JPS6326871B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to assays for the detection of bacterial endotoxins using novel synthetic substrates. A method for detecting trace amounts of bacterial endotoxins has been developed based on the phenomenon in which horseshoe crab blood cell extract (amebosite lysate) reacts with trace amounts of bacterial endotoxins and turns into a gel. used in the field of science. The present invention is based on the "Method for Detecting and Measuring Bacterial Endotoxins" previously disclosed by the applicant (Japanese Patent Application No. 70335/1989, Japanese Patent Application Laid-Open No. 54-1982).
This invention relates to an improved invention based on the principle of the invention of No. 15797), with the aim of putting the invention into practical use in the field of clinical diagnosis. That is, the principle of the invention disclosed above is that when horseshoe crab amebocyte lysate or the enzyme precursor (amidase precursor) component contained therein comes into contact with a specimen containing bacterial endotoxin, the enzyme precursor It utilizes the ability to specifically cleave a synthetic substrate with a certain chemical structure that is immediately stoichiometrically activated by a toxin, and detects and measures the residues that are liberated by cleavage using spectrophotometry. It was hot. By the way, when detecting and measuring bacterial endotoxins using this method, the type of free produced residue to be detected and measured must be selected as appropriate depending on the nature and properties of the specimen. In recent years, research into the clinical pathology of endotoxin (bacterial endotoxin) has become active, with the aim of understanding the pathophysiology of exogenous endotoxemia and endotoxin shock derived from diseases with clearly established infection foci. The Limulus Amebocyte Lysate Test has been used primarily in the surgical field. On the other hand, in the field of internal medicine, the effects of intestinal gram-negative bacterial endotoxins on liver damage and endogenous endotoxemia, such as a decline in the reticuloendothelial system processing function such as the liver's ability to detoxify and excrete bacterial endotoxins, are discussed. has become a problem, and it has become essential to clinically detect and measure endotoxins in the blood coagulation fibrinolytic system, kinin system, circulatory system, and immune reaction system. When applying the above method for measuring bacterial endotoxins for the purpose of such clinical diagnosis, patient body fluids, particularly blood, ascites, urine, pancreatic juice, brain fluid, bile, etc. are used as specimen samples. However, these body fluids have their own coloring, or pigments caused by diseases (e.g. dark yellow due to jaundice, red due to hemolysis, etc.)
The interference of these dyes often makes detection and quantification by spectrophotometry difficult. Furthermore, since there are many types of fluorescent substances in body fluids, interference with the measurement cannot be avoided even if a fluorescent substance is selected as the measuring residue of the synthetic substrate. As mentioned above, the present inventors have made extensive studies to search for a new synthetic substrate that can be applied to clinical diagnostic applications, to which it has been difficult to apply the above-mentioned bacterial endotoxin detection assay method. reached. That is, the object of the present invention is to employ a novel synthetic substrate that produces a measurement residue with a light absorption region that is not interfered with by the light absorption of the dye in the body fluid itself or in the specimen that occurs in response to clinical symptoms. Another object of the present invention is to provide a method that can more accurately detect and measure bacterial endotoxins. The present invention is based on the general 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.) p-(N,N-diethylamino)aniline produced by contacting one or more enzyme precursor components selected from the enzyme precursor components with a specimen containing bacterial endotoxin, and 1-naphthol-2-sulfonic acid. This is a method for detecting bacterial endotoxins, which is characterized by detecting and quantifying condensates produced by oxidative coupling by spectrophotometry, and is particularly suitable for clinical diagnosis of body fluids. Amebocyte lysate can be obtained by treating the amebocytes contained in horseshoe crab hemolymph with a hypotonic solution.
