IP-One HTRF^® Assay

IP1, a downstream metabolite of IP3, accumulates in cells following Gq recepetor activation and is stable in the presence of LiCl making it an ideal GPCR (Gq) functional assay. Cisbio Bioassays developed and patented a highly accurate HTRF assay for measuring IP1 in 96-, 384- and 1536-well formats providing for a HTS compatible assay.

Features:

Proven, cell-based functional assay
Simple and rapid assay protocol
Stable EC50 from 1 hour to 7 days
Miniaturization down to < 10µl
Large screening window with Z' greater that 0.85

Applications:

Gq coupled receptor activation
Assess agonists, antagonists, allosteric modulators and inverse agonists
Detection of slow acting agonist in functional assay

Assay Theory
Assay Principle
Product Specifications
Assay Performance
Ordering Info

Assay Theory:

GPCR Gq stimulation is known to induce phospholipase C (PLC) activation and trigger the Inositol phosphate (IP) cascade. Several metabolites in this pathway, including IP3, have extremely short half lives, making them difficult to accurately quantify. IP1, a downstream metabolite of IP3, accumulates in cells following Gq receptor activation and is stable in the presence of LiCl making it an ideal read out of receptor activation.

Assay Principle:

The IP-One assays are competitive immunoassays that use cryptate-labeled anti-IP1 MAb and d2-labeled IP1. LiCl is added to the cell stimulation buffer causing the accumulation of IP1 upon receptor activation. The assay can be run in a single microplate and requires only a single 1 hour incubation following cell stimulation.

Product Specifications:

Detection Limit:	5 nM
Dynamic Range:	5 - 3000 nM
S/B:	20 (depending on reader performance)
EC50:	100 nM
Z':	0.91 (20 µL, 384 wells)
Specificity:	No cross-reactivity with 50 µM Myo-inositol, PIP2, IP2, IP3, IP4 or PIP3
Sample types:	Living or frozen cells
Additive tolerance:	> 5% DMSO

IP1 standard curve performed using protocol outlined in IP-One Tb assay product insert.

Assay Performance:

The HTRF IP-One kit is a proven assay for assessing Gq coupled receptor activation. A side-by-side comparison was run between the HTRF IP-One kit and an isotopic inositol cascade method using a range of Gq coupled receptors and their associated agonist. The EC50 values calculated using HTRF and isotopic methods were comparable for all receptors that were tested (Table 1). With the introduction of Lumi4®-Tb (terbium cryptate) the IP-One Tb assay offers increased sensitivity and an enhanced assay window compared to the original IP-One assay.

IP-One Validated Receptors

			EC₅₀
GPCR target (G protein)	Cell line	Agonist	HTRF	Isotopic method
Muscarinic M1(Gq)	CHO-K1	Acetylcholine^a Carbachol^a	71 nM 296 nM	42 nM 300 nM
Vasopressin V1A (Gq)	CHO-K1	Vasopressin^a Vasopressin^b	1 nM 1.6 nM	0.4 nM 0.4 nM
Oxytocin OT (Gq)	CHO-K1	Oxytocin^a	13 nM	7 nM
Histamine H2 (G16)	CHO-K1	Amthamine^a	21 nM	16 nM
Purinergic P2Y1 (Gq)	1321N1	2-methylthio ADP^a	6.8 nM	ND
Cholecystokinine CCK1 (Gq)	1321N1	CCK8 Sulfate³	2 nM	43 nM
Chemokine CCRS (G16)	CHO-K1	RANTES^a MIP1 α^a	76 nM 48 nM	26 nM ND
HupCar (Gq)	CCL39	Calcium^a	2.9 nM	ND
Endothelin Etb (Gq)	CHO-K1	Endothelin 2^a Ala-endothelin³	82 nM 70 nM	83 nM 93 nM
TRHI (Gq)	CHO-K1	TRH^a	0.8 nM	ND
GB1+GB2 (Gqi9)	HEK293	GABA^a	980 nM	484 nM
mGluR 1 (Gq)	HEK293	Quisqualate^a	13 nM	9 nM
mGluR5 (Gq)	HEK293	Quisqualate^a	13 nM	9 nM
Muscarinic M3 (Gq)	HEK293	Acetylcholine^a	20 µM	ND
Purynergic P2Y1 (Gq)	HEK293	UTP^a ATP^a	2.1 µM 1.6 µM	ND ND
(a) Tested with adherent cells. (b) Tested with suspended cells.

Ordering Info:

IP-One Assay Reagents:

Description	Quantity*	Cat no.	Product Insert
IP-One Europium kits	1,000 tests	62P1APEB	pdf for batch numbers 01X pdf for batch numbers 02X
	20,000 tests	62P1APEC	pdf for batch numbers 01X pdf for batch numbers 02X
	100,000 tests	62P1APEJ	pdf for batch numbers 01X pdf for batch numbers 02X
IP-One Terbium kits	1,000 tests	62IPAPEB	pdf
	20,000 tests	62IPAPEC
	100,000 tests	62IPAPEJ
* based on 20 µl assay volume