Vasoactive Intestinal Peptide (Vip) ELISA Kit

Details for Product No. ABIN2506983, Supplier: Log in to see
Antigen
  • vip-A
  • VIP
  • vip1
  • zgc:171463
  • PHM27
  • PHI
  • vasoactive intestinal peptide
  • vasoactive intestinal polypeptide type II
  • vasoactive intestinal polypeptide
  • VIP peptides-like
  • VIP
  • vip-A
  • vip
  • vip2
  • Vip
  • LOC100352512
  • LOC100060155
Alternatives
Rat (Rattus) Vasoactive Intestinal Peptide ELISA Kit
Reactivity
Rat (Rattus)
Alternatives
Kits with alternative reactivity to:
21
19
19
9
7
6
6
4
3
2
1
1
Method Type
Competition ELISA
Detection Range
2.4-10000 pg/mL
Minimum Detection Limit
2.4 pg/mL
Application
ELISA
Options
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Purpose The kit is a competitive enzyme immunoassay for the in vitro quantitative measurement of VIP in rat serum, plasma, and other biological fluids not being tested
Brand EasyTest™
Sample Type Serum, Plasma
Analytical Method Quantitative
Detection Method Colorimetric
Specificity This assay has high sensitivity and excellent specificity for detection of rat VIP.
Sensitivity 0.5 pg/mL
Characteristics EasyTest Competitive ELISA is a simple and rapid technology for the quantitation of antigen in a range of sample matrices. The whole process takes 2.5 hours with high accuracy and precision.
Components
  • Assay plate (12x8 coated Microwells)-1
  • Primary antibody-2x 6μL
  • Standard peptide -2x 10μL
  • Biotinylated peptide-2x 10μL
  • HRP-Streptavidin concentrate-1x 60μL
  • Assay diluent (5x concentrate)-1 x15mL
  • Wash buffer (20x concentrate)-1x 25 mL
  • TMB substrate-1x12 mL
  • Stop solution-1x8 mL
  • Adhensive strip-2
  • Instruction manual-1.
Material not included 1. Distilled or deionized water, 2.Precision pipettes, with disposable plastic tips, 3.Beakers, flasks, cylinders necessary for preparation of reagents, 4.Microplate washing device (multichannel pipette or automated microplate washer), 5.Microplate shaker, 6.Microplate reader capable of reading at 450 nm.
Alternative Name VIP (Vip ELISA Kit Abstract)
Background Vasoactive intestinal peptide also known as VIP is a peptide hormone containing 28 amino acid residues. VIP is neuropeptide that belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. VIP is produced in many tissues of vertebrates including the gut, pancreas, and suprachiasmatic nuclei of the hypothalamus in the brain.
Molecular Weight 3325 Da
Gene ID 117064
NCBI Accession NP_446443
UniProt P01283
Research Area Neurology, Hormones, Signaling
Pathways Hormone Activity, cAMP Metabolic Process
Application Notes Optimal working dilution should be determined by the investigator.
Sample Volume 50 μL
Assay Time 2 h
Plate Pre-coated
Protocol The immunoplate in this kit is pre-coated with secondary antibody and the nonspecific binding sites are blocked. The secondary antibody can bind to the Fc fragment of the VIP antibody whose Fab fragment will be competitively bound by both biotinylated VIP peptide and peptide standard or targeted peptide in samples. The biotinylated VIP peptide interacts with streptavidin-horseradish peroxidase (SA-HRP) which catalyzes TMB substrate solution. The intensity of the yellow is directly proportional to the amount of biotinylated VIP peptide SA-HRP complex but inversely proportional to the amount of the peptide in standard solutions or samples. A standard curve of known VIP concentration can be established accordingly. The unknown VIP concentration in samples can be determined by extrapolation to this standard curve.
Reagent Preparation
  1. Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water (e.g. 10 mL plus 40 mL). 2. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water (e.g. 20 mL plus 380 mL). 3.Anti-VIP Antibody: Briefly centrifuge the vial before use. Add 1494 μL of 1x assay diluent to the vial, mix thoroughly. 4. Standard peptide: Briefly spin standard vial before use. Add 490 μL 1x Assay Diluent to prepare a 10 ng/mL standard, gently vortex to mix, this is standard #1. Label 5 tubes #2 through #6. Pipette 150 μL 1x assay diluent into tube #2, 300 μL 1x assay buffer into tube #3, 700 μL 1x assay diluent into tubes #4 through #6. Take 150 μL from standard #1 and add to tube #2, Mix thoroughly. Add 100 μL from tube #2 to tube #3, Mix thoroughly. Continue this for tubes #4 through #6. 5.Biotinylated peptide: Briefly centrifuge the vial before use. Add 1490 μL of 1x assay diluent to make the final concentration enough for 60 wells. 6. Streptavidin-HRP: The HRP-Streptavidin concentrate should be diluted 200- fold with 1x assay diluent.
Sample Collection Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles.
Plasma: Collect plasma using EDTA, or citrate or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g at 2-8 °C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles.
Sample Preparation

Levels of the target protein may vary among different specimens. Optimal dilution factors for each sample must be determined by the investigator,The dilution scheme is only suggestion: the recommended dilution for serum and plasma is 1:2.

Assay Procedure
  1. All reagents must be brought to room temperature (18-25 °C) prior to use 2. Prepare all reagents, primary anytibody, standard peptide, biotinylated peptide and samples as directed in the respective sections. 3.Determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4 °C. 4. Pipette 25 μL of anti-VIP antibody into all wells, 75 μL of 1x assay diluent into the blank wells, 50 μL of 1x assay diluent into the Bo (0 ng/mL standard) wells, 50 μL of Standards or samples to the appropriate wells ,then pipette 25 μL of biotinylated peptide into each well except the Blank wells, cover the plate with adhensive strip. Incubate for 1 hour at room temperature with gentle shaking. 5.Decant or aspirate contents of wells. Wash wells by filling with at least 300 μL/well prepared wash buffer followed by decanting/aspirating. Soak wells in wash buffer for 30 seconds to 1 minute for each wash. Repeat wash 3 times for a total of 4 washes. After the last wash, blot plate on absorbent paper to remove residual buffer. Thorough washing at this step is very important, complete removal of liquid is required for proper performance. 6.Pipette 100 μL of diluted streptavidin-HRP solution to each well, Seal the plate, ncubate for 45 min at room temperature with gentle shaking. 7. wash plate as step 5. 8. Pipette 100 μL of TMB Substrate Solution to each well. Incubate plate for 15 minutes at room temperature in the dark with gentle shaking. 9. Add 50μL of Stop Solution to each well, gently tap the plate to ensure thorough mixing. 10. Determine the optical density of each well within 15 minutes using a microplate reader set to 450nm. If wavelength correction is available, set to 570nm. Subtract readings at 570nm from the readings at 450nm. This subtraction will correct for optical imperfections in the plate.
Calculation of Results
  1. Average the duplicate readings for each standard and sample and subtract the average blank optical density. 2.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 (see calculation below) on the y-axis. Draw the best-fit curve through the standard points. Percentage absorbance = (B - blank OD)/ (B o - blank OD) *100 Where B = OD of sample or standard and Bo = OD of zero standard (total binding). 3. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Assay Precision intra CV<10%, inter CV <15%
Restrictions For Research Use only
Buffer 0.05 % Proclin 300
Preservative ProClin
Precaution of Use The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
Storage 4 °C/-20 °C
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ELISA image for Vasoactive Intestinal Peptide (Vip) ELISA Kit (ABIN2506983) Vasoactive Intestinal Peptide (Vip) ELISA Kit