High-Throughput Screening (HTS) by Immunoassay Tests Service de Marquage Moléculaire et de C himie Bioorganique CEA Saclay Service de P harmacologie et d ’ I mmunologie CEA Saclay (J. Grassi) Laboratoire de Synthèse Bioorganique UMR 7514 ULP / CNRS Illkirch High-Throughput Synthesis and Screening (HTS) HT Synthesis of molecules, catalysts, materials ... HT Screening for: molecules, macromolecules properties (biological, physical) (pure compounds or mixtures)
25
Embed
High-Throughput Screening (HTS) by Immunoassay · PDF fileHigh-Throughput Screening (HTS) by Immunoassay Tests ... Synthesis High-throughput screening ... Xanthohumol O O HO O
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
1
High-Throughput Screening (HTS) by Immunoassay Tests
Service de Marquage Moléculaire et de Chimie Bioorganique
CEA Saclay
Service de Pharmacologieet d ’Immunologie
CEA Saclay (J. Grassi)
Laboratoire de Synthèse BioorganiqueUMR 7514
ULP / CNRS Illkirch
High-Throughput Synthesis and Screening (HTS)
HT Synthesis of molecules, catalysts, materials ...
HT Screening for: molecules, macromoleculesproperties (biological, physical)(pure compounds or mixtures)
2
H T S for antioxidantsprotective agents against oxidative stressselection of Norbadione A
H T S for enantioselective catalysts : yields & ee’s
HTSby immunoassay tests
Total synthesis of Norbadione A
Structure of an antibody
3
Polyclonal responseSelection of the mouse with the best response
Generation of poly- and monoclonal antibodies (Ab)
Chemistry Biology
109 Ac.
Injection of the immunogen
109 Ac. 109 Ac.
Cell fusion~ 103 hybridomes
selection of anti- AbH
Multiplication by cloning~ 10 Monoclonal Ab
anti- H
Synthesis of a fonctionalderivative of
N
O
O
O
O
H
O
NH
BSA
Synthesis of the immunogen
NH2 BSA
H
Hapten H
H
Competitive EIA (Enzyme ImmunoAnalysis)
Capacity > 1000 analysis/ day
YY
Y E
E
E
H
H
H
Capture
S
Color (DO)
YY
Y E
WashH
H
H
H
= Acethylcholinesterase (AChE)E
Color (DO): Ellman reagent
4
Competitive EIA (Enzyme ImmunoAnalysis)
YE
H
0
25
50
75
100
0,001 0,01 0,1 1
Log [H]
B/B
o (
%)
H
= Acethylcholinesterase (AChE)E
Color (DO): Ellman reagent
+
Y
H
Y
EH
+
K*
K
B0DO max
B/B0
Color
(µm)
YELLOW COLOR
Principle of AChE detection with Ellman reagent
Reading of the DO
2 HO
O
S NS
NO
SS
O2N
O2CO2C
O2N
SO2C
O2N
O2N
O2C SS
N
+
+
+Acethylcholine
esterase
5
New chemoluminescent probes
Design and synthesis of chemoluminescentprobes for the detection of cholinesterase
Plasmide pUC18, phosphate buffer pH 7,4Irradiation 137Cs, 30 min. (60Gy)
Norbadione Analogue
Norbadione AnaloguepUC18
pUC18 + γ
O
OO OH
HO
O
CO2KKO2C
HO
OH
HOO
O
O
HO
O
KO2C
MeO
OMe
0
500
1000
1500
2000
2500
3000
SR
B a
rbitr
ary
units
of f
luor
esce
nce)
0 25 50 75 100 125
Cisplatine (µM)
NOR-03Cells : K1 wt
Test : SRB / 48 h
Nor : 20 µg/ml
SRB/NOR-B +
SRB/NOR-B -
SRB/NOR-A +
SRB/NOR-A -
CisplatineCisplatine
Cytotoxicity of cisplatinelowered by Norbadione A
Norbadione A +human thyroid carcinoma
Cisplatine(increasing conc.)
2 days
sulfo-rhodamine testliving cells count
RadioRadio-- & & chemoprotecting effects of Norbadionechemoprotecting effects of Norbadione A A
P. Bischoff (Strasbourg)X RaysX Rays
6 days
X Ray15 mV, 8Gy
RDM4 cells +increasing conc .Norbadione A
« uptiblue» testLiving cells count
Survival rate of cells increased with Norbadione A
0
250
500
750
1000
1250
1500
1750
Arb
itrai
ry u
nits
of f
luor
esce
nce
0 0,150,3 0,6 1,2 2,5 5 10 20
µg/ml
NOR-B/8Gy
NOR-A/8Gy
16
Mushrooms and radioactivity
Pisolithe (Pisolithus tinctorius )
Bolet bai (Xerocomus badius )
In 1986, radioactive particles from the electrical plan of Tchernobyl contaminated several european countries including several french regions. Bolet bai , a commestiblemushroom, contains high concentrations in cesium 137.In 1989, Steglich found that cesium 137 is selectively localised in the pigments present on the top of the mushroom in association with norbadione A.Norbadione A can be extracted in higher quantities from a other mushroom Pisolithe (M. Gill).