JPS6154400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for detecting bacterial endotoxins using a novel fluorescent substrate. A method for detecting trace amounts of endotoxin has been developed based on the phenomenon in which horseshoe crab blood cell extract (amebocyte lysate) reacts with trace amounts of bacterial toxins and turns into a gel.
and used in the environmental health field. Among these detection methods, a method that visually determines the hardness of the gel is often used, and other methods include a gelation time measurement method, a method that uses a photometer to track changes in turbidity, and a clot protein quantification method. However, since both methods are based on the gelation phenomenon, it is difficult to determine the end point of gelation and flocculation, which is even more difficult when dealing with highly viscous specimens. In addition, the sensitivity of detecting endotoxins by these detection methods varies somewhat depending on the activity of the amebocyte lysate, the type of endotoxin, and the criteria for determination, but approximately
It is estimated to be 10 ^-3 to ^{10 -4} μg/ml. Endotoxin detection methods that directly or indirectly measure the degree of gelation are difficult to detect when the sample contains gelation inhibitors or because gelation is affected by certain proteases. There has been a desire to develop a method for detecting endotoxins that does not, in principle, rely on gelation phenomena. The present invention is based on the invention of "Method for Detection and Measurement of Bacterial Endotoxins" (Japanese Patent Application No. 70335/1983) previously filed by the present applicant, and is based on a novel method different from the conventional gelation phenomenon. It relates to a method for sensitively detecting and measuring toxins. In other words, when a specimen is brought into contact with horseshoe crab amebocyte lysate or an enzyme component contained therein (amidase precursor) and a new fluorescent compound (substrate) having a certain chemical structure, the substrate increases the amount of bacterial endotoxin. Based on the phenomenon of proportionally specific cleavage, it has become possible to detect and quantify extremely small amounts of endotoxin by detecting and quantifying the fluorescent substance liberated at this time. To explain the method of the present invention in more detail, the present invention employs the following formula (): (In the formula, R ₁ represents an L-amino acid residue having a protecting group at the N-terminus or a peptide residue consisting of an L-amino acid.) A fluorescent substrate represented by the above, and a horseshoe crab amebosite lysate and/or the lysate. 7 produced by contacting the sample with the enzyme component separated by
This invention relates to a method for detecting and measuring bacterial endotoxins in a specimen, which is characterized by detecting or quantifying -amino-4-methyl-coumarin by a fluorescence analysis method. In the present invention, in order to release an amidase-like substance by contacting with bacterial endotoxin, amebocyte lysate in horseshoe crab blood or an enzyme component contained therein and separated from the lysate (amidase precursor: due to endotoxin It is activated and changes into an amidase-like substance).
Amebocyte lysate can be obtained by treating amebocytes contained in horseshoe crab blood with a hypotonic solution. It can be obtained by purifying and separating it by romatography or the like. A compound (substrate) that is specifically acted upon by an amidase-like substance produced by bacterial endotoxin acting on amebocyte lysate or an enzyme component (amidase precursor) separated from the lysate is expressed by formula (1). Among the peptidic fluorescent compounds shown above, R ₁ in the formula includes, for example, compounds having the following structure. That is, Boc－Val－Leu－, Boc－Leu－,
