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