JPH04285859A - Measuring agent for (1-3)-beta-d-glucan - Google Patents

Abstract

PURPOSE:To obtain a measuring agent for (1 3)-beta-D-glucan composed of lysate containing no endotoxin sensitive factor by bringing limulus amoebocyte lysate into contact with a polyamide type insoluble carrier. CONSTITUTION:A measuring agent for (1 3)-beta-D-glucan composed of lysate containing no endotoxin sensitive factor is obtained by bringing limulus amoebocyte lysate or a mixture of lysate and dextran into contact with a polyamide type insoluble carrier. Limulus amoebocyte lysate is brought into contact with a polyamide type insoluble carrier by a method wherein lysate is passed through the film like polyamide type insoluble carrier to collect the passed liquid. Further, by preliminarily mixing dextran with lysate at the time of the contact of lysate with the carrier, the adsorbing action of the endotoxin sensitive factor on the carrier can be further enhanced and the measuring sensitivity to (1 3)-beta-D-glucan can be also enhanced.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a measuring agent for (1→3)-β-D-glucan using horseshoe crab amebosite lysate.

0002

2. Description of the Related Art A method of measuring endotoxin using horseshoe crab amebocyte lysate (hereinafter simply referred to as lysate) is known. This method is based on the coagulation of the lysate by trace amounts of endotoxin, but subsequent biochemical elucidation has revealed that this reaction consists of the stepwise activation of several coagulation factors ( Takanori Nakamura et al., Japanese Journal of Bacteriology, 38,
781-803 (1983)).

[0003] This reaction is carried out using, for example, the Japanese horseshoe crab (T.
achypleus tridentatus), when endotoxin is added to the lysate, factor C (endotoxin-sensitive factor, molecular weight 1
23,000) is activated, and the generated activated factor C undergoes limited hydrolysis at a specific site of factor B (molecular weight 64,000) to produce active factor B, which is activated by proclotting enzyme ( (molecular weight 54,000) and converts it into clotting enzyme, and clotting enzyme is coagulogen (clotting protein, molecular weight 1
9,723) in the loop bridged by disulfide bonds, i.e., between Arg18-Thr19... and Arg46-Gly47... to obtain H-Thr19...Arg46-OH. This is a series of reactions (also called a cascade reaction) in which peptide C (28 amino acid residues) is released while the remaining part is converted to coagulin gel.

On the other hand, this cascade reaction not only produces endotoxin but also produces (1→3)-β-D- in the lysate.
When glucan is added, the G factor in Figure 1 is activated,
The generated activated G factor activates the pro-clotting enzyme into a clotting enzyme, which then reacts in the same manner as in the case of endotoxin to produce coagulin gel.

[0005] The clotting enzyme produced by the above-mentioned cascade reaction is also added to the reaction system separately, for example, t-butoxycarbonyl-leucyl-glycyl-arginine-para-nitroanilide (BOC-Leu
-Gly-Arg-pNA), endotoxin or (1→3)-β-D-glucan can be quantified by measuring the absorbance of the produced chromogenic substance paranitroaniline. . Utilizing this effect, (1
→3) Used for specific measurement of -β-D-glucan.

On the other hand, a method for measuring (1→3)-β-D-glucan using G factor in lysate has been reported (Obayashi T.
tal. , Clin. Chim. Acta,
149, 55-65 (1985)).

However, in this method, the lysate is fractionated by gel filtration or chromatography using an affinity carrier immobilized with heparin or dextran sulfate to remove factor C, which is an endotoxin-sensitive factor, and to separate factor G and chromatography. With Ting Enzyme (
1→3) A method for measuring -β-D-glucan, comprising:
This method requires extremely complicated operations.

[0008]

SUMMARY OF THE INVENTION The present invention is directed to a (1→3)-β-D-
The purpose of the present invention is to provide a measuring agent for glucan.

