The ProQuantum Human Leptin Immunoassay Kit is designed to provide quantitative measurements of human leptin in small sample volumes. Utilizing proximity-based amplification technology, the assay combines the analyte specificity of high-affinity antibody-antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a simple yet powerful next-generation protein quantitation platform. A user-friendly workflow combined with intuitive software for analytics enables sample-to-answer in just 2 hours.
• High sensitivity—detect low levels of protein with greater sensitivity than traditional methods
• Broad dynamic range—5 logarithmic units, minimizing sample dilutions to ensure they fall within the range
• Small sample consumption—use 2–5 µL of sample (compared to 75 µL for triplicate wells with other methods)
• Fast, easy workflow—2 hours from sample to answer, with no wash steps
• No proprietary instrument to purchase—runs on any real-time PCR instrument
ProQuantum immunoassays utilize a matched pair of target-specific antibodies, each conjugated to a DNA oligonucleotide. During antibody-analyte binding, the two DNA oligos are brought into close proximity, which allows for ligation of the two strands and subsequent creation of a template strand for amplification. This platform leverages the sensitivity and large dynamic range of Applied Biosystems TaqMan real-time PCR technology (Figure 1).
The assay workflow is fast and easy—2 steps in 2 hours. There are a total of 7 components in each kit (Figure 2). First, mix the antibody-conjugates, dilute the sample, and create the standard curve in a working plate. Then, using a multi-channel pipette, add the antibody-conjugates and sample (or standard) into the wells of a PCR plate and incubate for 1 hour. Combine the master mix and ligase and add to the wells of the PCR plate, then run the plate on any qPCR instrument. After the run is complete, import the results file into the ProQuantum software (cloud-based version at https://apps.thermofisher.com/apps/proquantum). Using this software, the data can be analyzed easily to obtain protein concentration values. The software allows you to set up standard curves, design plate layouts, apply a 5PL weighted algorithm, and obtain robust statistical group-wise comparisons.
Leptin is a 167 amino acid long protein hormone with important effects in regulating body weight, metabolism and reproductive function. Leptin is approximately 16 kDa in mass and encoded by the obese (ob) gene. Leptin is expressed predominantly by adipocytes, which fits with the idea that body weight is sensed as the total mass of fat in the body. Smaller amounts of leptin are also secreted by cells in the epithelium of the stomach and in the placenta. Leptin receptors are highly expressed in areas of the hypothalamus known to be important in regulating body weight, as well as in T lymphocytes and vascular endothelial cells. Leptin's effects on body weight are mediated through effects on hypothalamic centers that control feeding behavior and hunger, body temperature and energy expenditure. Leptin is involved in regulating food intake, energy expenditure, and adiposity through hypothalamic leptin receptors. Leptin promotes hematopoiesis, angiogenesis, wound healing, inflammation, immune responses, influences pubertal development and fetal growth. Studies have investigated the role of leptin in obesity, anorexia nervosa, insulin resistance, and hypertension. Leptin also has thermogenic actions and regulates enzymes of fatty acid oxidation. Severe hereditary obesity in rodents and humans can be caused by defects in leptin production.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.