Glucagon Assay Kit

The straightforward approach to measuring Glucagon in cell culture medium

Cisbio's glucagon assay offers superior sensitivity and ease of use for a reliable quantification of glucagon in cell culture media. Built on a truly homogeneous protocol, the assay does not require any washing or complex extraction steps, meaning significant time savings over Enzyme-linked immune-sorbent assay (ELISA) methods. Simply add the detection reagents, incubate and read.

Assay principle

Glucagon is measured using a sandwich immunoassay with two monoclonal antibodies, one labelled with Lumi4-Tb Cryptate (Donor) and the other with d2 (Acceptor). The intensity of the FRET signal obtained is proportional to the concentration of glucagon in the sample.

Designed for ease of use, the assay does not require lengthy extraction procedures or sample pre-treatment, as may be the case for other technologies. Simply add the detection reagents and read. The two detection reagents may be pre-mixed for a single dispensing step.

This cell-based assay is compatible with human, mouse, rat and pig cell & islet supernatants. 

Assay characteristics

Specificity

Cisbio has designed and optimized an assay with very high specificity and sensitivity for the quantification of glucagon in cell supernatant or plasma. Our assay has an extremely low cross-reactivity for oxyntomodulin and glicentin, and other glucagon related products such as miniglucagon, fragment 1-18, GLP-1, GLP-2 and GRPP, providing superior specificity for results you can trust.

Peptide Specificity ( % of recognition )
Glucagon 100%
Oxyntomodulin <0.07%
Glicentin <0.07%
Glucagon fragment 1-18 <1.81%
Glucagon fragment 19-29 <0.03%
GLP-1 (7-36) amide <0.06%
GLP-1 (7-37) <0.11%
GLP-2 <0.3%
GRPP (Glicentin-Related Pancreatic Peptide) <0.01%

Glucagon kit specificity: Cross-reactivity assessment on closely related peptides reveals an outstanding specificity profile.

Sensitivity

Typical calibration curves obtained by the dilution of glucagon standard in cell-culture media conditions for 20 µL final, using a white 384-well small volume plate, are presented below.

The detection limit was 6 pg/mL, as measured on PHERAstar FS reader (flash lamp excitation).

  In diluent #5
Analytical sensitivity (= dose of mean zero +2 SD) ~6 pg/mL
Assay range 15.6 to 2,000 pg/mL
Incubation time Overnight at room temperature

 

Linearity

Dilution linearity in cell culture media was assessed. Recovery results of between 88% and 100% were obtained, supporting the claims regarding linearity.

Serial dilutions of a mouse a-cell supernatant in diluent #5:

Mouse a-cell supernatant [Glucagon] measured (pg/mL) [Glucagon] expected (pg/mL) % of expected concentration
Undiluted 1460.0 - -
1:2 733.0 730.0 100.4 %
1:4 351.0 365.0 96.2 %
1:8 167.0 182.5 91.5 %
1:16 80.3 91.3 88.0 %

Mouse a-TC1-6 cells were seeded in 96-well culture treated plate (100k cells per well) in complete culture medium containing 25mM glucose, and incubated for 24 h at 37 °C, 5 % CO2. Following incubation, the media was removed and the wells washed twice with KRB buffer before being incubated 1 h in presence of KRB buffer containing low glucose (1 mM). Supernatants were then collected and serially diluted in diluent #5. The results are expressed as percent observed from expected.

Intra and Inter assay variability

Intra-assay and inter-assay variability in cell culture media conditions were assessed. Typical CVs of less than 5% were obtained, supporting the features required for a robust and reproducible assay.

Intra-Assay - n = 24

CV

Standard 2 (= 31.25 pg/mL)

2.8%

Standard 6 (= 500 pg/mL)

2.4%

Standard 8 (= 2,000 pg/mL)

3.0%

 

Inter-Assay - n = 6

CV

Standard 2 (= 31.25 pg/mL)

8.1%

Standard 6 (= 500 pg/mL)

4.4%

Standard 8 (= 2,000 pg/mL)

5.4%

 

Assay validation

Glucagon secretion on isolated pancreatic islets

15 pancreatic islets isolated from a C57BL/6J mouse were exposed to increasing concentrations of Glucose or Adrenaline. Following exposure, supernatant were collected and their Glucagon levels quantified. In the presence of glucose, Glucagon levels were readily detected using as few as 15 islets per well. All results were similar to those previously published by other investigators.

Courtesy of: Physiopathology of pancreatic BETA cell research team, Institute de Génomique Fonctionnelle, Montpellier, France.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Glucagon kit - 500 tests62CGLPEG
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Glucagon kit - 10,000 tests62CGLPEH
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Companion products

DescriptionCat. noProduct insertMSDS
Glucagon kit standard62GLCCDA
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