Z―Leu―, Boc―Ileu―Glu―, Boc―Val―Ser
-, Boc-Ser-, Boc-Ser(-Bz)-, (wherein, Boc represents a tert-butoxycarbonyl group, Z represents a carbobenzoxy group, and Bz represents a ben(d)yl group). . All compounds represented by formula (1) are new substances, and 7-amino-4-methylcoumarin is R ₁ -
Arg in peptidic residues denoted by Gly-Arg-
The C-terminus and the amino group at the 7-position are linked by an acid amide bond, but under the action of the amidase-like substance, this acid amide bond is easily enzymatically hydrolyzed to form 7-amino- 4-Methylcoumarin (hereinafter abbreviated as AMC) is released. By detecting and quantifying released AMC with high sensitivity using a fluorescence analysis method, particularly a fluorescence analysis photometer, we can quantitatively detect and measure extremely small amounts of endotoxins that could not be reached using conventional methods. I can do it. Taking advantage of the fact that AMC obtained by enzymatic hydrolysis is completely different from the fluorescence spectrum of the substrate, it is possible to reliably detect and measure liberated AMC.
That is, the AMC released is at the excitation wavelength
By selecting 380nm and measurement wavelength 460nm,
It is almost unaffected by substrate-derived fluorescence and has high quantitative properties. The substrate used in the present invention is characterized by the peptide residue represented by -Gly-Arg- in formula (),
It is essential that amino acids are linked in the order of L-arginine and glycine from the C-terminus. In addition, the substrate represented by formula () is highly specifically hydrolyzed by an amidase-like substance that exists in horseshoe crab amebocyte lysate and is activated by bacterial endotoxin, so it coexists in amebocyte lysate. Other proteases that
When a foreign protease is present in the sample, etc.
The conventional gelation phenomenon makes it possible to overcome the drawbacks that exist when detecting endotoxins. The present invention will be explained in further detail with reference to Preparation Examples and Examples below. Preparation example 1 Preparation of amebosite lysate Japanese horseshoe crab according to Special Publication No. 51-40131
Approximately 100 ml of hemolymph is collected from Tachypleue tridentatus (weighing approximately 2 kg) while strictly avoiding contamination. The amebocytes are separated by centrifugation and washed with 3% sodium chloride solution to obtain an amebocyte pellet. Add distilled water or buffer solution (tris-HCl,
0.05M; CaCl ₂ , 0.001M; NaCl 0.15M; PH7.2) was added to 1/10 volume of the original blood lymph, stirred well with a sterilized homogenizer, frozen and thawed, and then heated at 5000 r.p.
The supernatant was obtained by centrifugation at m. for 15 minutes. This is Amebosite Lysate Tachypleus (hereinafter referred to as ALT).
). This ALT is NSYoung
J. Clin. Invest., 51 1790 (1972), gel filtration was performed using Sephadex G50 (trade name of Pharmacia Fine Chemical Co., Ltd.) to obtain a fraction containing the amidase precursor (Fraction-1). (hereinafter abbreviated as ALT-F1) is obtained. Preparation Example 2 The hemolymph of the North American horseshoe crab Limulus polyphemus was treated in the same manner as in Preparation Example 1 to obtain amebocyte lysate.
Limulus (Amoebocyte.Lysate・Limulus: below)
(abbreviated as ALL). Preparation Example 3 General formula () used in the present invention (Hereinafter, base

