Peptide YY ELISA Kit

Catalog No. ABIN1979239

Human, Mouse and Rat Peptide YY ELISA Kit, Colorimetric assay for quantification of Human, Mouse and Rat Peptide YY, that has been mentioned in 2+ publications.

Quick Overview for Peptide YY ELISA Kit (ABIN1979239)

Target: Peptide YY (PYY)

Reactivity: Human, Mouse, Rat

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0.1 pg/mL - 1000 pg/mL

Application: ELISA

Sample Type: Plasma, Serum

Product Details Peptide YY ELISA Kit

Minimum Detection Limit: 0.1 pg/mL

Purpose: Human/Mouse/Rat PYY EIA Kit optimized for serum and cell culture supernatants. Competition-based ELISA

Analytical Method: Quantitative

Specificity: Cross Reactivity: This EIA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC. This kit detects the 1-36 form PYY.

Cross-Reactivity (Details): This ELISA kit shows no cross-reactivity with any of the cytokines tested: Ghrelin, Nesfatin, Angiotensin II, NPY and APC.

Sensitivity: 5.6 pg/mL

Characteristics:

• 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

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

,

• 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

Material not included:

• 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)

Application Details

Application Notes: Suggested Plasma Dilution: Human: 2x / Mouse: 2x / Rat: 2x

Sample Volume: 100 μL

Assay Time: 5 h

Plate: Pre-coated

Protocol:

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.

Reagent Preparation:

1. Keep kit reagents on ice during steps. Equilibrate plate to room temperature before opening the sealed pouch.
   2. Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled water.
   3. Briefly centrifuge the Anti-PYY Antibody vial (Item N) and reconstitute with 5 µL of ddH2O before use. Add 50 µL of 1x Assay Diluent B into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently.
   4. The antibody concentrate should then be diluted 100-fold with 1x Assay Diluent B. This is your anti-PYY antibody working solution, which will be used in step 2 of the Assay Procedure. NOTE: the following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure).
   5. Briefly centrifuge the vial of biotinylated PYY peptide (Item F) and reconstitute with 20 µL of ddH2O before use. Add 5 µL of Item F to 5 mL of Assay Diluent A. Pipette up and down to mix gently. The final concentration of biotinylated PYY will be 40 pg/mL. This solution will only be used as the diluent in step 6 of Reagent Preparation.
   6. Preparation of Standards: Label 6 microtubes with the following concentrations: 1000 pg/mL, 250 pg/mL, 62.5 pg/mL, 15.6 pg/mL, 3.91 pg/mL and 0 pg/mL. Pipette 300 mL of biotinylated PYY solution into each tube, except for the 1000 pg/mL (leave this one empty). It is very important to make sure the concentration of biotinylated PYY is 40 pg/mL in all standards. a. Briefly centrifuge the vial of standard PYY peptide (Item C) and reconstitute with 6 µL of ddH2O. In the tube labeled 1000 pg/mL, pipette 6 µL of Item C and 594 µL of 40 pg/mL biotinylated PYY solution (prepared in step 5 above). This is your PYY stock solution (1000 pg/mL PYY, 40 pg/mL biotinylated PYY). Mix thoroughly. This solution serves as the first standard. b. To make the 250 pg/mL standard, pipette 100 µL of PYY stock solution the tube labeled 250 pg/mL. Mix thoroughly. c. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 300 mL of biotinylated PYY and 100 mL of the prior concentration until 3.91 pg/mL is reached. Mix each tube thoroughly before the next transfer. d. The final tube (0 pg/mL PYY, 40 pg/mL biotinylated PYY) serves as the zero standard (or total binding).
   7. Prepare a 10-fold dilution of Item F. To do this, add 2 mL of Item F to 18 mL of the appropriate Assay Diluent. This solution will be used in steps 8 and
   10.
   8. Positive Control Preparation: Briefly centrifuge the positive control vial and reconstitute with 100 µL of ddH2O before use (Item M). To the tube of Item M, add 101 µL 1x Assay Diluent B. Also add 2 µL of 10-fold diluted Item F (prepared in step 7) to the tube. This is a 2-fold dilution of the positive control. Mix thoroughly. The positive control is a cell culture medium sample that is meant to be a system control (to verify that the detection & kit components are working). It may be diluted further if desired, but be sure the final concentration of biotinylated PYY is 40 pg/mL.
   9. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1X Wash Buffer. 100 mL 1000 250 62.5 15.63 3.91 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL 100µl 100µl 100µl
   10. Sample Preparation: Use Assay Diluent A + biotinylated PYY to dilute serum samples. For cell culture medium and other sample types, use 1X Assay Diluent B + biotinylated PYY as the diluent. It is very important to make sure the final concentration of the biotinylated PYY is 40 pg/mL in every sample.
   Example: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 7), 185 mL of appropriate Assay Diluent, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated PYY to a final concentration of 40 pg/mL.
   Example: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.
   11. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. The HRP-Streptavidin concentrate should be diluted 60- fold with 1X Assay Diluent B. Note: Do not use Assay Diluent A for HRP-Streptavidin preparation in Step 11.

