Human Leptin Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human Leptin Immunoassay is a 3.5 hour solid phase ELISA designed to measure soluble human Leptin in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human Leptin and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate recombinant Leptin accurately. Results obtained measuring natural human Leptin showed dose-response curves that were parallel to the standard curves obtained using the recombinant Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for natural human Leptin.

Preparation and Storage

Background: Leptin/OB

Leptin is a product of the mouse obese gene. Mice with mutations in the obese gene that block the synthesis of leptin are obese and diabetic, and have reduced activity, metabolism, and body temperature. Rat leptin shares approximately 96% and 82% sequence identity with the mouse and human protein, respectively. The expression of leptin mRNA is restricted to adipose tissu.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.