Human Neuropeptide Y EIA

RayBio® Human/Mouse/Rat Neuropeptide Y EIA Kit optimized for serum, plasma and cell culture supernatants. Competition-based ELISA on a 96-well strip plate.

EIA-NPY-1
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Antigen Information

Accession Number:
  • P01303
Gene ID:
  • 4852
Gene Symbols:
  • NPY
Protein Name & Synonyms:
Pro-neuropeptide Y [Cleaved into: Neuropeptide Y (Neuropeptide tyrosine) (NPY) C-flanking peptide of NPY (CPON)]

Assay Format

Detection Range:
0.1-1,000 ng/ml
Recommended Dilution (serum & plasma):
Human: 2X / Mouse: 2X / Rat: 8X
Sensitivity:
0.2 ng/ml
Specificity:
This EIA kit is designed to detect human, mouse, and rat active NPY (1-36).
Number of Targets Detected:
1
Species Detected:
  • Human
  • Mouse
  • Rat
Compatible Sample Types:
  • Cell Culture Supernatants
  • Plasma
  • Serum
Design Principle:
  • Competition-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Microplate Kit

Introduction

Neuropeptide Y (NPY) is a 36 amino acid peptide hormone found in the neural system and has an important role in obesity.

The main effect of Neuropeptide Y is increased food intake and decreased physical activity. It also increases the proportion of energy stored as fat and blocks nociceptive signals to the brain. In addition to its role in obesity, Neuropeptide Y has been associated with a number of other physiologic processes in the brain, including the regulation of energy balance, memory and learning, and epilepsy.

Animal studies strongly demonstrate that the stimulation of Neuropeptide Yergic activity via the administration of certain Neuropeptide Y agonists increases food intake compared to control animals. The effects of Neuropeptide Yergic activity on food intake is also demonstrated by the blockade of certain Neuropeptide Y receptors (Y1 and Y5 receptors), which expectedly inhibited Neuropeptide Yergic activity; thus, decreases food intake. For its role in obesity, an increase in Neuropeptide Y is caused by high levels of glucocorticosteriods through directly activating type II glucocorticosteriods receptors and indirectly, by abolishing the negative feedback of CRF on Neuropeptide Y synthesis and release. Meanwhile, obesity-induced insulin resistance and the mutation of the leptin receptor (ObRb) results in the abolishment of other negative feedback mechanisms to regulate Neuropeptide Yergic activity and ultimately food intake. High levels of Neuropeptide Y were also found in the cerebrospinal fluid of patients with anorexia nervosa.

The 291 bp coding sequence of the NPY gene synthesizes the pre-pro form of NPY, a 97 aa precursor peptide (see diagram below). Cleavage of the n-terminal 29 aa results in pro-NPY (69 aa), which then undergoes a further cleavage, yielding NPY 1-39 along with the 30-aa C-terminal peptide known as C-flanking peptide of NPY (CPON). NPY 1-39 is further processed to the mature form (NPY 1-36). The function of CPON is not currently understood. This EIA kit is designed to detect active NPY (1-36).
Pro-NPY / CPON amino acid diagram

Product Features

  • Strip plates and additional reagents allow for use in multiple experiments
  • Quantitative protein detection
  • Establishes normal range
  • The best products for confirmation of antibody array data

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Standard Peptide
  • Assay Diluent(s)
  • Biotinylated Peptide
  • HRP-Streptavidin
  • TMB One-Step Substrate
  • Stop Solution
  • Assay Diagram
  • Positive Control Sample
  • Capture Antibody
  • User Manual
Other Materials Required
  • Distilled or deionized water
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare standard and sample dilutions
  • Orbital shaker
  • Aluminum foil
  • Saran Wrap
  • Absorbent paper
  • Microplate reader capable of measuring absorbance at 450nm
  • SigmaPlot software (or other software that can perform four-parameter logistic regression models)
Protocol Outline
  1. Prepare all reagents, samples and standards as instructed.
  2. Add 100 µl detection antibody to each well.
  3. Incubate 1.5 h at RT or O/N at 4°C.
  4. Add 100 µl standard or sample to each well.
  5. Incubate 2.5 h at RT.
  6. Add 100 µl prepared streptavidin solution.
  7. Incubate 45 min at RT.
  8. Add 100 µl TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl Stop Solution to each well.
  11. Read plate at 450 nm immediately.

