Glucagon ELISA Kit

Catalog No. ABIN956063

Product Details Glucagon ELISA Kit

Target: Glucagon (GCG)

Reactivity: Human, Mouse, Rat

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Purpose: The Rat, Mouse and Human Glucagon EIA is for the quantitative determination of Glucagon in rat, mouse and human plasma.

Analytical Method: Quantitative

Specificity: Does not cross-react with intestinal glucagons, GLP-1 and GLP-2.

Characteristics: According to many studies on glucagon immunoassays, it has been established that the antibody against the C-terminal fragment (19-29) of glucagon specifically binds with pancreatic glucagon, whereas the antibody against the N-terminal fragment (1-19) of glucagon specifically binds with both pancreatic and intestinal glucagon (total glucagon). Once, 30K by Unger et al. had been widely used as an antibody specific for the C-terminal fragment of glucagon, but Nishino, Shima and Yanaihara et al. have succeeded in producing pancreatic glucagon-specific antibody using a synthetic peptide with the C-terminal fragment (19-29) of glucagon as the immunogen (in 1981). This EIA kit has been developed by using a polyclonal antibody against glucagon (19-29), a synthetic glucagon as the calibrator, and biotinylated glucagon as the labeled antigen for the measurement of rat, mouse or human glucagon in plasma. Advantages of this assay include sensitive quantification, high specificity, no interference from other components in plasma and no need for sample pre- treatment. The Glucagon Calibrator is a highly purified synthetic product (purity: > 98%) and the biotinylated peptide is purified by HPLC.This EIA kit is based on a competitive enzyme immunoassay using a combination of a highly specific antibody to Glucagon and a biotin-avidin affinity system. The 96-well plate is coated with rabbit anti-Glucagon and Glucagon calibrator or samples, and biotinylated Glucagon are added to the wells for competitive immuno-reaction. After rinsing excess rat, mouse or human Glucagon, HRP-labeled streptoavidin is added to bind to the antigen-antibody complex so that HRP- labeled streptoavidin-biotinylated glucagon-antibody complexes are formed on the surface of the wells. Finally, excess HRP-labeled streptoavidins are rinsed, HRP enzyme activity is determined by OPD and the concentration of rat, mouse or human pancreatic Glucagon is calculated.

Components: 1. Antibody-Coated Plate MTP: 1 plate (96-well) Rabbit anti-Glucagon
2. Glucagon Calibrator Lyophilized: 1 vial (10 ng/vial) Synthetic Glucagon
3. Labeled Antigen Lyophilized: 1 vial Biotinylated pancreatic Glucagon
4. SA-HRP Solution Liquid: 1 bottle (12 mL) HRP-labeled streptoavidin
5. Substrate Buffer Liquid: 1 bottle (26 mL) 0.015% Hydrogen peroxide
6. OPD Tablet: 2 tablets o-Phenylenediamine dihydrochloride
7. Stop Solution Liquid: 1 bottle (12 mL) 1M H2SO4
8. Buffer Solution A Liquid: 1 bottle (10 mL) Phosphate buffer with serum
9. Buffer Solution B Liquid: 1 bottle (10 mL) Phosphate buffer
10. Wash Solution Liquid: 1 bottle (50 mL) Concentrated saline Concentrate
11. Plate Seal: 4 sheets

Material not included: Photometer for microtiter plate (plate reader), which can read absorbance up to 2.5 at 490 nm
Rotator for microtiter plate
Washing device for microtiter plate and dispenser for ~ 0.3 mL with aspiration system
Micropipettes, multi-channel pipettes for 8 wells or 12 wells and their tips
Test tubes for preparation of Calibrator Solution
Graduated cylinder (1,000 mL)
Distilled water or de-ionized water

Application Details

Plate: Pre-coated

Reagent Preparation:

1. Preparation of Calibrator Solutions: Reconstitute the Glucagon Calibrator (lyophilized rat/human/mouse Glucagon, 10 ng/ vial) with 1 mL of Buffer Solution A, giving a 10,000 pg/mL Calibrator Solution after reconstitution. 0.5 mL of the reconstituted Calibrator Solution is diluted with 1.0 mL of Buffer Solution A to yield a 3,333 pg/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 1,111, 370, 123, and 41 pg/mL. Buffer Solution A is used as the zero calibrator (0 pg/mL). Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   
   2. Preparation of Labeled Antigen: Reconstitute Labeled Antigen with 6 mL of Buffer Solution
   b. Note: Labeled Antigen must be prepared immediately before assay. Use clean test tubes or vessels.
   
   3. Preparation of Substrate Solution: Dissolve one OPD Tablet in 12 mL of Substrate Buffer. Note: Substrate Solution must be prepared immediately before assay. Use clean test tubes or vessels.
   
   4. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled or de-ionized water. Note: During storage of the Wash Solution Concentrate at 4°C, precipitates may be observed, however, they will dissolve when diluted.
   
