B. Specificity The antibodies in this kit are highly specific for mouse Adiponectin protein. No cross-reactivity with sera of other animal species (rat, hamster, rabbit, sheep, goat, cattle, swine, horse) has been observed. The assay recognizes natural and recombinant mouse Adiponectin. No cross-reactivity has been observed for mouse cytokines

RELM- , RELM- , Leptin, Leptin-receptor, Resistin; as well as for rat Leptin and human Adiponectin at 100ng/ml.

very little cross-reactivity (0.3%) has been measured for rat recombinant adiponectin at 100ng/ml.

C. Precision • Intra-assay (Within-Run, n=8)

Sample Mean(ng/ml) SD(ng/ml) CV(%) 1 2.708 0.063 2.31 2 1.487 0.040 2.66 3 1.123 0.022 1.96 4 0.871 0.031 3.53

• Inter-assay (Run-to-Run, n=5) Sample Mean(ng/ml) SDng/ml) CV(%) 1 2.571 0.121 4.71 2 1.462 0.056 3.82 3 1.297 0.093 7.14 4 0.785 0.044 5.65

D. Dilution Linearity Serum samples were further serially diluted with Dilution Buffer, after primary dilution 1

10 000, and assayed.

Sample Dilution Observed Expected Recovery

(ng/ml) (ng/ml) O/E(%)

- 2.61 - -

1 : 2 1.15 1.24 92.6

1 : 4 0.51 0.62 82.4

1 : 8 0.26 0.31 83.4


- 5.26 - -

1 : 2 2.55 2.63 96.8

1 : 4 1.22 1.32 92.9

1 : 8 0.53 0.66 80.7

E. Spiking Recovery Serum samples were spiked to different levels of mouse Adiponectin, diluted with Dilution Buffer 1

10 000, and assayed.

Sample Addition Observed Expected Recovery

(ng/ml) (ng/ml) O/E (%)


- 0.90 - -

0.5 1.34 1.40 98.5

1.0 1.82 1.90 97.2

2.0 2.65 2.90 97.8


- 2.00 - -

0.5 2.52 2.50 101.0

1.0 3.09 3.00 103.2

2.0 3.94 4.00 98.6

F. Effect of freezing/thawing on the concentration of mouse adiponectin in undiluted serum

No significant decline was observed in concentration of mouse Adiponectin in serum samples after repeated (5x) freezing/thawing cycle. Analyte concentration varies within the limit of 10%. Sodilum azide and -aminocaproic acid are used as a conservans.

However, we recommend researchers avoid using a sample that has been frozen and thawed more than once. Number Of Serum Sample f/t Cycles (ng/ml) (%) 1 0 1.245 100.0% 1x 1.238 99.4% 3x 1.217 97.8% 5x 1.239 99.5% 2 0 5.975 100.0% 1x 6.208 103.9% 3x 5.585 93.5% 5x 5.634 94.3% 3 0 4.881 100.0% 1x 5.420 111.0% 3x 5.252 107.6% 5x 4.952 101.5% 4 0 3.215 100.0% 1x 3.411 106.1% 3x 3.386 105.3% 5x 3.507 109.1%

Mean values for 13 samples

Number of Number of serum samples f/t cycles Mean n=13 0 100.0% n=13 1x 97.5% n=13 3x 99.5% n=13 5x 100.6%

G. Stability of undiluted samples at 4C No significant decline was observed in the concentration of mouse adiponectin in serum samples after 14 days storage at 4C. Analyte concentration varies usually within the limit of 5% and rarely up to 15%. There is no difference between serum with and without preservatives. Sodilum azide and epsilon-aminocaproic acid are used as preservatives. In spite of this stability, we recommend storing samples frozen.

Temperature, Serum Sample Period (ng/ml) (%) 1 - 20C 2.213 100.0% 4C, 1 day 2.107 95.2% 4C, 7 days 2.097 94.8% 4C, 14 days 2.139 96.7% 2 - 20C 1.524 100.0% 4C, 1 day 1.418 93.0% 4C, 7 days 1.574 103.3% 4C, 14 days 1.485 97.4% 3 - 20C 1.630 100.0% 4C, 1 day 1.508 92.5% 4C, 7 days 1.625 99.7% 4C, 14 days 1.721 105.6% 4 - 20C 1.858 100.0% 4C, 1 day 1.892 101.8% 4C, 7 days 1,840 99.0% 4C, 14 days 1.888 101.6%

Mean values for 18 samples, in per cent units, are reported in the table below.

Number of Temperature, serum samples Period Mean n=18 -20C 100.0% n=18 4C, 1 day 96.8% n=18 4C, 7 days 102.3% n=18 4C, 14 days 107.4%
Troubleshooting and FAQs 1. Weak signal in all wells

Possible explanations:

Ommiting a reagent or a step

Improper preparation or storage of a reagent

Assay performed before reagents were allowed to reach room temperature

2. High signal and background in all wells Possible explanations

Improper or inadequate washing

Overdeveloping; incubation time should be reduced before addition of Stop Solution

3. High coefficient of variation (CV) Possible explanation

Improper or inadequate washing


Product Note

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.

Related Tags Adiponectin, Mouse Bioassay, Acrp30 Protein, Aminoacid Protein, Globular Domain, Glucose, Insulin, Triglyceride, Conjugated Polyclonal, Antibody Coated Microtiter Strips, Dilution Linearity Serum




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