[0009]

[Means for Solving the Problems] The present invention is directed to horseshoe crabs.
Measurement for (1→3)-β-D-glucan consisting of a lysate substantially free of endotoxin-sensitive factors obtained by contacting an amebosite lysate or a mixture of the lysate and dextran with a polyamide-based insoluble carrier It is a drug.

[0010] As the lysate, Limulus polyphemus (L. polyphemus, produced in America),
Tachypleus tridentatus (T. trident
atus, from Japan and China), Tachypleus gigas (T.
．． gigas, from Thailand and Peninsular Malaysia), Carcinoscorpius rotundicauda (C. rotun)
Examples include blood cell extracts prepared from horseshoe crab hemolymph such as P. dicauda (produced in Thailand and Peninsular Malaysia) by a conventional method.

[0011] Horseshoe crab
The method of contacting amebosite lysate is as follows:
A method in which the lysate is passed through a membrane-like carrier and the passing liquid is collected; a method in which the lysate is passed through a column packed with a granular carrier and the passing liquid is collected; Examples include a method in which the lysate is brought into contact with a carrier (like a carrier) and then the carrier is removed by techniques such as centrifugation and filtration.

[0012] Furthermore, when the lysate is brought into contact with the carrier, by mixing dextran with the lysate, it is possible to further increase the adsorption effect of endotoxin-sensitive factors onto the carrier, and (1→3)- β-D-
The measurement sensitivity for glucans can also be increased.

The dextran used has an average molecular weight of 5
,000-5,000,000, preferably 10,00
A range of 0 to 100,000 can be mentioned.

[0014] Dextran having an average molecular weight of less than 5,000 cannot be used because its adsorption effect on endotoxin-sensitive factors is weak. Moreover, those having a molecular weight of more than 5,000,000 are too viscous to be used.

The polyamide-based insoluble carrier used in the present invention is a crystalline linear polymeric substance whose main chain is composed of repeated acid amide bonds in the form of films, particles, chips, etc. and dicarboxylic acids, and compounds synthesized by polycondensation from ω-aminocarboxylic acids or corresponding lactams (for example, nylon 6, nylon 66, etc.).

According to the present invention, biological samples for measuring (1→3)-β-D-glucan include blood, plasma, serum,
Body fluids such as cerebrospinal fluid, ascites, joint fluid, pleural effusion, milk, urine,
Exudates, excreted fluids, etc. can be mentioned.

Using the measuring agent of the present invention, (1→3)-β-
To measure D-glucan, methods such as coexisting the aforementioned chromogenic synthetic substrate or fluorescent synthetic substrate in the reaction solution and measuring the amidase activity of the clotting enzyme, or testing gel formation due to the coagulation reaction are adopted. can do.

[0018]

[Operation] The present invention provides a polyamide-based insoluble carrier (1→3
)-β-D-glucan-sensitive factor is adsorbed and removed without impairing the activity of the endotoxin-sensitive factor, thereby obtaining a lysate that reacts only with (1→3)-β-D-glucan. . Further, when the lysate is brought into contact with the carrier, dextran is mixed with the lysate to increase the viscosity of the lysate, thereby increasing the adsorption and removal effect.

[0019]

[Examples] The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 500 ml of horseshoe crab (T. tridentatus) hemolymph was centrifuged at 4° C. and 1,500 rpm for 10 minutes, and approximately 20 g of the precipitated portion (amebocytes) was divided into 10
0ml of 0.02M Tris-HCl buffer (pH 8.0)
) and homogenizer (Polytron R PT10
(trade name), manufactured by Kinematica), and centrifuged at 10,000×G for 30 minutes to collect the supernatant and precipitate. The obtained precipitate was further extracted twice with 60 ml of buffer, and finally extracted with 20 ml of buffer.
0 ml of lysate was obtained.

Five types of reagents were prepared by the following method, and their reactivity towards endotoxin and (1→3)-β-D-glucan was compared and examined.