   5. Other reagents are ready for use.

Assay Procedure:

1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Add 100 µL of the prepared Calibrator Solutions (0, 41, 123, 370, 1,111, 3,333, 10,000 pg/mL) or samples to the wells. Then add 50 µL of Labeled Antigen to the wells. If only 50 µL sample volume is available, please see Protocol for 50 µL Sample Volume following this Assay Protocol.
   3. Cover the plate with a Plate Seal and incubate at 4°C overnight (20-24 hours).
   4. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells three times with approximately 0.35 mL/well of Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to insure blotting free of most residual washing solution.
   5. Pipette 100 µL of SA-HRP Solution into each of the wells.
   6. Cover the plate with a Plate Seal and incubate at room temperature for 1 hour. During the incubation, the plate should be rotated on a plate rotator.
   7. Remove the Plate Seal, aspirate and wash the wells three times with approximately 0.35 mL/well of Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to insure blotting free of most residual washing solution.
   8. Add 100 µL of Substrate Solution into the wells, cover the plate with a Plate Seal and incubate for 20 minutes at room temperature for the color reaction.
   9. Add 100 µL of Stop Solution into the wells to stop the color reaction.
   10. Read the optical absorbance of the wells at 490 nm. The optical absorbance of reaction solution in wells should be read as soon as possible after stopping the color reaction. Note: Perform all determinations in duplicate. During incubation except the case of 4°C incubation and color reaction, the plate should be shaken gently by a microtiter plate shaker to promote immunoreaction.
   
   Protocol for 50 µL Sample Volume
   1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Add 50 µL of the prepared Calibrator Solutions (0, 41, 123, 370, 1,111, 3,333, 10,000 pg/mL) or samples to the wells. Then add 50 µL of Labeled Antigen to the wells.
   3. Cover the plate with a Plate Seal and incubate at 4°C for two nights (44-48 hours). 4.-10. Follow instructions 4. -10. in the protocol above.

Calculation of Results:

1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Add 100 µL of the prepared Calibrator Solutions (0, 41, 123, 370, 1,111, 3,333, 10,000 pg/mL) or samples to the wells. Then add 50 µL of Labeled Antigen to the wells. If only 50 µL sample volume is available, please see Protocol for 50 µL Sample Volume following this Assay Protocol.
   3. Cover the plate with a Plate Seal and incubate at 4°C overnight (20-24 hours).
   4. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells three times with approximately 0.35 mL/well of Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to insure blotting free of most residual washing solution.
   5. Pipette 100 µL of SA-HRP Solution into each of the wells.
   6. Cover the plate with a Plate Seal and incubate at room temperature for 1 hour. During the incubation, the plate should be rotated on a plate rotator.
   7. Remove the Plate Seal, aspirate and wash the wells three times with approximately 0.35 mL/well of Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to insure blotting free of most residual washing solution.
   8. Add 100 µL of Substrate Solution into the wells, cover the plate with a Plate Seal and incubate for 20 minutes at room temperature for the color reaction.
   9. Add 100 µL of Stop Solution into the wells to stop the color reaction.
   10. Read the optical absorbance of the wells at 490 nm. The optical absorbance of reaction solution in wells should be read as soon as possible after stopping the color reaction. Note: Perform all determinations in duplicate. During incubation except the case of 4°C incubation and color reaction, the plate should be shaken gently by a microtiter plate shaker to promote immunoreaction. Protocol for 50 µL Sample Volume 1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Add 50 µL of the prepared Calibrator Solutions (0, 41, 123, 370, 1,111, 3,333, 10,000 pg/mL) or samples to the wells. Then add 50 µL of Labeled Antigen to the wells.
   3. Cover the plate with a Plate Seal and incubate at 4°C for two nights (44-48 hours). 4.-10. Follow instructions 4. -10. in the protocol above. RESULTS Calculate mean absorbance values of wells containing the Calibrators and plot a calibration curve on semilogarithmic graph paper (abscissa: concentration of Calibrators, ordinate: absorbance values of Calibrators). Use the calibration curve to read Glucagon concentrations in samples from the corresponding absorbance values. When a sample value exceeds 10,000 pg/mL (10 ng/mL), it must be diluted with Buffer Solution A and re-assayed until the sample value is within the assay range.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store kit at 4°C. Reconstituted reagents (calibrator and labeled antigen solution) should be stored at or below -30°C if not assayed on the same day.

Target Details for Glucagon

Target: Glucagon (GCG)

Alternative Name: Glucagon (GCG Products)

Synonyms: GLP1 ELISA Kit, GLP2 ELISA Kit, GRPP ELISA Kit, GLP-1 ELISA Kit, Glu ELISA Kit, PPG ELISA Kit, GCG ELISA Kit, gcg-A ELISA Kit, gcg1 ELISA Kit, glucagon ELISA Kit, glucagon L homeolog ELISA Kit, GCG ELISA Kit, Gcg ELISA Kit, gcg.L ELISA Kit

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