The Glucagon kit is intended to provide significant time-saving over ELISA, in the quantification of glucagon in serum and plasma samples.
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  • Low sample consumption Low sample consumption
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Scalable from 96 to 1536 format Scalable from 96 to 1536 format
The Glucagon kit is intended to provide significant time-saving over ELISA, in the quantification of glucagon in serum and plasma samples.
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Overview

The Glucagon offers superior specificity and sensitivity for a reliable quantification of glucagon in serum and plasma samples. 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!

Benefits

  • HIGH SPECIFICITY
  • HIGH SENSITIVITY

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.
Glucagon Serum kit principle

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.
Glucagon Serum kit protocol

Specificity

PeptideSpecificity ( % of recognition )
Glucagon100%
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 #6. The assay ranges from 15,6 to 2000 pg/mL and the detection limit measured was 12 pg/mL (PHERAstar FS reader - flash lamp excitation).
Glucagon standard curve with serial dilution in diluent #6

Linearity

Dilution linearity in cell culture media was assessed, with recovery results between 100% and 117%. Human serum samples were collected and serially diluted in diluent #6. The results are expressed as percent observed from expected.
Glucagon serum kit linearity curve with serial dilutions of human sample in diluent #6
Serial dilutions of a human serum sample in diluent #6: Human serum sample [Glucagon] measured (pg/mL) [Glucagon] expected (pg/mL) % of expected concentration Undiluted 1666.7 - - 1:2 846.3 833.3 101.6 % 1:4 420.7 416.7 101.0 % 1:8 221.7 208.3 106.4 % 1:16 121.8 104.2 116.9 %

Intra- and inter-assay variability

Intra-assay and inter-assay variability in cell culture media conditions were assessed. Typical CVs of less than 4% for intra- and less than or around 10% for inter- were obtained, supporting claims regarding the features of a robust and reproducible assay.

Intra-Assay - n = 24

CV

Standard 2 (= 31.25 pg/mL)

1.8%

Standard 6 (= 500 pg/mL)

1.9%

Standard 8 (= 2,000 pg/mL)

3.6%

Intra-Assay - n = 6

CV

Standard 2 (= 31.25 pg/mL)

11.2%

Standard 6 (= 500 pg/mL)

6.6%

Standard 8 (= 2,000 pg/mL)

6.4%

Normal and pathological levels of glucagon

Glucagon concentrations for 16 pathological and normal EDTA-plasma samples were determined using the HTRF Serum Glucagon kit reagents. The results obtained show the good discrimination of the glucagon assay, clearly differentiating between normal and pathological levels of glucagon.
Glucagon in pathologic and normal EDTA-plasma samples
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