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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.
Sample Values
Serum - Samples from apparently healthy volunteers were evaluated for the presence of human Leptin in this assay. No medical histories were available for the donors used in this study.| Sample Type | Range (pg/mL) | Mean (pg/mL) |
| Male Serum (n=16) | 2205-11,419 | 4760 |
| Female Serum (n=36) | 3877-77,273 | 20,676 |
Five additional male serum samples fell below the lowest standard,15.6 pg/mL, when diluted 100-fold.
Note: Values in EDTA and heparin plasma have been found to be comparable to paired serum samples. Values in citrate plasma have been found to be slightly decreased compared to paired serum, EDTA or heparin plasma samples.
Cell Culture Supernates:
Human peripheral blood mononuclear cells (5 x 106 cells/mL) were cultured in RPMI supplemented with 5% fetal bovine serum, 50 μM beta -mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cells were cultured unstimulated or stimulated with 10 μg/mL PHA. Aliquots of the cell culture supernates were removed on days 1 and 5 and assayed for levels of human Leptin.
| Condition | Day 1(pg/mL) | Day 5 (pg/mL) |
| Unstimulated | ND | ND |
| Stimulated | 152 | 75.9 |
BeWo human choriocarcinoma cells (1 x 106 cells/mL) were cultured in F-12 media supplemented with 15% fetal bovine serum. The cells were cultured unstimulated or stimulated with 2 μM forskolin and 20 μM forskolin. Aliquots of the cell culture supernates were removed on days 1, 2, and 3 and assayed for levels of human Leptin.
| Condition | Day 1 (pg/mL) | Day 2 (pg/mL) | Day 3 (pg/mL) |
| Unstimulated* | 849 | 1549 | 1667 |
| 2 μM forskolin* | 1231 | 1699 | 2054 |
| 20 μM forskolin* | 1137 | 1725 | 2747 |
Recovery
The recovery of Leptin spiked to three different levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=5) | 98 | 94-102 |
| Citrate Plasma (n=5) | 95 | 87-105 |
| EDTA Plasma (n=5) | 99 | 85-112 |
| Heparin Plasma (n=5) | 90 | 81-100 |
| Serum (n=5) | 95 | 89-109 |
Linearity
To assess the linearity of the assay, cell culture media samples spiked with high concentrations of Leptin and natural serum/plasma samples were diluted with Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Scientific Data
Assay Procedure
Refer to the product Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Add 100 µL of Assay Diluent to each well.
- Add 100 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
- Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
- Add 200 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 1 hour.
- Aspirate and wash 4 times.
- Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.
- Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
100 µL Assay Diluent
100 µL Standard, Control, or Sample
200 µL Conjugate
200 µL Substrate Solution
50 µL Stop Solution
Human Leptin Quantikine ELISA Kit Summary
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
3.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (100 uL), Serum (10 uL), EDTA Plasma (10 uL), Heparin Plasma (10 uL), Citrate Plasma (10 uL)
Sensitivity
7.8 pg/mL
Assay Range
15.6 - 1,000 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Citrate Plasma)
Specificity
Natural and recombinant human Leptin
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules.< 50% cross-species reactivity observed with species tested.
Interference
Interference observed with 1 or more available related molecules.
背景
别名
FLJ94114,LEP,leptin (murine obesity homolog),leptin (obesity homolog, mouse),Leptin,OB,Obese protein,obese, mouse, homolog of,Obesity factor,OBOBS
背景
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 tissue.
Entrez Gene IDs:
3952 (Human); 16846 (Mouse); 25608 (Rat)
Alternate Names:
FLJ94114; LEP; leptin (murine obesity homolog); leptin (obesity homolog, mouse); Leptin; OB; Obese protein; obese, mouse, homolog of; Obesity factor; OBOBS
研究领域
制备和贮存
保存方式
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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危险品化学品经营许可证(不带存储) 许可证编号：沪(杨)应急管危经许[2022]202944(QY) 
营业执照(三证合一)