[0022] Agent A is 0.8M magnesium chloride 20
0μl and 6mM Boc-Leu-Gly-Arg-p
This is an untreated lysate prepared by adding 880 μl of lysate to a 200 μl NA mixture (hereinafter referred to as MS mixture) and freeze-drying.

[0023] For agent B, 1.2 ml of lysate was mixed with 0.20 ml of lysate.
The nylon membrane treatment of the present invention was prepared by passing 880 μl of the filtrate through a nylon membrane (Nalgene Syringe Filter (trade name), diameter 25 mm, manufactured by Nalge Co., Ltd.) with a diameter of It is lysate.

[0024] For agent C, 1.2 ml of lysate was mixed with 0.22 ml of lysate.
μm polyvinylidene fluoride film (Millex GV
(trade name) (diameter: 25 mm, manufactured by Millipore), 880 μl of the filtrate was added to the MS mixture, and the mixture was freeze-dried to prepare a comparative lysate.

[0025] For agent D, 1.2 ml of lysate was mixed with 0.20 ml of lysate.
μm polytetrafluoroethylene membrane (Millex F
A comparative lysate was prepared by adding 880 μl of the filtrate to the MS mixture and freeze-drying the lysate.

[0026] For agent E, 1.2 ml of lysate was mixed with 0.20 ml of lysate.
μm polysulfone membrane (Acrodisc (product name)
This is a comparative lysate prepared by adding 880 μl of the filtrate to the MS mixture and freeze-drying it.

Add and dissolve 2.2 ml of 0.2 M Tris-HCl buffer (pH 8.0) to each of the above five types of reagents, and add endotoxin (25 ml) to 0.1 ml of the solution.
Pg/ml) and (1→3)-β-D-glucan (3
0 Pg/ml) was added and heated at 37° C. for 30 minutes. The reactivity of the sample with respect to the five reagents was determined by comparing the pNA (paranitroaniline) generated after 30 minutes with 0.5 ml of 0.04% sodium nitrite (0.48
M hydrochloric acid solution), 0.3% ammonium sulfamate, and 0.7% N-(1-naphthyl)ethylenediamine dihydrochloride were sequentially added to develop color, which was expressed as an absorbance value at 545 nm.

The results are shown in Table 1. from this result,
By using a lysate that has passed once through a nylon membrane, which is a polyamide membrane, (1→3)-β-D-glucan can be specifically quantified without being affected by endotoxin.

[0029]

[Table 1]

*...(Preparation method of (1→3)-β-D-glucan) According to International Patent Publication No. 90/02951 (1990), 1 g of curdlan (Wako Pure Chemical Industries, Ltd.) was mixed with approx.
Suspend in 00 ml of 5mM NaOH aqueous solution and use a sonic generator, Sonicator TM (Otake Seisakusho, Model 5) under ice cooling.
202PZT, Tokyo) was subjected to sonication at 20 KHz and 80 W for 12 minutes to reduce the molecular weight. 5 of processing liquid
Gel permeation chromatography using M NaOH aqueous solution to make a final 0.3M aqueous solution; GPC column: TSK gel G3000PW x L 2
Book, G2500PW×L 1 piece, mobile phase: 0.3M
Aqueous NaOH solution, flow rate 0.5 ml/min) was used to collect fractions, and re-chromatography showed that the molecular weight was 216.0.
00 GPC fractionated and purified standard ((1→3)-β-D-glucan standard) was obtained.

Example 2 The same amount of distilled water was added to 40 ml of the raw material lysate prepared in Example 1 (hereinafter referred to as lysate+DW). In addition, the same amount of 15% (W/V) dextran (
molecular weight 40,000) aqueous solution and 3,500 rpm.
The supernatant was obtained by centrifugation for 10 minutes (hereinafter referred to as lysate + Dx).

Four types of reagents were prepared by the following method, and their reactivity towards endotoxin and (1→3)-β-D-glucan was compared and examined.