Sample Preparation:

Use Assay Diluent A + biotinylated PYY to dilute serum samples. For cell culture medium and other sample types, use 1X Assay Diluent B + biotinylated PYY as the diluent. It is very important to make sure the final concentration of the biotinylated PYY is 40 pg/mL in every sample. EXAMPLE: to make a 4-fold dilution of sample, mix together 2.5 µL of 10-fold diluted Item F (prepared in step 7), 185 mL of appropriate Assay Diluent, and 62.5 µL of your sample, mix gently. The total volume is 250 µl, enough for duplicate wells on the microplate. Do not use Item F diluent from Step 5 for sample preparation. If you plan to use undiluted samples, you must still add biotinylated PYY to a final concentration of 40 pg/mL. EXAMPLE: Add 2.5 mL of 10-fold diluted Item F to 247.5 mL of sample.

Assay Procedure:

1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL anti-PYY antibody (see Reagent Preparation step 4) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycles/sec). You may also incubate overnight at 4 degrees C.
   3. Discard the solution and wash wells 4 times with 1x Wash Buffer (200-300 µL each), Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of each standard (see Reagent Preparation step 6), positive control (see Reagent Preparation step 8) and sample (see Reagent Preparation step 10) into appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) or overnight at 4 °C.
   5. Discard the solution and wash 4 times as directed in Step
   3.
   6. Add 100 µL of prepared HRP-Streptavidin solution (see Reagent Preparation step 11) to each well. Incubate with gentle shaking for 45 minutes at room temperature or overnight at 4 °C. It is recommended that incubation time should not be shorter or longer than 45 minutes.
   7. Discard the solution and wash 4 times as directed in Step
   3.
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec). 1
   9. Add 50 µL of Stop Solution (Item I) to each well. Read absorbances at 450 nm immediately.

Calculation of Results:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance on the y-axis. Draw the best-fit curve through the standard points.

Assay Precision: Intra-Assay: CV < 10 %
Inter-Assay: CV < 15 %

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze/thaw cycles.

Storage: -20 °C

Storage Comment: 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.

Expiry Date: 6 months

References for Peptide YY ELISA Kit (ABIN1979239)

Hand, Giblin, Green: "Hormone profiling in a novel enteroendocrine cell line pGIP/neo: STC-1." in: Metabolism: clinical and experimental, Vol. 61, Issue 12, pp. 1683-6, (2012) (PubMed).

Batterham, Cohen, Ellis, Le Roux, Withers, Frost, Ghatei, Bloom: "Inhibition of food intake in obese subjects by peptide YY3-36." in: The New England journal of medicine, Vol. 349, Issue 10, pp. 941-8, (2003) (PubMed).

Target Details for Peptide YY

Target: Peptide YY (PYY)

Alternative Name: PYY

Background:

Gene Symbols: PYY

Background: Peptide YY is a 36 amino acid peptide released by cells in the ileum and colon in response to feeding. It is also known as PYY, Peptide Tyrosine Tyrosine, or Pancreatic Peptide YY3-36.

There are two major forms of Peptide YY: PYY1-36 and PYY3-36 which is the most common form of circulating PYY. Peptide YY3-36 (PYY) is a linear polypeptide consisting of 36 amino acids with structural homology to NPY and pancreatic polypeptide. Circulating PYY concentration increases postprandially and decreases by fasting.

PYY exerts its action through NPY receptors, inhibits gastric motility and increases water and electrolyte absorption in the colon. PYY may also suppress pancreatic secretion. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite. PYY works by slowing the gastric emptying, hence, it increases efficiency of digestion and nutrient absorption after meal.

PYY has been shown to play an important role in obesity. Animal studies have shown that acute peripheral administration of PYY3-36 inhibits feeding of rodents and primates. Studies on Y2R-knockout mice have revealed that there is no anorectic effect on Y2R-knockot mice (Y2R is the receptor for PYY). These findings indicate that PYY3-36 has anorectic effect which is suggested to be mediated by Y2R. Studies on PYY-knockout mice have shown that they have higher fat mass and lower glucose tolerance when compared to control mice, indicating that PYY also plays very important role in energy homeostasis by balancing the food intake.

Studies have also shown that obese people secrete less PYY than non-obese people. The anorectic effect of PYY represents a possible anti-obesity therapy in the future.

Protein Name (Synonyms): Peptide YY (PYY) (PYY-I) (Peptide tyrosine tyrosine)

Gene ID: 5697

UniProt: P10082