Human Leptin DuoSet ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.	-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Upstream tips
-Bring all reagents to room temperature before use. Allow all components to sit for a minimum of 15 minutes with gentle agitation after initial reconstitution. -Working dilutions should be prepared and used immediately.
Protocol tips
-A standard curve should be generated for each set of samples assayed. -It is suggested to start Reagent Diluent optimization for serum and plasma samples by using PBS supplemented with 10-50% animal serum. -Do not use buffers with animal serum to reconstitute or dilute the Detection Antibody or Streptavidin-HRP B.

Publication protocol

Venous blood samples obtained by venipuncture from overnight fasting individuals underwent centrifugation within 1 h after collection according to the standard protocol. Plasma samples were separated and stored in aliquots at −80 °C until usage. Following our working hypothesis presented in Fig. 1, circulating levels of five soluble markers were detected by ELISA using the DuoSet kits (R&D systems, Minneapolis, MN, USA). The detection limits were as follows: 7.8 pg/ml for GDF-15, 46.9 pg/ml for follistatin, 16.7 pg/ml for chemerin, 31.2 pg/ml for leptin, and 62.5 pg/ml for adiponectin. The intra- and inter-assay coefficients of variation were between 2.3 and 8.1%. Due to the significant deviation of the respective distribution from the normality assumptions, the original GDF-15 and leptin measurements were subjected to log-normal transformation to approximate normality prior to analysis.

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Manufacturer protocol

Download the product protocol from R&D Systems for Human Leptin DuoSet ELISA below.

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