[0033] The F agent is MS mixed solution with lysate + DW1.7.
This is untreated lysate + DW prepared by adding 6 ml and freeze-drying.

[0034] Agent G consists of MS mixture containing lysate + Dx1.
This is untreated lysate + Dx prepared by adding 76 ml and freeze-drying.

[0035] For agent H, 5.0 ml of lysate + DW was
．． This is the nylon membrane-treated lysate +DW of the present invention, which was prepared by passing through a 20 μm nylon membrane (Nalgene syringe filter), adding 1.76 ml of the filtrate to an MS mixture, and freeze-drying.

[0036] For agent I, 5.0 ml of lysate + Dx was
．． The nylon membrane-treated lysate +Dx of the present invention was prepared by passing through a 20 μm nylon membrane (Nalgene syringe filter), adding 1.76 ml of the filtrate to an MS mixture, and freeze-drying.

Add and dissolve 2.2 ml of 0.2 M Tris-HCl buffer (pH 8.0) to each of the above four types of reagents, and add endotoxin (25 ml) to 0.1 ml of the solution.
Pg/ml) and (1→3)-β-D-glucan (3
0.1 ml of each sample (0 Pg/ml) was added and heated at 37° C. for 30 minutes, and the reactivity of the sample with respect to various reagents was examined in the same manner as in Example 1. The results are shown in Table 2. This result shows that by allowing dextran to coexist in the lysate in advance, it is possible to enhance the adsorption effect of endotoxin-sensitive factors and to significantly increase the sensitivity to (1→3)-β-D-glucan.

[0038]

[Table 2]

Example 3 600 ml of horseshoe crab (L. polyphemus) hemolymph was centrifuged at 4° C. and 1,500 rpm for 10 minutes, and about 23 g of the precipitated portion (amebocytes) was mixed with 110 ml of hemolymph.
Add 0.02M Tris-HCl buffer (pH 8.0), homogenize and extract using a homogenizer (Polytron R PT10), cool and centrifuge at 10,000 x G for 30 minutes to separate the supernatant and precipitate. I collected things. The obtained precipitate was further extracted twice with 65 ml of buffer to finally obtain 220 ml of lysate. Lysate 20
ml of the same amount of 15% (W/V) dextran (molecular weight 7
After centrifugation at 3,500 rpm for 10 minutes, the supernatant was used as D-lysate. Using this,
Two types of reagents were prepared by the following method, and their reactivity towards endotoxin and (1→3)-β-D-glucan was compared and examined.

[0040] For agent J, 1.76% of D-lysate was added to the MS mixture.
This is untreated lysate + Dx prepared by adding ml and freeze-drying.

[0041] For the K agent, 10 ml of D-lysate was mixed with 0.2
After passing through a 0 μm nylon membrane (Nalgene Media Plus Filter Unit (trade name), diameter 90 mm, manufactured by Nalge), 1.76 ml of the filtrate was added to the MS mixture and freeze-dried. Nylon membrane treated lysate + Dx.

Add and dissolve 2.2 ml of 0.2 M Tris-HCl buffer (pH 8.0) to each of the above two types of reagents, and add endotoxin (25p) to 0.1 ml of the solution.
g/ml) and (1→3)-β-D-glucan solution (3
0 pg/ml) at 37°C.
After heating for 0 minutes, the reactivity was compared and examined in the same manner as in Example 1. The results are shown in Table 3. It is clear that the lysate passed through the nylon membrane, which is a polyamide-based membrane, does not react with endotoxin, but reacts only with (1→3)-β-D-glucan. In addition, when lysate was treated with dextran, (1→3)-β-
It can be seen that the sensitivity to D-glucan is significantly increased.

[0043]

[Table 3]

Example 4 Using commercially available gelation Limulus test reagent (1→3)
- Preparation of measuring agent for β-D-glucan Using a commercially available lysate product, that is, a gelling Limulus test reagent, the target measuring agent for (1→3)-β-D-glucan was prepared as follows. .

