ImmunoDiagnostics Human Adiponectin ELISA kit

Reagents Supplied

Each kit is sufficient for one 96-well plate and contains the following components:

1. Microtiter strips (96 wells)-Coated with a mouse monoclonal antibody against human adiponectin, sealed
2. 10xwash buffer, 30ml
3. 5xAssay buffer, 30ml
4. 100xDetection antibody solution – A mouse monoclonal antibody against human adiponectin conjugated with horseradish peroxidase, 0.12ml
5. Human adiponectin standard, 100ng of recombinant human adiponectin in a buffered protein base, lyophilized
6. Substrate solution, 12ml, ready for use
7. Stop solution, 12ml, ready for use

Other Materials Required, But Not Provided

1. Pipettes and pipette tips
2. 96-well plate or manual strip washer
3. Buffer and reagent reservoirs
4. Paper towels or absorbent paper
5. Plate reader capable of reading absorbency at 450nm
6. Distilled water or deionized water

Storage

The kit should be stored at 2-8°C upon receipt, and all reagents should be equilibrated to room temperature before use. Remove any unused antibody-coated strips from the Human adiponectin microplate, return them to the foil pouch and reseal. Once opened, strips may be stored at 2-8°C for up to one month.

Reagent Preparation

Bring all reagents and materials to room temperature before assay.

1. 1xAssay Buffer
   Prepare 1xAssay buffer by mixing the 5xAssay buffer (30ml) with 120ml of distilled water or deionized water. If precipitates are observed in the 5x Assay buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1xAssay buffer may be stored at 2-8°C for up to one month.
2. 1xWash Buffer
   Prepare 1xWash buffer by mixing the I0xWash buffer (30ml) with 270ml of distilled water or deionized water. If precipitates are observed in the 10x Wash buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1xWash buffer may be stored at 2-8°C for up to one month.
3. 1xDetection antibody solution
   Spin down the 100xDetection antibody solution briefly and dilute the desired an1ount of the antibody 1: 100 with 1 xAssay buffer, 100µl of the 1xDetection antibody solution is required per well. Prepare only as much 1xDetection antibody solution as needed. Return the l00xDetection antibody solution to 2-8°C immediately after the necessary volume is removed.

Preparation of Standard and Samples

Human Adiponectin Standards: Reconstitute the lyophilized standard with 1ml of 1xAssay buffer to generate a standard stock solution of 100ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare serially diluted standards using 1xAssay buffer as follows.

1xAssay buffer serves as the zero standard (0ng/ml). The reconstituted standard stock should be aliquoted and frozen at -20°C for one month. Avoid repeating freezing/thawing cycles. Please do not store the diluted standard solutions.

Sample preparation

Serum or plasma sample is generally required a 1000-fold dilution in this assay. A two-step dilution is suggested.

Step 1: Add 10µl of sample to 490µl of 1xAssay buffer to give a 50-fold diluted sample solution.

Step 2: Add 20µl of the 50-fold diluted sample solution to 380µl of 1xAssay buffer to give a final 1000-fold diluted sample solution. Cellular extract and culture media dilutions will vary and need to be optimized by the user, also use 1xAssay buffer to prepare these samples.

Assay Principle

This assay is a quantitative sandwich ELISA using monoclonal antibodies against human adiponectin. The immunoplate is pre-coated with a monoclonal antibody specific for human adiponectin. Standards and samples are pipetted into the wells and any human adiponectin present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase (HRP)-linked monoclonal antibody specific for human adiponectin is added to the wells. After a final wash step, an HRP substrate solution is added and color develops in proportion to the amount of human adiponectin bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured human adiponectin, the unknown sample concentration can be interpolated from a reference curve included in each assay.

Assay Procedure

It is recommended that all standards and samples be assayed in duplicate.

1. Add 100µl of standard or sample per well, incubate at room temperature for 1 hour.
2. Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300µl of 1xwash buffer to each well and incubate for 1 minute. Discard the 1xwash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes.
3. Add 100µl of 1xDetection antibody solution to each well, incubate at room temperature for 1 hour.
4. Wash each well 3 times as in step 2.
5. Add 100µl of Substrate solution to each well, incubate at room temperature for 15 minutes. Protect from light.
6. Add 100µl of Stop solution to each well, gently tap the plate frame for a few seconds to ensure thorough mixing.
7. Measure absorbance of each well at 450nm immediately.

Calculation

1. Subtract the absorbance of the blank from that of standards and samples.
2. Generate a standard curve by plotting the absorbance obtained (y-axis) against human adiponectin concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Any curve of 4-parameter or log-log curve fitting can be used for calculation.
3. Determine human adiponectin concentration of samples from standard curve and multiply the value by the dilution factor.

Assay Characteristics

Sensitivity

The lowest level of human adiponectin that can be detected by this assay is 1.56ng/ml.

Specificity

The antibody pair used in this assay is specific to human adiponectin and does not cross-react with mouse and rat adiponectin, and other cytokine or hormone molecules including human resistin, TNFα, ANGPTL4, insulin, leptin and IL6.

Precision

The assay variations of this ELISA kits were studied on four human serum samples with varying concentrations of endogenous adiponectin. The mean within variation was calculated from results of five duplicate determinations in each assay of the indicated samples. The mean between variations of each sample was calculated from results of four separate assays with duplicate samples in each assay.

Recovery

Varying amounts of human adiponectin were added to three human serum samples and the Adiponectin content was determined in three separate assays. The % of recovery = observed adiponectin concentrations/expected adiponectin concentrations x 100%.

Assay Procedure Summary

Add 100µl of Standard or sample per well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well 3 times.
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Add 100µl of 1xDetection antibody solution to each well.
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Incubate at room temperature for 1 hour.
↓
Aspirate and wash each well 3 times.
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Add 100µl of Substrate solution to each well.
↓
Incubate at room temperature for 15 minutes.
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Add 100µl of Stop solution to each well.
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Measure absorbance of each well at 450nm.
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Calculation

Typical Standard Curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.

Human adiponectin standard curve (4-parameter)

Adiponectin, also known as apMl, Acrp30, GBP28 and adipoQ, is a circulating hormone predominantly produced from adipose tissue. Many pharmacological studies demonstrated that this protein possesses potent anti-diabetic, anti-atherogenic and anti-inflammatory functions. Supplement of adiponectin protein can decrease blood glucose, improve insulin sensitivity, alleviate fatty liver and prevent atherosclerosis. The protein is post-translationally modified by hydroxylation and glycosylation, and forms three different oligomeric complexes in the circulation.

Many clinical studies demonstrated that plasma adiponectin is a useful biomarker for metabolic syndrome, nonalcoholic steatohepatitis and certain type of cancers. Decreased circulating levels of plasma adiponectin (hypoadiponectinaemia) are associated with increased body mass index (BMI), decreased insulin sensitivity, less favorable plasma lipid profiles, increased levels of inflammatory markers and increased risk for the development of type 2 diabetes, hypertension, and coronary heart diseases. Low adiponectin concentrations were found to be predictive of a future reduction in insulin sensitivity and cardiovascular disorders. Administration of the anti-diabetic drugs thiazolidinediones (TZDs) raises circulating adiponectin levels. In addition, low plasma adiponectin levels are also associated with nonalcoholic steatohepatitis (NASH) and certain types of cancers.

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