[Formula] is abbreviated as MCA. ) will be described, and the physical properties of the various compounds obtained are shown in the table. 1 N-carbobenzoxy-L-arginine (N-Z-L-Arg) and 7-amino-4-
Methyl coumarin (AMC) was stirred in dimethylformamide (DMF) at room temperature and dehydrated and condensed with dicyclohexylcarbodiide (DCCI) to obtain 7-(N〓-carbobenzoxy-L-arginyl)amino-4-. Obtain methylcoumarin. The N-carbobenzoxyl group of this compound was reductively cleaved with H ₂ (palladium black/H ₂ ) under a palladium carbon catalyst, and 7-L-arginylamino-4-methylcoumarin (Arg-
MCA)...obtain (1). 2 Dissolve (1) in DMF-H ₂ O, add carbobenzoxy and glycine N-hydroxysuccinimide ester (Z-Gly-OSu), and react.7-(N〓-carbobenzoxyglycyl- L-
arginyl)amino-4-coumarin is obtained. N
〓-The protective group was reductively cleaved with palladium black/H ₂ to form 7-(glycyl-L-arginyl)amino-
4-Methylcoumarin (Gly-Arg-MCA)…(2)
get. 3 (1) is N〓-tert-butoxycarbonyl-L-
N-hydroxysuccinimide ester of valyl-L-leucine-glycine (Boc-Val-Leu
-Gly-OSu) to react 7-(N〓-tert
-butoxycarbonyl-L-valyl-L-leucyl-glycyl-L-arginyl)amino-4
-Methylcoumarin (Boc-Val-Leu-Gly-
Arg−MCA)…(3) is obtained. 4 (2) is N〓-tert-butoxycarbonyl-L-
N-hydroxysuccinimide ester of leucine is added and reacted to form 7-(N〓-tert-butoxycarbonyl-L-leucine-glycyl-L
-Arginyl)amino-4-methylcoumarin (Boc-Leu-Gly-Arg-MCA)...(4) is obtained. 5 Add N-hydroxysuccinimide ester of N〓-carbobenzoxy-L-leucine to (2) and react to form 7-(N〓-carbobenzoxy-
L-leucyl-glycyl-L-arginyl)amino-4-methylcoumarin (Z-Leu-Gly-
Arg−MCA)…(5) is obtained. 6 N〓-Carbobenzoxy-N ^G -Tosyl-L
-Dissolve arginine in DMF to form 4-methyl-7
7-(N〓-carbobenzoxy-N ^G -tosyl-L-arginyl)amino-4 is obtained by adding aminocoumarin and dehydration condensation using DCCI.
- Obtain methylcoumarin. The N-carbobenzoxyl group of this compound was reductively cleaved with palladium black/ _H2 to form 7-(N ^G -tosyl-L
-Arginyl)amino-4-methylcoumarin is obtained. This compound was reacted with N-hydroxysuccinimide ester of N-carbobenzoxyglycine to produce 7-(N-carbobenzoxyglycyl-N ^G -tosyl-L-arginyl)amino-4-methylcoumarin. get. Next, the N-carbobenzoxyl group was reductively cleaved with palladium black/H ₂ to form 7-(glycyl-N ^G -tosyl-L-arginyl)amino-
4-Methylcoumarin is obtained. This compound and N
7-(N〓-
tert-butoxycarbonyl-γ-benzyl-L
-Glutamine-Glycyl-N ^G -Tosyl-L-
arginyl)amino-4-methylcoumarin is obtained. This compound was reacted with anhydrous hydrogen fluoride at low temperature to cleave the N-tert-butoxycarbonyl group and γ-benzyl group of glutamic acid and the N ^G -tosyl group of arginyl, and 7-(L-glutamine-glycyl- L-arginyl)amino-4-methylcoumarin is obtained. This compound was reacted with N-hydroxysuccinimide ester of N-tert-butoxycarbonyl-L-isoleucine, and 7-(N
-tert-butoxycarbonyl-L-isoleucyl-L-glutamyl-glycyl-L-arginyl)amino-4-methylcoumarin (Boc-ILeu
-Glu-Gly-Arg-MCA)...(6) is obtained. 7 (1) N〓-tert-butoxy-L-valyl-L
-N-hydroxysuccinimide ester of serylglycine is added and reacted to form 7-(N
〓-tert-butoxycarbonyl-L-valyl-
L-seryl-glycyl-L-arginyl)amino-4-methylcoumarin (Boc-Val-Ser-
Gly−Arg−MCA)…(7) is obtained. 8 (2) is N〓-tert-butoxycarbonyl-O-
2-hydroxysuccinimide ester of benzyl-L-serine was added and reacted, and 7-(N〓
-tert-butoxycarbonyl-O-benzyl-
L-seryl-glycyl-L-arginyl)amino-4-methylcoumarin

