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HTRF Glucagon Detection Kit, 500 Assay Points

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The Glucagon kit is intended to provide significant time-saving over ELISA, in the quantification of glucagon in cell culture media.

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Feature	Specification
Application	Protein Quantification
Sample Volume	10 µL

The Glucagon kit is intended to provide significant time-saving over ELISA, in the quantification of glucagon in cell culture media.

View product information

Product Variants

Unit Size: 500 Assay Points

Part #:

62CGLPEG

Unit Size: 10,000 Assay Points

Part #:

62CGLPEH

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

Overview

The 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 ELISA methods. Simply add the detection reagents, incubate, and read!

Specifications

Other Specifications

Application	Protein Quantification
Brand	HTRF
Detection Modality	HTRF
Product Group	Kit
Sample Volume	10 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Biomarkers
Technology	TR-FRET
Therapeutic Area	Metabolism/Diabetes
Unit Size	500 Assay Points

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Citations

View product citations for 62CGLPEG on CiteAb

How it works

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.

Assay protocol

10 µL of cell supernatants are supplemented with 5 µL of acceptor-labelled antibodies and 5 µL of donor-labelled antibodies (or 10 µL of pre-mixed reagents for a single dispensing step). The mix is then incubated from 4h to overnight at RT and results are read on an HTRF compatible reader.

Assay details

Specificity

Peptide	Specificity (% of recognition)
Glucagon	1
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%

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. Samples were incubated overnight at RT and diluted in diluent #5. The assay ranges from 15,6 to 2000 pg/mL and the detection limit measured was 6 pg/mL (PHERAstar FS reader - flash lamp excitation).

Linearity

Dilution linearity in cell culture media was assessed. Recovery results of between 88% and 100% were obtained, supporting the claims regarding linearity. Mouse a-TC1-6 cells were seeded in 96-well culture treated plates (100k cells/well) in complete culture medium containing 25mM glucose and incubated for 24 h at 37°C - 5% CO2. The media was then removed and the wells washed twice with KRB buffer before being incubated 1 h with KRB buffer containing low glucose (1 mM). Supernatants were collected and serially diluted in diluent #5. Results are expressed as percent observed from expected.

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 %

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

Validation on mouse isolated pancreatic islets

15 pancreatic islets isolated from a C57BL/6J mouse were exposed to increasing concentrations of Glucose or Adrenaline. Following exposure, supernatants 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.