Serum was collected from blood obtained by cardiocentesis under anesthesia and stored at −80 °C until measurement. Leptin (Leptin ELISA, Millipore, St. Charles, MO, USA), insulin (Mouse Insulin ELISA kit, Shibayagi, Gunma, Japan), glucose (Glucose CII-test, Wako, Osaka, Japan), and non-esterified fatty acid (NEFA) (NEFA C-test, Wako) levels in the sera were measured according to the manufacturers’ protocols.
Leptin elisa
Manufactured by Merck Group
Sourced in United States
The Leptin ELISA is a quantitative sandwich enzyme-linked immunosorbent assay designed to measure the concentration of leptin, a hormone involved in regulating energy balance and body weight. The assay utilizes a pair of high-specificity antibodies to capture and detect leptin in biological samples. The core function of the Leptin ELISA is to provide a sensitive and reliable method for the quantitative determination of leptin levels.
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6 protocols using leptin elisa
1
Murine Metabolic Biomarker Analysis
2
Serum Metabolite Quantification Protocol
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Serum was collected from blood obtained by cardiocentesis under anesthetization and stored at −80 °C. Insulin (Mouse Insulin ELISA kit, Shibayagi, Gunma, Japan), leptin (Leptin ELISA, Millipore, St. Charles, MO), glucose (Glucose CII-test, Wako, Osaka, Japan), and non-esterified fatty acid (NEFA) (NEFA C-test, Wako) levels in the sera were measured according to the manufacturers’ protocols.
3
Estradiol and Leptin Quantification
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Estradiol concentration in plasma was measured using an estradiol ELISA (Calbiotech) according to the manufacturer’s instructions. Plasma leptin was measured using a leptin ELISA (Millipore), according to the manufacturer’s protocol.
4
Plasma Leptin and Triglyceride Quantification
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On P7 or P14, we rapidly decapitated the animals and collected trunk blood for later assessment of plasma leptin and triglycerides. Whole blood was collected in EDTA-coated tubes, kept on ice and quickly centrifuged to separate the plasma. The plasma samples were aliquoted and stored at −20°C avoiding freeze–thaw cycles until use.
To determine leptin concentrations in our samples, we performed a standard commercial leptin ELISA, following the manufacturer’s instructions (Millipore, Ballerica, MA, USA). Intra-assay variability was 1.9–2.5% CV, inter-assay variability, 3.0–3.9% CV, and lower limit of detection, 0.04 ng/ml. All compared samples were assayed in duplicates and processed in the same assay.
To determine triglyceride concentrations in our samples, we performed a triglyceride assay (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Intra-assay coefficient of variation was 1.34%, inter-assay coefficient of variation was 3.17%, and the lower limit of detection for this assay was 0.5 mg/dl. All samples were assessed in duplicates and under the same conditions.
To determine leptin concentrations in our samples, we performed a standard commercial leptin ELISA, following the manufacturer’s instructions (Millipore, Ballerica, MA, USA). Intra-assay variability was 1.9–2.5% CV, inter-assay variability, 3.0–3.9% CV, and lower limit of detection, 0.04 ng/ml. All compared samples were assayed in duplicates and processed in the same assay.
To determine triglyceride concentrations in our samples, we performed a triglyceride assay (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Intra-assay coefficient of variation was 1.34%, inter-assay coefficient of variation was 3.17%, and the lower limit of detection for this assay was 0.5 mg/dl. All samples were assessed in duplicates and under the same conditions.
5
Fasting and Metabolic Markers in Mice
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Blood was removed from the tail of mice that were fasted overnight, ad libitum fed, or fasted overnight followed by 4-h refeeding. Blood was collected via heparinized capillary tubes and centrifuged to isolate serum. Serum was analyzed using mouse insulin ELISA (Mercodia, Uppsala, Sweden); leptin ELISA (Millipore, Billerica, MA); active ghrelin ELISA (Millipore, St. Charles, MO); and kits for cholesterol E (Wako, Richmond, VA), Infinity triglycerides (TGs) (Thermo Scientific, Middletown, VA), and nonesterified fatty acid (NEFA) (Wako) according to the manufacturer’s instructions.
6
Leptin Regulation in Neonatal Overfeeding
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As our results showed significant effects of neonatal overfeeding on the neonatal leptin system, we assessed a further cohort of animals in adulthood to determine if effects were maintained past the neonatal period. We allowed a cohort of CL and SL rats to grow to ~P70 and assessed leptin receptor expression, weight and hypothalamic responses to leptin, POMC-, AgRP-and NPY-immunoreactivity as described above. For food intake measurements, we singly housed CL and SL adult rats and injected 3 mg/kg i.p. leptin (or saline) immediately prior to lights off (i.e. between 18:30 and 18:50). Each rat was given access to a pre-weighed amount of pelleted rat chow. We monitored food intake and body weight at 12 and 24 h post injection. After 7 days recovery, we assessed neuronal activation in these rats in response to 1 mg/kg i.p. leptin, having determined in pilot experiments that this dose is sufficient to activate the ARC and VMH; and based on Sachot and colleagues (Sachot et al. 2007 (link)). We assessed plasma leptin levels using a standard leptin ELISA (Millipore) following the manufacturer's instructions. Intra-assay variability was 1.9-2.5% CV, inter-assay variability, 3.0-3.9% CV and lower limit of detection, 0.04 ng/mL. Samples were assayed in duplicate and were all processed in the same assay.
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