Neuropeptide Y (NPY) ELISA Kit

Antigen: Neuropeptide Y (NPY)

Synonyms: PYY4, NPY02, RATNPY, RATNPY02, si:dkey-22m8.5, 0710005A05Rik, NPY, preproNPY, npya, pyy4, xnpy, MGC86288, prepronpy

Reactivity: Rat (Rattus)

Application: ELISA

Certificates: ISO 9001:2008, ISO 13485:2003

Catalog no.: ABIN431547

Additional Information

Alternative name: Neuropeptide Y (NP-Y)

Description: The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of NPY in rat serum, plasma, tissue homogenates, cell culture supernates and other biological fluids.

Specificity: This assay has high sensitivity and excellent specificity for detection of rat NPY. No significant cross-reactivity or interference between rat NPY and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between rat NPY and all the analogues, therefore, cross reaction may still exist.

Sensitivity: The minimum detectable dose of rat NPY is typically less than 9.68pg/mL. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

Detection Range: 24.69-2,000pg/mL. The standard curve concentrations used for the ELISA's were 2,000pg/mL, 666.67pg/mL, 222.22pg/mL, 74.07pg/mL, 24.69pg/mL.

Application Details

Principle: This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for rat NPY has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled rat NPY and unlabeled rat NPY (Standards or samples) with the pre-coated antibody specific for rat NPY. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of NPY in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of NPY in the sample.

Reagent Preparation: 1. Bring all kit components and samples to room temperature (18-25°C) before use.
2. Standard - Reconstitute the Standard with 2.0mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 2,000pg/mL. Please prepare 5 tubes containing 0.6mL Standard Diluent and produce a triple dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 5 points of diluted standard such as 2,000pg/mL, 666.67pg/mL, 222.22pg/mL, 74.07pg/mL, 24.69pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL. Tube 1 2 3 4 5 6 pg/mL 2,000 666.67 222.22 74.07 24.69 0
3. Assay Diluent A and Assay Diluent B - Dilute 6mL of Assay Diluent A or B Concentrate(2x) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B. The prepared working dilution can't be frozen. (In fact, more than 6mL Assay Diluent A and Assay Diluent B are contained in the bottles. Therefore, in every test, please precisely pipette required amount of Diluent and make double dilution in a new container. The prepared working dilution can't be frozen.)
4. Detection Reagent A and Detection Reagent B - Briefly spin or centrifuge the stock Detection A and Detection B before use. Dilute to the working concentration with working Assay Diluent A or B, respectively (1:100).
5. Wash Solution - Dilute 20mL of Wash Solution concentrate (30x) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1x).
6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again.
Note:
1. Making serial dilution in the wells directly is not permitted.
2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37°C directly.
3. Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors.
4. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10µL for once pipetting.
5. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once.
6. If crystals have formed in the Wash Solution concentrate (30x), warm to room temperature and mix gently until the crystals are completely dissolved.
7. Contaminated water or container for reagent preparation will influence the detection result.

Sample Collection: Serum - Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000xg within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS(0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10 mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000xg. Remove the supernate and assay immediately or aliquot and store at -20°C. Cell culture supernates and other biological fluids - Centrifuge samples for 20 minutes at 1000xg. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
Note:
1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C ( 1 x month) or -80°C ( 2 months) to avoid loss of bioactivity and contamination.
2. Sample hemolysis will influence the result, so hemolytic specimen can not be detected.
3. When performing the assay, bring samples to room temperature.

Sample Preparation: 1. The manufacturer is only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

Assay Procedure: 1. Determine wells for diluted standard, blank and sample. Prepare 5 wells for standard points, 1 well for blank. Add 50µL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50µL of Detection Reagent A to each well immediately. Shake the plate gently (using a microplate shaker is recommended). Cover with a Plate sealer. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
2. Aspirate the solution and wash with 350µL of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper.
3. Add 100µL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37°C after covering it with the Plate sealer. 4. Repeat the aspiration/wash process for total 5 times as conducted in step
2.
5. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 25 minutes at 37°C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution.
6. Add 50µL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
7. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately.
Note:
1. Assay preparation: Keep appropriate numbers of wells for 1 experiment and remove extra wells from microplate. Rest wells should be resealed and stored at -20°C.
2. Samples or reagents addition: Please use the freshly prepared Standard. Please carefully add samples x x x x to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent.
3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be controlled.
4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading.
5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading.
6. TMB Substrate is easily contaminated. Please protect it from light.
7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.
Summary:
1. Prepare all reagents, samples and standards,
2. Add 50µL standard or sample to each well. And then add 50µL prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37°C,
3. Aspirate and wash 3 times, 4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C,
5. Aspirate and wash 5 times,
6. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37°C,
7. Add 50µL Stop Solution. Read at 450 nm immediately.

Calculation of Results: This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between NPY concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of NPY concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Handling Advice: The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

Components: Pre-coated, ready to use 96-well strip plate: 1
Plate sealer: for 96 wells: 4
Standard (lyophilized): 2
Standard Diluent: 1x20mL
Detection Reagent A (green): 1x120µL
Assay Diluent A (2 x concentrate): 1x6mL
Detection Reagent B (red): 1x120µL
Assay Diluent B (2 x concentrate): 1x6mL
TMB Substrate: 1x9mL
Stop Solution: 1x6mL
Wash Buffer (30 x concentrate): 1x20mL

Material not included: 1. Microplate reader with 450 x 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution

Storage: 1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4 °C.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20°C upon receipt while the others should be at 4 °C.
2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.
Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.
Stability:The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 3 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 1 day storage at 37°C can be considered as 2 months at 4°C, which means 3 days at 37°C equaling 6 months at 4°C).
Note: To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Research Area: Hormones

Restrictions: For Research Use only