ImmunoDiagnostics Rapid 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. 10×Wash buffer, 20ml.
3. 5×Assay buffer, 30ml.
4. 100×Detection antibody solution – A mouse monoclonal antibody against human adiponectin conjugated with horseradish peroxidase, 0.25ml.
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.
8. Plate cover

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
7. Horizontal micro-plate shaker capable of 600rpm.

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, the strips may be stored at 2-8°C for up to one month.

Preparation of Reagents

Bring all reagents and materials to room temperature before assay.

1. 1×Assay buffer
   Prepare 1×Assay buffer by mixing the 5×Assay buffer (30ml) with 120ml of distilled water or deionized water. If precipitates are observed in the 5×Assay buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. 1×Assay buffer may be stored at 2-8°C for up to one month.
2. 1×Wash buffer
   Prepare 1×Wash buffer by mixing the 10×Wash buffer (20ml) with 180ml of distilled water or deionized water. If precipitates are observed in the 10×Wash buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. 1×Wash buffer may be stored at 2-8°C for up to one month.
3. 1×Detection antibody solution
   Spin down the 100×Detection antibody solution briefly and dilute the desired amount of the antibody 1:100 with 1×Assay buffer, 200µl of the 1×Detection antibody solution is required per well. Prepare only as much 1×Detection antibody solution as needed. Return the 100×Detection Antibody to 2-8°C immediately after the necessary volume is removed.

Preparation of Standards and Samples

Human adiponectin standards: Reconstitute the lyophilized standard with 200µl of 1×Assay buffer to generate a standard stock solution of 500ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare serially diluted standards using 1×Assay buffer as follows:

1×Assay buffer serves as the zero standard (0ng/ml). The reconstituted standard stock should be aliquoted and stored at -20ºC 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 100-fold dilution in this assay. A suggested dilution step is to add 10µl of sample to 990µl of 1×Assay buffer. Cellular extract and culture media dilutions will vary and need to be optimized by the user, also use 1×Assay buffer to prepare these samples.

Assay Characteristics

Assay Range

3.9-500ng/ml

Sensitivity

The lowest level of adiponectin that can be detected by this assay is 3.9ng/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 tested, including human resistin, TNFα, ANGPTL4, insulin, leptin and IL6.

Precision

Intra-assay Precision (Precision within an assay) C.V <10%.

Inter-assay Precision (Precision between assays) C.V <10%.

Recovery

The recovery of the assay was determined by adding various amounts adiponectin to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 91%.

Assay Principle

This assay is a sandwich ELISA designed for the quantitative detection of human adiponectin in samples in 1 hour. A mouse monoclonal antibody specific to human adiponectin has been pre-coated onto a microtiter plate. The user pipettes standards and samples into the wells and any human adiponectin present is sandwiched by the immobilized antibody and a second horseradish peroxidase (HRP)-linked monoclonal antibody specific to human adiponectin that is co-incubated with the samples. After wash step to remove any unbound reagents, 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 10µl of standard or sample to its respective well.
2. Add 200µl of the 1×Detection antibody solution to each well, seal the plate with a plate cover. Incubate at room temperature for 30 minutes, shaking the plate at 600rpm on a horizontal micro-plate shaker.
3. Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300µl of 1×Wash buffer to each well and incubate for 30 seconds. Discard the 1×Wash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes.
4. Add 100µl of Substrate solution to each well. Incubate for 15 minutes at room temperature. Protect from light.
5. Add 100µl of Stop solution to each well. Gently tap the plate frame for a few seconds to ensure thorough mixing.
6. 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 adiponectin concentrations (x-axis). The best fitting line can be generated with any curve-fitting software, any curve of 4-parameter or log-log curve fitting can be used for calculation.
3. Determine adiponectin concentration of samples from the standard curve and multiply the value by the dilution factor.

Assay Procedure Summary

Add 10µl of Standard or sample per well.
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Add 200µl of 1×Detection antibody to each well.
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Incubate at room temperature for 30 minutes (600rpm).
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Aspirate and wash each well 3 times.
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Add 100µl of Substrate solution to each well.
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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

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 apM1, Acrp30, GBP28 and adipoQ, is a circulating hormone predominantly produced from adipose tissue. Many pharmacological studies demonstrated that this protein possesses potent anti-diabetic, antiatherogenic and anti-inflammatory functions. Supplement of adiponectin protein can decrease blood glucose, improve insulin sensitivity, alleviate fatty liver and prevent atherosclerosis. The protein is posttranslationally 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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