This assay utilizes both the competitive ELISA and sandwich ELISA formats to measure hormones/cytokines in samples.

 

Competitive ELISA

 

The hormones insulin, amylin, glucagon, and GLP-1, are analyzed using a competitive ELISA assay. In this assay, the plate is coated with the target hormone conjugated to a protein, which competes with the free hormones in the samples that are added to the plate. After the samples containing free hormone are added to the plate, an anti-hormone antibody labeled with biotin is added to the samples and binds to both the free hormones and conjugated hormones immobilized on the plate. The plate is then washed to remove any sample and antibodies not binding to the hormones bound on the plate. The antibodies that are bound to the immobilized hormones on the plate are measured by adding Streptavidin-HRP, which binds to the biotin-labeled antibodies. To detect the HRP, TMB substrate is added, which produces a blue color when it interacts with HRP. After the blue color fully develops, the reaction is terminated with an acidic Stop solution, which changes the blue color to yellow. The intensity of the yellow color is measured at an absorbance of 450 nm and is used to determine the hormone level in the sample. Because this is a competitive ELISA assay, the signal is inversely proportional to the hormone concentration, since higher free hormone concentrations will prevent the labeled antibody from binding to the plate.

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Sandwich ELISA

 

The peptides leptin, adiponectin, IL-6, and TNFα, are analyzed using a sandwich ELISA assay. In this assay, peptides present in the sample bind to specific capture antibodies pre-coated on a plate. After unbound components are washed away, biotin-labeled detection antibodies are added to bind the immobilized hormones. Streptavidin-HRP is then introduced, forming a complex with the biotin-labeled antibodies. Then, TMB substrate is added, producing a blue color when it interacts with HRP. After the blue color fully develops, the reaction is terminated with an acidic Stop solution, which changes the blue color to yellow. The intensity of the yellow color is measured at an absorbance of 450 nm and is used to determine the hormone level in the sample.​

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The assay can be used to analyze serum, cell, tissue, and other liquid samples from human, mouse, and rat models.

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The Obesity Hormone/Adipokine ELISA Combo Kit measures 8 analytes with one 96-well plate (12 wells per analyte) and enables simultaneous and quantitative detection of multiple targets in a single assay.

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Limit of Detection

• Insulin: 1–10 pg/mL

• Amylin: 1–10 pg/mL
• Glucagon: 1–10 pg/mL
• GLP-1: 1–10 pg/mL

• Leptin: 1–10 pg/mL

• Adiponectin: 1–10 pg/mL
• IL-6: 1–10 pg/mL
• TNFα: 1–10 pg/mL