VASP/P2Y12 assay demonstrating similar IC20, IC50, and IC90 by each assay. 2. IC50 values for prasugrel active metabolite-treated samples were comparable ...
PP-MO-787
COMPARISON OF A NOVEL ELISA-BASED VASP WHOLE BLOOD (WB) ASSAY WITH THE FLOW CYTOMETRIC (FC) TECHNIQUE J. A. Jakubowski1, N. Bourguet2, D. Boulay-Moine2, A. Sugidachi3, P. Barragan4, and M. Moulard2 BioCytex
1Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, United States; 2Biomarkers, BioCytex, Marseille, France; Research Laboratories II, Daiichi Sankyo Company, Limited, Tokyo, Japan; and 4Département de Cardiologie Interventionnelle, Polyclinique les Fleurs, Ollioules, France
3Biological
INTRODUCTION
ELISA VS FC COMPARISON
STABILITY OF FROZEN SAMPLES
STABILITY OF WB
A new ELISA-based technique for the measurement of Ser 239-phosphorylated VASP in whole blood (WB) is presented. Adapted from the commercially available (BioCytex) "PLT VASP/P2Y12" flow cytometry (FC) assay, the test is based on the calculation of the Platelet Reactivity Index (PRI, %), which reflects the activity of anti-platelet agents targeting the P2Y12 ADP receptor. We also tested the stability of VASP levels in samples stored frozen and subsequently analyzed by the ELISA assay.
WB samples from healthy subjects were incubated with 0.1 to 30 µM prasugrel active metabolite (R-138727) for 30 min. at 37°C and then analyzed (triplicates) with both FC-based and ELISA-based assays in parallel. IC20, IC50 and IC90 were determined using SigmaPlot's four-parameters logistic regression.
WB samples from healthy subjects (A, B, C and D) were incubated with R-138727 (A and B) for 30 min. at 37°C or MRS2395 (C and D) for 20 min. at RT, activated with PGE1 or PGE1+ADP, lyzed, and analyzed (triplicates) immediately or after 1 to 3 weeks of storage at -20°C.
WB samples from healthy subjects (+/- ASA) were collected at T=0. Tubes were stored unopened at RT until treatment with R-138727 and analysis (triplicates). IC50 determined at T= 24, 48 and 72H were consistent with data at T = 0 in the ELISA vs. FC comparison.
PGE1
WITHOUT Thienopyridine
Gi
P2Y12
cAMP
ATP
100%
ADP
PG-R
AC
Gi
P2Y12
cAMP
ATP
VASP
VASP
PKA
PKA
VASP
VASP
P E1 PG
PGE1
AD
P
P
ADP
PGE1
PG-R
AC
ATP
Gi
P2Y12
PG-R
cAMP
AC
ATP
Gi
Platelet Reactivity Index (PRI)
AC
E1 PG
WITH Thienopyridine
ADP
PGE1
PG-R
P2Y12
cAMP
VASP
PG-R
Adenosine diphosphate
ATP
Adenosine triphosphate
cAMP
Cyclic adenosine monophosphate Thienopyridine
P2Y12
P
VASP
P
Gi protein
AC
Adenylate cyclase
PKA
cAMP-dependent protein kinase
VASP
Vasodilator-stimulated phosphoprotein
VASP
0%
Platelet ADP Receptor
Gi
PKA
VASP
Prostaglandin Receptor
ADP
VASP
PKA
Prostaglandin E1
P
Phosphorylated VASP protein
10
0,1
30
1
10
SUBJECT 4
SUBJECT 5
SUBJECTS 1 to 5 (MEAN)
100
40 20 0
60 40 20 0 -20
-20 0,1
1
10
60%
40%
40%
20%
20%
0%
0%
1
10
30
R-138727 concentration (µM)
60 40 20 0
0,1
1
10
30
R-138727 concentration (µM)
SUBJECT 2
SUBJECT 3
SUBJECT 4
SUBJECT 5
SUBJECTS 1 to 5 (MEAN)
IC20
2.1 µM / 2.6 µM
1.2 µM / 1.6 µM
1.6 µM / 1.3 µM
1.4 µM / 1.2 µM
1.4 µM / 1.3 µM
1.5 µM / 1.4 µM
IC50
3.4 µM / 4.2 µM
2.2 µM / 2.2 µM
2.2 µM / 2.0 µM
2.2 µM / 2.0 µM
2.1 µM / 2.1 µM
2.4 µM / 2.4 µM
IC90
7.0 µM / 8.9 µM
5.4 µM / 3.8 µM
3.9 µM / 4.1 µM
4.4 µM / 4.3 µM
4.0 µM / 4.1 µM
4.9 µM / 5.1 µM
IC50 values are consistent with aggregation data published by Hasegawa et al. (2005)*, reporting an IC50 = 1.8 µM when incubating human PRP with R-138727 for 30 min. at 37°C.
