1
2
3
(LAL)
Benzoyl-Leu-Gly-Arg-COOHBenzoyl-Leu-Gly-Arg-pNA
Activated Factor BFactor B
Activated Factor CFactor C
ProClotting Enzyme Clotting Enzyme
pNA+
Gram negative Bacteria
Coagulogen
LAL
4
0.01 0.1 1.0 10 100
0 50 100
5
Signal / Noise
6
Arg530
Gly436Leu423Asp436
Ile423
Leu530
Arg218
Mutant typeWild type
Arg218
(Arg218)
7
Mutant type
Wildtype
Wild type
Mutant type
0
100,000
200,000
300,000
400,000
500,000
600,000
0 5 10 15Time(s)
Lum
ines
cenc
e in
tens
ity (c
.p.s
.) (Luciferase FM)
15
8
Benzoyl-Leu-Gly-Arg-COOHBenzoyl-Leu-Gly-Arg-Luciferin
Activated Factor BFactor B
Activated Factor CFactor C
ProClotting Enzyme Clotting Enzyme
Amino-luciferin
N
S
N
S
COOHH2N
+
Gram negative Bacteria
( )
9
:
:
10
LAL
LAL 1/2 2
-LAL
+LAL
LAL
11
37ºC,10 min 37ºC,5 min
- -
(LAL )
LAL ATP
or
A405
- -
37ºC,10 min 37ºC,5 min(LAL )
LAL
37ºC,10 min 37ºC,5 min
- -
(LAL )
LAL ATP
37ºC,10 min 37ºC,5 min
- -
(LAL )
LAL ATP
or
A405
- -
37ºC,10 min 37ºC,5 min(LAL )
LAL
A405
- -
37ºC,10 min 37ºC,5 min(LAL )
LAL
A405
- -
37ºC,10 min 37ºC,5 min(LAL )
LAL
12
15 0.1pg/ml ( EU 0.0005 EU/ml)
( )( )
LAL 10
5
13
N
S
N
S
COOHH2N
COOH
N
S
N
S
X-Leu-Gly-Arg
Endotoxin
14
LAL
( or (120 ) 2 )
0.1pg/ml
15
FDA
Endotoxin Activity Assay (EAA )20pg/ml(0.017EU/ml)
16
~120 min2800-0.1 pg/ml
20-0.0075 EU/ml)
15 min150-10 pg/ml
(1-0.1 EU/ml)
17
•
• 0.1pg/ml 120
15 0.1pg/ml
• LAL
18
15 200
~120 min2800-0.1 pg/ml
15 min150-10 pg/ml
15 min10-0.05 pg/ml
19
•
•
20
21
• 0.1pg/ml
•
•
( )
22
•ATP
• ATP
•
23
• PCT/JP2008/070450 (2008)
••
24