(Limulus Amebosite Lysate Test)
You may also purchase commercially available products. For example, Pregel (Imperial Organ), Pyrostat (Worthington)
Biochem.corp.USA.), Pyrotest (Difco
Lab.USA.), Pyrozient (Mallinckrodt,
Amebosite lysate under trade names such as Inc.USA.) is known. Enzyme precursor components can be obtained by purifying and separating amebosite lysate by column chromatography, electrophoresis, electrofocusing, affinity chromatography, or the like. These amebocyte lysates or enzyme precursors separated from the lysates are activated by bacterial endotoxins to become activated enzymes, and the formula ()
It specifically acts on the synthetic substrate, which is a peptidic compound shown in In the formula, R ₁ includes, for example, those having the structure shown below. That is, Boc, −Leu−, Boc−Val−Leu−,
Boc−Ser−, Boc−Val−Ser−, Bz−Leu−,
Bz−Val−Leu−, Bz−Ser−, Bz−Val−Ser
-, (where Boc is a tert-butoxycarbonyl group,
Bz represents a benzoyl group. ) etc. All of the synthetic substrates represented by the formula () are new substances, and p-(N,N-diethylamino)aniline forms an anilide bond with the C-terminus of Arg in the peptidic residue represented by R ₁ -Gly-Arg-. However, under the action of the activating enzyme, this anilide bond is easily enzymatically hydrolyzed to liberate p-(N,N-diethylamino)aniline (hereinafter abbreviated as DEAA). do. liberated
DEAA has weak absorption in the visible wavelength range of 510 nm and 550 nm, giving it a pale pink color, but it is not suitable as a target for absorption analysis because it is interfered with by the color tone of body fluids. DEAA is 1-naphthol- as a coupler
When oxidative condensation is carried out in a system in which 2-sulfonic acid coexists, a condensation product having the following formula as the main reaction exhibits a deep blue color. Since this condensate has a maximum absorption at 675 nm, it can be detected and quantified by spectrophotometry. Furthermore, since body fluids do not absorb similar colors, the presence of bacterial endotoxins can be clearly detected and measured without any interference. A feature of the present invention is that by using a novel peptidic substrate represented by the formula (), bacterial endotoxins in body fluids as samples for clinical tests can be detected in a jaundiced manner according to the original color tone of the body fluid and the symptoms of the disease. Bacterial endotoxins can be detected and measured accurately by avoiding interfering color tones such as deep yellow and red due to hemolysis, and by utilizing blue color tones that are not naturally found in body fluids or the corresponding light absorption. The present invention will be explained in more detail below using Preparation Examples, Examples, and Comparative Examples. Preparation Example 1 Preparation of amebosite lysate Japanese horseshoe crab according to Special Publication No. 51-40131
Approximately 100 ml of hemolymph fluid is collected from Tachypleus tridentatus (weighing approximately 2 kg) while strictly avoiding contamination. Amebosites are separated by centrifugation and washed with 3% sodium chloride solution to obtain amebosite pellets. A 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 fluid, stirred well with a sterilized homogenizer, frozen and thawed, and then centrifuged at 5000 rpm for 15 minutes to obtain a supernatant. This is called Amebosite Lysate Tachypleus (hereinafter abbreviated as ALT). This ALT method of Young et al. [NSYoung
et al; J. Clin. Invest., 51 1970 (1972)]
Gel filtration was performed using Sephadex G-50 (trade name of Pharmacia Huain Chemical Co., Ltd.), and the fraction containing the amitase precursor (Fraction-1) (hereinafter referred to as Fraction-1)
It is abbreviated as ALT-F1. ) was 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.
Amoebocyte Lysate Limulus:
(abbreviated as ALL) was obtained. Preparation Example 3 General formula () used in the present invention (Hereinafter, [Formula] is abbreviated as -DEAA.) An outline of the preparation of the novel peptidic substrate represented by the formula is described below, and the physical properties of the various novel substrates are shown in Table 1. (1) H.Arg(NO ₂ )-DEAA Boc-Arg(NO ₂ )-OH and p-(N,N-diethylamino)aniline are dehydrated and condensed with water-soluble carbodiimide (WSC) to form Boc- Arg
(NO ₂ )-DEAA is obtained. The tert-butyloxycarbonyl group (Boc-) of this compound was
Hydrolysis is carried out using an AcOEt (Ac represents an acetyl group, Et represents an ethyl group, and hereinafter the same will be expressed hereinafter) to obtain H.Arg(NO ₂ )-DEAA...(1). (2) Boc-Leu-Gly-OH Boc-Leu-OH and glycine ethyl ester are dehydrated and condensed with water-soluble carbodiimide,
Obtain Boc-Leu-Gly-OEt. this compound
Perform ester hydrolysis with NaOH alkali and Boc
-Leu-Gly-OH is obtained. (3) Boc−Leu−Gly−Arg−DEAA Boc−Leu−Gly−OH and H・Arg(NO ₂ )−
DEAA was dehydrated and condensed in butanol in the presence of water-soluble carbodiimide, and Boc-Leu-Gly
-Arg( _NO2 )-DEAA is obtained. Through palladium-catalyzed reduction of this compound, the -NO ₂ group was cleaved and Boc
-Leu-Gly-Arg-DEAA is obtained. The result of elemental analysis (f) is shown below. The calculated value (c) is
It was determined as C ₂₉ H ₅₀ C ₂ N ₈ ·AcOH·H ₂ O. [Table] (4) Bz-Leu-Gly-OH Bz-Leu-OH and H-Gly-OEt are dehydrated and condensed with a water-soluble carbodiimide in butanol to obtain Bz-Leu-Gly-OEt. This compound was subjected to ester hydrolysis with NaOH alkalinity, and Bz
−Leu−Gly・OH is obtained. (5) Bz−Leu−Gly−Arg−DEAA Bz−Leu−Gly−OH and H・Arg(NO ₂ )−
Bz-Leu-Gly-Arg was dehydrated and condensed with DEAA using water-soluble carbodiimide in butanol.