Obtain [formula]. 9 The O-benzyl group of (8) was reductively cleaved with palladium black/H ₂ to give 7-(N〓-tert-butoxycarbonyl-L-seryl-glycyl-L-arginyl)amino-4-methylcoumarin (Boc
-Ser-Gly-Arg-MCA)...(9) is obtained.

[Table] Example 1 Horseshoe crab ALT-F1 obtained in Preparation Example 1
In, H. Niwa et al. Japan J. Med. Sci. Biol. 26, 20
Preparation Example 3
The water lysis rate of the endotoxin-activating enzyme for each substrate was determined using the newly synthesized substrates obtained in . These values are shown in Table 2. Various substrates are 0.05MTris-HCl buffer PH8.0,
A 0.1 mM solution of 0.1M NaCl; 0.01MCaCl ₂ was prepared and used. Substrates that were difficult to dissolve were dissolved by adding a small amount of dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). The water dissolution rate is determined by prewarming 2.5 ml of the substrate solution at 37°C for 2.5 minutes, adding 10 μl of the enzyme solution, and calculating the amount of 7-amino-4-methylcoumarin (AMC) released from the substrate after 100 seconds using the excitation wavelength. 380nm and 460nm as the measurement wavelength, and measured using a fluorescence spectrophotometer (manufactured by Hitachi, MPF-2A type).
The value per absorbance (A ₂₈₀ ) of the enzyme was calculated. Example 2 Horseshoe crab ALT-F1 obtained in Preparation Example 1 and Boc-Val-Leu-Gly-Arg shown in Preparation Example 3
-MCA 0.05M Tris-HCl buffer PH8.0,
E. using 0.1M NaCl; 0.01MCaCl ₂ , 0.1mM solution.
coli0111: _B4 endotoxin was quantified. The measurement is
Add 0.5 ml of endotoxin solution of each concentration to ALT-F-110 μl (0.D ²⁸⁰ = 0.3/ml), incubate at 37°C for 15 minutes, add 2.5 ml of substrate solution, and remove the AMC fireflies generated 100 seconds later. This was done by measuring light intensity. The results are shown in Figure 1. The conditions for fluorescence measurement were the same as in Example 1.

[Table] Example 3 American horseshoe crab (Limulus)
The commercial product pyrostat (Worthington
Using an aqueous solution (1 vial/5 ml) of Biochemicals, the rate of water dissolution for various fluorescent substrates was investigated. Various substrates are 0.1M Tris HCl (PH8.0)
Dissolve it in 0.025M _MgCl2 to a concentration of 0.1mM.
0.5 ml of it was used. Add 20 μl of 0.1% endotoxin (Salmonella minnesota R595),
Prewarmed to 37° C. for 3 minutes, added 0.5 ml of lysate, incubated for an additional 15 minutes, and measured fluorescence increase. The measurement conditions were the same as in Example 1.
The results are shown in Table 3. From Table 3,
It can be seen that the amidase-like enzyme within ALP is activated by endotoxin and exhibits hydrolysis activity against various fluorescent substrates. As shown in the Examples above, by using horseshoe crab amebocyte lysate and a novel fluorescent synthetic substrate, it is possible to detect and measure bacterial endotoxins rapidly, with high sensitivity, and quantitatively. In the present invention, the endotoxin detection sensitivity reached 10 ^-6 to ^{10 -9} μg/ml by using fluorophotometry.

【table】 [Brief explanation of the drawing]

Figure 1 shows the relationship between the amount of endotoxin and the amount of released AMC.

Claims (1)

[Claims] 1 formula [In the formula, R ₁ is Boc-Val-Leu-, Boc-Leu-, Z-Leu, Boc-ILeu-Glu-, Boc-Val-Ser-,
[Formula] and Boc-Ser- (wherein Boc is a tert-butoxycarbonyl group,
Z represents a carbobenzoxy group, and Bz represents a benzyl group. ) represents a residue selected from the group consisting of ] 7 produced by contacting a specimen with a fluorescent substrate represented by horseshoe crab amebosite lysate and/or an enzyme component separated from the lysate.
-A method for detecting and measuring bacterial endotoxin in a specimen, which comprises detecting or quantifying amino-4-methyl-coumarin by a fluorescence analysis method.