Formulation L was prepared by dissolving Pregel-M (Lysate sold by Seikagaku Corporation, Lot. AB-01) in 2.6 ml of distilled water for injection, and then using a 0.20 μm nylon membrane (Tissue Culture Filter Unit TC (commercial product)). name), diameter 47m
This is the nylon membrane-treated lysate +DW of the present invention, which was obtained by passing the lysate through a nylon membrane-treated lysate (Nalge Inc.) and using the filtrate.

The M agent is untreated Pregel-M.

The N agent is Limulus HSII-Test Wako (
Wako Pure Chemical Industries Sales Lysate, Lot. EMM090
) was dissolved in 5.0 ml of distilled water for injection, and passed through the tissue culture filter unit TC in the same manner, and the filtrate 2
．． This is nylon membrane-treated lysate + DW prepared by freeze-drying 6 ml.

The O agent was untreated Limulus HSII-Test Wako.

Using distilled water for injection, the M agent and N agent were
．． The O agent was dissolved in 5.0 ml. 0 of L~O agent
．． Distilled water for injection (blank), endotoxin (derived from E. coli0111:B4) and (1→
3) Add 0.1 ml of -β-D-glucan separately,
The mixture was left to warm at ℃ for 60 minutes, and the presence or absence of gel formation was examined. The results are shown in Table 4. In the table, + indicates that a gel was formed, and - indicates that no gel was formed. As is clear from Table 4, agents L and N are lysates suitable for the purpose of reacting only with (1→3)-β-D-glucan.

[0050]

[Table 4]

(1→3)-β-D-glucan is also known as a constituent polysaccharide of fungal cell walls, and (1→3)-β-D-glucan
- By measuring glucan, the presence of fungi in the body can be detected. In Examples 5 to 7, examples of fungal detection by the method of the present invention will be given below.

Example 5 (Measurement of plasma samples) Eleven patients with severe blood diseases (acute lymphocytic leukemia, acute myeloid leukemia, multiple myeloma, etc.) who were hospitalized and suspected to have sepsis due to fungi were tested. Heparinized plasma, which had been aseptically collected, was used as a sample and centrifuged at 150×G at 4° C. for 10 minutes to obtain platelet-rich plasma. 0.32M in that 0.1ml
Add 0.2 ml of perchloric acid and heat at 37°C for 20 minutes.
The precipitate was removed by centrifugation (3,000 rpm, 10 minutes), and 0.18 M NaOH was added to 0.05 ml of the supernatant.
05 ml was added to neutralize it and use it as a test solution.

Subsequently, 0.1 ml of the (1→3)-β-D-glucan assay agent (Agent I) of the present invention prepared by the method described in Example 2 was added, and the mixture was heated at 37° C. for 30 minutes. The reaction solution contained 0.04% sodium nitrite (0.48M hydrochloric acid solution), 0.3% ammonium sulfamate, and 0.04% sodium nitrite (0.48M hydrochloric acid solution).
Diazo coupling was performed by sequentially adding 0.5 ml of 07% N-1-naphthylethylenediamine dihydrochloride to 545n
The absorbance was measured at m and a separately prepared calibration curve (Figure 2
) and expressed as (1→3)-β-D-glucan equivalent amount. As shown in Table 5, all cases (No. 1 to No. 1
High concentrations of (1→3)-β-D-glucan were detected in 1) (healthy subjects: 0.2 ± 0.3 pg/ml), and in 5 cases (No. 1 to No. 5), In blood culture,
Candida albicans
ans), Candida guglielmondi (Candida
a guilliermondii), Candida tropicalis
, Candida krusei
) and Cryptococcus neoformans (Cry
Two cases (No. 6 and No. 7) were negative in blood culture, but histopathological examination at post-mortem autopsy revealed Aspergillus fumigatus (Aspergillus neoformans).
llus fumigatus) was detected. Furthermore, for the remaining four cases (No. 8 to No. 11), although fungal infection was strongly suspected based on clinical symptoms, progress, drug sensitivity, etc., blood cultures were negative, but antifungal agents (amphotericin, Miconazole, fluconazole) administration resulted in significant clinical improvement. Therefore, it can be seen that the measuring agent of the present invention can be appropriately used as a rapid diagnostic method for fungal infections, particularly deep fungal infections that are extremely difficult to diagnose using conventional testing methods.