(NO ₂ )-DEAA is obtained. This compound was subjected to palladium-catalyzed reduction to cleave the -NO ₂ group and form Bz-
Obtain Leu−Gly−Arg−DEAA. The result of elemental analysis (f) is shown below. The calculated value (c) is
It was determined as C ₃₁ H ₄₆ O ₄ N ₈ ·AcOH·H ₂ O. [Table] (6) Boc-Val-Leu-Gly-OH Boc-Val-OH and H-Leu-Gly-OEt were dehydrated and condensed with water-soluble carbodiimide in butanol, and Boc-Val-Leu-Gly-OEt get. This compound was subjected to NaOH alkaline hydrolysis and Boc−Val
-Leu-Gly-OH is obtained. (7) Boc−Val−Leu−Gly−Arg−DEAA Boc−Val−Leu−Gly−OH and H・Arg
(NO ₂ )-DEAA was dehydrated and condensed with water-soluble carbodiimide in butanol to form Boc-Val-Leu.
-Gly-Arg( _NO2 )-DEAA is obtained. This compound is subjected to palladium-catalyzed reduction to cleave the _-NO2 group to obtain Boc-Val-Leu-Gly-Arg-DEAA. The result of elemental analysis (f) is shown below. Calculated value (c)
was determined as C ₃₄ H ₅₉ O ₆ N ₉ ·AcOH·H ₂ O. [Table] (8) Bz-Val-Leu-Gly-Arg-DEAA Bz-Val-Leu-Gly-OEt obtained by dehydration condensation of Bz-Val-OH and H-Leu-Gly-OEt
is subjected to alkaline hydrolysis, then H.Arg(NO ₂ )-
Dehydration condensation with DEAA and reductive cleavage of the _-NO2 group yields Bz-Val-Leu-Gly-Arg-DEAA. The result of elemental analysis (f) is shown below. Note that the calculated value (c) was obtained as C ₃₆ H ₅₅ C ₅ N ₉ ·AcOH·H ₂ O. [Table] (9) Boc-Ser(OBz)-Gly-OH Boc-Ser(OBz)-OH and glycine ethyl ester are dehydrated and condensed, Boc-Ser(OBz)-
Obtain Gly−OEt. This compound is subjected to alkaline hydrolysis to obtain Boc-Ser(OBz)-Gly-OH. (10) Bz−Ser(OBz)−Gly−OH Bz−Ser(OBz)−OH and glycine ethyl ester are dehydrated and condensed, and Bz−Ser(OBz)−Gly−
Get OEt. This compound is subjected to alkaline hydrolysis,
Obtain Bz-Ser(OBz)-Gly-OH. (11) Boc-Val-Ser(OBz)-Gly-OH Boc-Ser(OBz)-Gly-OEt was removed with HCl in ethyl acetate, and then Boc-
Boc−Val−Ser is dehydrated and condensed with Val−OH.
Let (OBz)−Gly−OEt. this compound
NaOH alkaline saponification in MeOH, Boc-
Val−Ser(OBz)−Gly−OH is obtained. (12) Bz−Val−Ser(OBz)−Gly−OH Boc−Ser(OBz)−Gly−OEt was hydrolyzed with HCl in ethyl acetate solvent, and H・Ser(OBz)−Gly
-Obtain OEt. This compound and Bz-Val-OH were dehydrated and condensed in butanol/chloroform using WSC to obtain Bz-Val-Ser(OBz)-Gly-OEt, which was then hydrolyzed with NaOH in MeOH to form Bz-Val-Ser(OBz)-Gly-OEt.
(OBz)-Gly-OH is obtained. (13) Boc−Val−Ser−Gly−Arg−DEAA Boc−Val−Ser(OBz)−Gly−OH and H.
Arg(NO ₂ )-DEAA was dehydrated and condensed in dimethylformamide/butanol solvent using WSC to form Boc-Val-Ser(OBz)-Gly-Arg(NO ₂ ).