Storage/Stability

Standard, biotinylated peptide, and positive control should be stored at -20°C after arrival. Avoid multiple freeze-thaws. The remaining kit components may be stored at 4°C. Opened Microplate Wells and antibody (Item N) may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack and reseal along entire edge.
  1. Lin N, Cai DL, Jin D, Chen Y, Shi JJ. Ginseng Panaxoside Rb1 Reduces Body Weight in Diet-Induced Obese Mice. Cell Biochem Biophys. Cell Biochem Biophys (2014) 68:189-194 DOI 10.1007/s12013-013-9688-3
    Species: Mouse
    Sample type: Serum
  2. Kaya A., Orbak Z., Polat I., et al. Leptin and neuropeptide Y levels in newborns. J Pediatr Endocrinol Metab. 2016 Jan;29(1):21-5. doi: 10.1515/jpem-2015-0201.
    Species: Human
    Sample type: Plasma
  3. Bilir C., Engin H., Can M., et al. The prognostic role of inflammation and hormones in patients with metastatic cancer with cachexia. Med Oncol. 2015 Mar;32(3):56. doi: 10.1007/s12032-015-0497-y.
    Species: Human
    Sample type: Serum
  4. Wysokinski A. Fasting Serum Levels of Neuropeptide Y in Patients With Schizophrenia on Clozapine Monotherapy. Clin Neuropharmacol. 2015 Jan-Feb;38(1):18-22. doi: 10.1097/WNF.0000000000000062
    Species: Human
    Sample type: Serum
  5. Gao B., Li L., Zhu P., et al. Chronic administration of methamphetamine promotes atherosclerosis formation in ApoE-/- knockout mice fed normal diet. Atherosclerosis. 2015 Nov;243(1):268-77. doi: 10.1016/j.atherosclerosis.2015.09.001.
    Species: Mouse
    Sample type: Plasma
  6. Symons F., et al. Can Biomarkers Differentiate Pain and No Pain Subgroups of Nonverbal Children with Cerebral Palsy? A Preliminary Investigation Based onNoninvasive Saliva Sampling. Pain Medicine Sept 2014, epub Ahead of print. DOI: 10.1111/pme.12545
    Species: Human
    Sample type: Saliva
  7. Duncan, Clare, et al. "Copper modulates the large dense core vesicle secretory pathway in PC12 cells." Metallomics 5.6 (2013): 700-714.
    Species: Rat
    Sample type: Cell Lysate
  8. Sgourakis, George, et al. "The value of plasma neurotensin and cytokine measurement for the detection of bowel ischaemia in clinically doubtful cases: a prospective study." Experimental Biology and Medicine 238.8 (2013): 874-880.
    Species: Human
    Sample type: Serum
  9. Bae B., Oh S., Park C., et al. Progressive Muscle Relaxation Therapy for Atopic Dermatitis: . Objective Assessment of Efficacy.Acta Derm Venereol. 2012 Jan;92(1):57-61. doi: 10.2340/00015555-1189
    Species: Human
    Sample type: Serum
  10. Matic I., Matthews B., Kizivat T., et al. Bone-specific overexpression of NPY modulates osteogenesis. J Musculoskelet Neuronal Interact. 2012 Dec;12(4):209-18.
    Species: Human
    Sample type: Serum
This assay worked as expected with a good standard curve range. Unknown values had a low % CV. The protocol itself, is easy to follow and understand. However, I had to stop the reaction after 10-15 minutes instead of the recommended 30 minutes as written in the protocol.

University of MN
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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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