Active GLP-1 Assay Kit

HTRF active GLP-1 kit is a rapid sandwich assay for accurate quantification of the glucagon-like peptide-1 active forms, GLP-1(7-36)NH2 and GLP-1(7-37) on cell supernatants. The simple and robust procedure gives increased throughput compared to ELISA. The extended dynamic range allows crude sample to be analyzed with no need of a pre-dilution step.

GLP-1 has become a key biomarker in treatment of type 2 diabetes. The main actions of GLP-1 are stimulation of insulin secretion and  inhibition of glucagon secretion and food intake. In vivo, the active forms are rapidly degraded into inactive forms (9-36)NH2 and (9-37) by the dipeptidyl peptidase IV (DPP-IV).

Active GLP-1 assay complements other HTRF technology based assays in Cisbio’s offering for diabetes investigation, such as insulin, cortisol, cAMP and GLP-1 receptor ligand binding assay.

 

Assay principle

Active GLP-1 is measured using sandwich immunoassay involving two antibodies, one labelled with Lumi4Tb-Cryptate (Donor) and the second with d2 (Acceptor). The intensity of the signal is proportional to the concentration of GLP-1 active forms present in the sample.

Active GLP-1 test can be run on cell supernatants using a simple “addition and read” procedure described on the right (no wash steps). The HTRF conjugates may be pre-mixed and added in a single dispensing step to further streamline the protocol. An assay can be run in 96- to 384-well plates by simply resizing each addition volume proportionally. The active GLP-1 assay protocol is described here, using a white 384-well small volume plate.

Product specifications

  Active GLP-1
Incubation ON at RT
Linear range 43. 8 to 1,400 pg/mL
Detection limit 25 pg/mL*
Species reactivity Rat, mouse, human, porcine

Active GLP-1 standard curve was performed in a final 20µL assay volume and read on a Pherastar instrument (BMG LABTECH).

* Caution: all HTRF compatible readers, but SpectraMax M5e  and FlexStation reach this sensitivity criteria.

Peptide Cross reactivity
GLP-1 (7-36) amide
GLP-1 (7-37)
GLP-1 (1-36) amide
GLP-1 (1-37)
GLP-1 (9-36) amide
GLP-1 (7-17)
GLP-2
Glucagon
100%
98.4%
0.39%
0.37%
<0.01%
<0.01%
<0.01%
<0.01%

Assay performance

Comparaison between HTRF and ELISA assay formats

HTRF active GLP-1 Fluorescent  ELISA
1 assay step mix and read (no washing)  - <1hrs assay time 6 sequential steps - > 4hrs assay time

HTRF active GLP-1 assay correlates well with classical ELISA assay while offering handling easiness and labor time saving.

Side by side comparison of HTRF and ELISA assays for GLP-1 quantification was run in white 384-w small volume plate using crude NCI-H176 cell supernatants. A correlation factor of 0.97 was obtained (n=6).

Linearity-of-dilution assessment

Dilutions of the NCI-H716 cell supernatants 1 and 2 were performed using the HTRF assay diluent. GLP-1 concentration was then determined in crude supernatants and in diluted samples (1:2, 1:4, 1:8 and 1:16). Figure on the right indicates the means (+/- SD) of 3 independent experiments. The strong correlations obtained between measured and expected GLP-1 concentrations demonstrate the linearity of dilution within the assay range.

Glucose dose-response for active GLP-1 secretion in NCI-H716 cells

A time-course was determined for active GLP-1 secretion in human enteroendocrine NCI-H716 cells after glucose stimulation. Cells (2.10cells/well) were seeded into 96-well culture plates pre-coated with Matrigel, and cultured for two days with DMEM 10% FBS medium. On the experiment day, cells were washed and incubated for 2 hours with increasing  concentration of glucose  in 100 µL of KRB supplemented with DPP IV inhibitor . At the end of glucose stimulation, 10 µL of supernatants were transferred to a 384 small volume-well plate, and assayed for active GLP-1 level as described in “Assay protocol”. As expected, active GLP-1 was induced with Glucose dose dependence. The maximum amplitude of active GLP-1 triggered by 10% glucose versus basal production was 2.1.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Active GLP1 kit - 500 tests62GLPPEG
-
Active GLP1 kit - 10,000 tests62GLPPEH
-

Companion products

DescriptionCat. noProduct insertMSDS
Active GLP1 kit standard62GLPCDA
-
-

ドキュメント