- Obtain DEAA. This compound is reduced under palladium catalyst to cleave the _-NO2 and -OBz groups to obtain Boc-Val-Ser-Gly-Arg-DEAA. The result of elemental analysis (f) is shown below. Note that the calculated value (c) was obtained as C ₃₁ H ₅₃ O ₇ N ₉ ·AcOH·H ₂ O. [Table] (14) Bz−Val−Ser−Gly−Arg−DEAA Bz−Val−Ser(OBz)−Gly−OH and H・Arg
WSC of (NO ₂ )-DEAA in DMF/HOBt solvent
Bz−Val−Ser(OBz)−Gly
-Arg( _NO2 )-DEAA is obtained. This compound was reduced under palladium catalyst to produce -NO ₂ groups and -OBz
By cutting the group, Bz−Val−Ser−Gly−Arg−
Get DEAA. The result of elemental analysis (f) is shown below.
The calculated value (c) is C ₃₃ H ₄₉ O ₆ N ₉・AcOH3/2・
Obtained as _H2O . [Table] [Table] Example 1 Japanese horseshoe crab (Tachypleus tridentatus) lysate obtained in Preparation Example 1, ALT-
Method of M. Niwa et al. (Japan.J.Med.Sci.Biol.
26, 20, 1973) was applied to the newly synthesized substrate obtained in Preparation Example 3 to activate endotoxin for each substrate. The water decomposition rate of the enzyme was determined. These values are shown in Table 2. Various substrates are 0.2Mtris−HCl PH8.0, 0.005M
A 2.5mM solution containing _CaCl2 was also prepared and used. The water dissolution rate was determined by prewarming 0.2 ml of the substrate solution at 37°C for 2.5 minutes, adding 20μ of the enzyme solution, and measuring the amount of p-(N,N-diethylamino)aniline released from the substrate after 30 minutes with 0.6 mM of the reagent. -Naphthol-2-sulfonic acid sodium salt, 0.05M sodium borate-NaOH buffer PH10.0 1ml, reagent 0.2%
Using 2ml of NaIO ₄ 0.05M borate buffer PH8.6
The absorbance at 675 nm was measured, and the water decomposition rate per enzyme protein absorbance (A ₂₈₀ ) was calculated. [Table] Example 2 Comparative Example 1 Using the North American horseshoe crab (Limulus polyphemus) amebocyte lysate (ALL) obtained in Preparation Example 2, endotoxin in patient plasma was detected. 0.2M Tris-0.02M CaCl ₂ , PH8 to 0.1ml
Add 20μ of ALL, then add 20μ of a physiological saline solution of known concentration endotoxin (0.1-0.5ng/ml) and 0.4mM Boc-Leu-Gly-Arg- as a substrate.
After adding 0.1 ml of DEAA and incubating at 37°C for 30 minutes, add 1 ml of 0.6M potassium 1-naphthol-2-sulfonate-0.05M borate PH8.6 and 2 ml of 0.2% periodic acid-0.05M Borate PH8.6. In addition,
After 30 minutes of reaction, the calibration curve shown in FIG. 1 was obtained by measuring the absorbance at 675 nm. Next, in the same procedure as above, patient plasma was added as a test solution instead of endotoxin with a known concentration, the absorbance was measured, and the amount of endotoxin in the plasma was calculated using a calibration curve. The results are shown in Table 3. As a comparative example, the substrate Boc-Leu-Gly-Arg-
A similar operation was performed using Boc-Leu-Gly-Arg-pNA instead of DEAA, as shown in FIG. 2 and Table 3. [Table] It is well known that there are factors in plasma that inhibit the enzymatic activity of the amidase precursor in amebocyte lysate, and various methods have been devised to remove the inhibitory factors. In this example, inhibitory factors were removed by diluting plasma 3 times and heating it at 100°C for 10 minutes (Lancet, June 7th issue).
1272 pages 1975). As seen in Table 3, the color of plasma varies depending on the disease or symptom, from light yellow to dark yellow, and when this is measured with a PNA substrate, the blank value will be high, and therefore the sample The absorbance reading will also be higher, which will undesirably increase the error in the colorimeter reading. However, if the DEAA substrate is used, it is clear that the blank will be uniformly small to the extent that it can be ignored in any plasma, and the detection accuracy will be improved, and in this respect, the effect of the DEAA substrate is clearly demonstrated.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a calibration curve of absorbance versus endotoxin concentration according to an example of the present invention, and FIG.
FIG. 3 is a diagram showing a calibration curve when using a substrate having a pNA residue.

Claims (1)

[Claims] 1. General 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 new substrate is isolated from horseshoe crab amebosite lysate and the lysate. p-(N,N-diethylamino)aniline, which is produced by contacting one or more enzyme precursor components selected from the above enzyme precursor components with a specimen containing bacterial endotoxin, is added to 1-naphthol-2-sulfonic acid. 1. A method for detecting and quantifying bacterial endotoxins, which comprises detecting and quantifying a condensate produced by oxidative coupling with a condensate by spectrophotometry.