80 60 40 20 0
20
40
60
80
WB
0 µM (DMSO)
100
FC-based PRI (%)
*Hasegawa M, et al. Thromb Haemost 2005;94(3):593-598
International Society on Thrombosis and Haemostasis, XXII Congress: Boston, MA, USA; July 11-16, 2009
0,5 µM
2 µM
3 µM
10 µM
60 40
T = 24H
20
T = 48H
0
Subject C IC50= 176 µM IC50= 174 µM IC50= 159 µM IC50= 155 µM
80%
60%
40%
100
IC50= 1.7 µM IC50= 2.3 µM IC50= 1.9 µM
80 60 40
T = 24H
20
T = 48H
0
T = 72H
T = 72H -20
0,1
1
10
R-138727 concentration (µM)
100%
0,1
1
IC50= 196 µM IC50= 180 µM IC50= 191 µM IC50= 188 µM
80%
60%
INTER-LOT REPRODUCIBILITY
40%
20%
20%
0% 0 µM (DMSO)
200 µM
400 µM
600 µM
800 µM
WB
0 µM (DMSO)
200 µM
400 µM
600 µM
800 µM
MRS2395 concentration (µM) T=0
T = 1 week
T = 2 weeks
T = 3 weeks
Additionally, samples from clopidogrel-treated patients (n=7) were stored at -20°C after activation and lysis, and analyzed (duplicates) using the ELISA-based assay after T = 2, 4, 6 and 9 weeks. Stability of frozen samples from treated patients (n=7)
Results show that frozen samples are stable for up to 9 weeks if stored at -20 °C.
100%
80%
60%
40%
20%
0% Sample A
Sample B
T = 2 weeks
Sample C
Sample D
T = 4 weeks
Sample E
Sample F
T = 6 weeks
10
R-138727 concentration (µM)
Subject D
120%
Correlation between PRI (n=44, assayed in triplicate) measured using both the new ELISA-based assay and the existing FC-based PLT VASP/P2Y12 assay was significant (R² = 0.93, p < 0.0001).
y = 1.0 x + 10,5 R² = 0.93
0
10 µM
0%
ELISA-based vs FC-based assay comparison 120
-20 -20
3 µM
100%
ELISA-based assay (CY-QUANT ELISA VASP/P2Y12)
SUBJECT 1
100
2 µM
Stability of MRS2395-treated frozen samples
IC50= 2.4 µM IC50= 2.4 µM
80
0,5 µM
80
-20
WB
FC-based assay (PLT VASP/P2Y12)
FC / ELISA
0 µM (DMSO)
IC50= 1.8 µM IC50= 1.9 µM IC50= 1.7 µM IC50= 1.9 µM
80%
60%
WB
Subject B
100%
R-138727 concentration (µM)
-20
0,1
30
R-138727 concentration (µM)
30
IC50= 3.1 µM IC50= 3.1 µM IC50= 3.0 µM IC50= 3.1 µM
80%
100
IC50= 2.1 µM IC50= 2.1 µM
80
PRI (%, mean ± SD)
60
PRI (%, mean ± SD)
IC50= 2.2 µM IC50= 2.0 µM
FC / ELISA
PGE1
1
PRI (%, mean ± SD)
E1 PG
0,1
R-138727 concentration (µM)
80
ELISA-based PRI (%)
E1 PG
30
0 -20
R-138727 concentration (µM)
FC / ELISA
PGE1+ADP
10
20
Subject A
100%
IC50= 2.4 µM IC50= 2.9 µM IC50= 2.6 µM
Sample G
T = 9 weeks
Control and clopidogrel-treated WB samples (n=32) were analyzed for PRI in parallel using CY-QUANT™ ELISA VASP/P2Y12 kits from 2 different lots. PRI values from both lots were highly correlated (R² = 0.99, p < 0.0001).
LOT TO LOT COMPARISON (n = 32) y = 1.06 x - 0.04 R² = 0.99
100
PRI (%) - Lot B
1
40
R-138727 concentration (µM)
100
PRI (%, mean ± SD)
Approx. 1H45
STEPS OF THE PROTOCOL
0,1
6. WASHING 7. COLOUR DEVELOPMENT (TMB substrate) 8. PRI CALCULATION from OD450nm PGE1
0 -20
METHODS & ASSAY PRINCIPLE 1. WB ACTIVATION with PGE1 or PGE1+ADP 2. CELL LYSIS 2'. OPTIONAL FREEZING 3. ANTIGEN CAPTURE (Anti-Total VASP mAb) 4. WASHING 5. ANTIGEN DETECTION (Anti-VASP-P mAb)
20
60
100
PRI (%, mean ± SD)
0 -20
40
IC50= 2.2 µM IC50= 2.0 µM
80
PRI (%, mean ± SD)
20
60
PRI (%, mean ± SD)
40
IC50= 2.2 µM IC50= 2.2 µM
80
PRI (%, mean ± SD)
60
PRI (%, mean ± SD)
PRI (%, mean ± SD)
IC50= 3.4 µM IC50= 4.2 µM
80
100
STABILITY AFTER SAMPLING, SUBJECT F (ASA treated)
STABILITY AFTER SAMPLING, SUBJECT E
Stability of R-138727-treated frozen samples
SUBJECT 3
SUBJECT 2 100
PRI (%, mean ± SD)
SUBJECT 1 100
80 60 40 20 0 0
20
40
60
80
100
PRI (%) - Lot A
SUMMARY & CONCLUSIONS 1. A new ELISA-based VASP assay significantly correlated with the established FC-based PLT VASP/P2Y12 assay demonstrating similar IC20, IC50, and IC90 by each assay 2. IC50 values for prasugrel active metabolite-treated samples were comparable with previously published data 3. The ELISA assay is sensitive to both reversible (MRS2395) and irreversible P2Y12 antagonists (prasugrel, clopidogrel) 4. Frozen samples appear stable for up to 9 weeks 5. WB samples are stable for 3 days prior to VASP analysis 6. Inter-lot results were consistent