[0054]

[Table 5]

Example 6 (Measurement of urine specimen) A case of urinary tract infection during hospitalization revealed Candida albicans and Candida albicans in urine culture.
In three cases where Candida glabrata was detected, (
1→3)-β-D-glucan was quantified. For urine, collect midstream urine aseptically into a sterile urine collection cup, and collect 0.005 ml of it.
After adding 0.1 ml of distilled water for injection, add the (1→3)-β-D-glucan measuring agent (B
0.1 ml of agent) was added and heated at 37°C for 30 minutes. After diazo coupling in the same manner as above, the absorbance of the solution was measured at 545 nm, and from a separately prepared calibration curve (1→3
)-β-D-glucan equivalent value. As shown in Table 6, high concentrations of (1→3)-β-D were observed in all three cases.
-Glucan detected (healthy person: 10 pg/ml or less)
It can be seen that the measuring agent of the present invention can be appropriately used as a rapid diagnostic method for fungal urinary tract infections.

[0056]

[Table 6]

Example 7 (Measurement of cerebrospinal fluid specimen) Meningitis was suspected during hospitalization, and Cryptococcus neoformans was detected in the cerebrospinal fluid.
For the three cases of fungal meningitis in which ans) was detected, 0.05 ml of cerebrospinal fluid was aseptically collected by lumbar puncture, 0.05 ml of distilled water for injection was added, and 0.05 ml of distilled water for injection of the present invention described in Example 3 was added. (1→3)-β-D-glucan measuring agent (K agent) 0
．． 1 ml was added and heated at 37°C for 30 minutes. After diazo coupling in the same manner as above, the absorbance of the solution was measured at 545 nm, and (1→3)-β was determined from a separately prepared calibration curve.
It was expressed as a -D-glucan equivalent value. As shown in Table 7, high concentrations of (1→3)-β-D-glucan were detected in all three cases (healthy subjects: 1 pg/ml or less), and the measuring agent of the present invention It can be understood that this method can be appropriately used as a rapid diagnostic method for inflammation.

[0058]

[Table 7]

[0059]

[Effect of the invention] The present invention uses lysate (1→3)
- Provides a measuring agent that does not contain endotoxin-susceptible factors for specifically measuring β-D-glucan. )-β-D-glucan can be measured quickly, easily, and with high accuracy. It can be used to quickly diagnose deep-seated fungal infections that are difficult to diagnose using conventional testing methods such as bacterial culture, as well as to determine appropriate treatment methods and therapeutic effects.

Furthermore, the measuring agent of the present invention includes distilled water for injection,
(1→3)-β-D- mixed in injection drugs and medical equipment
It enables rapid and accurate measurement of glucan, and can also be used to screen for antitumor polysaccharides typified by (1→3)-β-D-glucan.

[Brief explanation of the drawing]

FIG. 1 shows the reaction mechanism of horseshoe crab amebocyte lysate with endotoxin and (1→3)-β-D-glucan.

FIG. 2 shows a calibration curve for (1→3)-β-D-glucan of agent I.

Claims (2)

[Claims] 1. A measuring agent for (1→3)-β-D-glucan comprising a lysate substantially free of endotoxin-sensitive factors obtained by contacting a horseshoe crab amebosite lysate with a polyamide-based insoluble carrier. [Claim 2] Claim 1, wherein the horseshoe crab amebosite lysate is a lysate containing dextran.
Measuring agent.