Glucagon-like peptide 1 (GLP-1) ELISA Kit ELISA Kit
GLP-1 Reactivity: Cow Colorimetric Competition ELISA 24.4-100000 pg/mL Serum
Catalog No. ABIN2801478
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- Detection Method
- Method Type
- Competition ELISA
- Detection Range
- 24.4-100000 pg/mL
- Minimum Detection Limit
- 24.4 pg/mL
- The kit is a competitive enzyme immunoassay for the in vitro quantitative measurement of GLP-1 in bovine serum, plasma, and other biological fluids not being tested
- Sample Type
- Serum, Plasma
- Analytical Method
- This assay has high sensitivity and excellent specificity for detection of bovine GLP-1.
- 90 pg/mL
- 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.
- 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.
- Sample Volume
- 50 μL
- Assay Time
- 2 h
- 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 GLP-1 antibody whose Fab fragment will be competitively bound by both biotinylated GLP-1 peptide and peptide standard or targeted peptide in samples. The biotinylated GLP-1 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 GLP-1 peptide SA-HRP complex but inversely proportional to the amount of the peptide in standard solutions or samples. A standard curve of known GLP-1 concentration can be established accordingly. The unknown GLP-1 concentration in samples can be determined by extrapolation to this standard curve.
- Reagent Preparation
- 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-GLP-1 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 100 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 400- 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, plasma and serum need to be extracted before use this kit.
- Assay Procedure
- 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-ANGII 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
- 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%
- For Research Use only
- 0.05 % Proclin 300
- 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.
- 4 °C/-20 °C
- Alternative Name
- GLP-1 (GLP-1 ELISA Kit Abstract)
- Glucagon-like peptide-1 (GLP-1) is an incretin derived from the transcription product of the proglucagon gene. The major source of GLP-1 in the body is the intestinal L cell that secretes GLP-1 as a gut hormone. The biologically active forms of GLP-1 are: GLP-1-(7-37) and GLP-1-(7-36) NH2. Those peptides result from selective cleavage of the proglucagon molecule.GLP-1 secretion by ileal L cells is dependent on the presence of nutrients in the lumen of the small intestine. The secretagogues (agents that cause or stimulate secretion) of this hormone include major nutrients like carbohydrate, protein and lipid. Once in the circulation, GLP-1 has a half-life of less than 2 minutes, due to rapid degradation by the enzyme dipeptidyl peptidase-4. It is a potent antihyperglycemic hormone, inducing glucose-dependent stimulation of insulin secretion while suppressing glucagon secretion. Such glucose-dependent action is particularly attractive because, when the plasma glucose concentration is in the normal fasting range, GLP-1 no longer stimulates insulin to cause hypoglycemia. GLP-1 appears to restore the glucose sensitivity of pancreatic β-cells, with the mechanism possibly involving the increased expression of GLUT2 and glucokinase. GLP-1 is also known to inhibit pancreatic β-cell apoptosis and stimulate the proliferation and differentiation of insulin-secreting β-cells. In addition, GLP-1 inhibits gastric secretion and motility. This delays and protracts carbohydrate absorption and contributes to a satiating effect.
- Molecular Weight
- 3298 Da
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