Model 680 benchmark plus

Leptin concentration in serum was determined by ELISA technique, utilizing a kit (Quantikine^® ELISA Human Leptin Immunoassay, R&D Systems Inc., Minneapolis, MN, USA) for the quantitative measurement of human leptin concentration in serum and plasma and a microplate reader for absorbance (ELISA Bio-Rad, model 680 Benchmark Plus, Bio-Rad, Hercules, CA, USA).
The assay was performed according to manufacturer’s instructions. The range of the assay was between 1.0 and 100 ng/mL. The sensitivity of the procedure was 0.5 ng/mL. The experimental variation and the variation coefficient were 2.3–6.2% and 2.1–5.3%, respectively.

During gestational weeks 28, 32, and 36, 5 mL of blood samples were collected between 7 to 8 AM, after an 8–10 hour fast. Serum samples were frozen at -70°C until processing. We calculated the mean of the three measurement times of each hormone—leptin, IGF-I, and estradiol—and used the mean to represent the level of hormones during the third trimester of pregnancy. Leptin was measured with an ELISA technique using an absorbance microplate reader (ELISA Bio-Rad, model 680 Benchmark Plus, Bio-Rad, Hercules, CA, USA) and the ELISA Human Leptin Immunoassay kit (Quantikine® ELISA Human Leptin Immunoassay, R&D Systems Inc., Minneapolis, MN, USA), following manufacturer’s instructions. The range of the assay was between 1.0 and 100 ng/mL, with a sensitivity of 0.5 ng/mL. The experimental variation and the intra-assay variation coefficient were 2.0–6.0% and 2.0–5.0%, respectively. IGF-I was processed using a two-step sandwich immunoassay amplified by enzymes using the IGF-I ELISA kit (ADSL-10-2800 ACTIVE). The sensitivity was 0.01 ng/mL, and the coefficient of variation was <5%. Estradiol (estradiol-17 beta, E2) serum concentration was calculated using Immulite-1000 estradiol assay (Siemens Healthcare). The detection range was from 20 to 2000 pg/mL, the sensitivity was 15 pg/mL, and the variation coefficient was <5%.