KR20020093612A - Composition for detecting peptidoglycan, and diagnostic kit detecting peptidoglycan - Google Patents

Abstract

PURPOSE: A composition for detecting peptidoglycan and a kit for detecting peptidoglycan using the same are provided, thereby detecting a trace amount of peptidoglycan in a sample, and consequently diagnosing infection of gram positive bacteria easily. CONSTITUTION: The composition for detecting peptidoglycan comprises an extract of insect body fluids which shows phenoloxidase activity due to peptidoglycan in the absence of calcium. Wherein, the extract of insect body fluids is a plasma solution separated from the body fluids; the extract of insect body fluids contains a plasma solution and blood corpuscle lysate of insect body fluids; the extract of insect body fluids is derived from the larva of Galleria mellonella; the extract of insect body fluids is obtained by treating a plasma of the larva of Galleria mellonella with a solvent or a buffer solution to obtain a fraction and adding blood corpuscle lysate into the fraction; the solvent or buffer solution contain a chelating agent; and the fraction is obtained by column chromatography packed with sugar or vinyl resin. The kit for detecting peptidoglycan contains the composition for detecting peptidoglycan.

Description

Composition for detecting peptidoglycan, and peptidoglycan detection kit using the same {composition for detecting peptidoglycan, and diagnostic kit detecting peptidoglycan}

The present invention relates to a composition capable of detecting peptidoglycan, and a kit for detecting peptidoglycan using the same.

Pathogenic Gram-positive bacteria infections are a common infection that accounts for a large part of both sensitizing and widespread infections in hospitals, and food poisoning and bacterial pneumonia caused by pathogenic Gram-positive bacteria are life-threatening diseases. There is a need for a system for rapidly detecting pathogenic Gram-positive bacteria present in clinical specimens or foods such as blood, tissue, and urine. It takes several days to detect pathogenic bacteria by traditional methods, and food can be circulated during this period and consumed by consumers, which poses a risk.

Gram-positive bacteria present in trace amounts in various types of samples can be identified by detecting and measuring peptidoglycan, a cell wall component of the bacterium. Peptidoglycan is a component of the bacterial cell wall and is a glycoprotein polymer comprising N-acetylmuramic acid or N-glycosylmuramic acid and D-amino acid which forms a thin layer inside the outer membrane of the cell wall.

Therefore, it is possible to detect and measure peptidoglycan and apply it to the stability test of medicines, microbiological test of water and food, and diagnosis of infectious diseases.

During insect bioprotective reactions, the prophenoloxidase system selectively recognizes trace amounts of lipopolysaccharides, peptidoglycans, and beta-1,3-glucans so that the zymogen form of the prophenoloxidase is activated. It is known that the signal is amplified more than 1000 times by the cascade reaction transformed with phenol oxidase. However, since the prophenoloxidase system recognizes all lipopolysaccharides, peptidoglycans, and beta-1,3-glucans, there is a need for a system that selectively recognizes only certain substances.

Prophenolic oxidase is present in the in vivo of morphotropic insects and is activated with phenol oxidase by a cascade reaction activated by beta-1,3-glucan or lipopolysaccharide. This reaction system, which consists of a series of cascade steps, is easily activated by an invading pathogen, foreign substance, or an internal factor induced by the degranulation reaction of its own blood cells, and is converted into phenol oxidase to use melanin using catechol amines. Will form. Therefore, it was difficult to isolate this reaction system in vitro (Ashida and Yoshida, (1988), Insect. Biochem. 18, 11-19).

Ashida et al. Eur. J. Biochem, 188, 507-515 (1990) presented a composition that recognizes beta-1,3-glucan, isolated from mosquito larvae, and revealed that divalent ions play an important role in the activity of prophenoloxidase. Prophenol oxidase activation systems are known to require calcium ions.

As an example of a composition and method for detecting peptidoglycan, US Pat. No. 4,970,152 describes specifically detecting peptidoglycan by removing proteins reacting with beta-1,3-glucan from the plasma solution of silkworm moth larvae. Although reports have been made on the composition, the composition requires the addition of calcium ions for the phenol oxidase activity to be exerted by the peptidoglycan. That is, according to U.S. Patent 4,970,152, when collecting bodily fluids of insects, phenol oxidase is inhibited by calcium ions to obtain phenol oxidase composition, and calcium ions are added in order to cause color development using peptidoglycan as a substrate. It is required.

U. S. Patent No. 5,747, 277 reports SLP reagents but does not specifically react to peptidoglycans because they simultaneously detect beta-1,3-glucan and peptidoglycan.

In view of the problems of the prior art as described above, the present invention is to provide a composition for detecting peptidoglycan that can selectively detect only peptidoglycan.

Another object of the present invention relates to a method for producing a composition for detecting peptidoglycan.

Another object of the present invention relates to a method for detecting peptidoglycan using a composition for detecting peptidoglycan capable of selectively detecting only the peptidoglycan.

Another object of the present invention relates to a peptidoglycan detection kit using a peptidoglycan detection composition capable of selectively detecting only the peptidoglycan.

FIG. 1 is a graph illustrating phenol oxidase activation of lipopolysaccharide, beta-1,3-glucan or peptidoglycan with honey bee moth larva plasma solution under conditions with or without calcium addition.

2 is a graph showing the degree of phenol oxidase activation using lipopolysaccharide, beta-1,3-glucan or peptidoglycan as a substrate of plasma fraction B of honeybee larva moth larvae.

3 is a graph showing the phenol oxidase activation according to the peptidoglycan concentration in the plasma fraction B of honeybee moth larvae.

4 is a graph showing the degree of activation of phenol oxidase using beta 1,3-glucan, or peptidoglycan as a substrate with plasma fraction B, hemolytic hemolytes, and mixtures thereof of honeybee moth larvae.

FIG. 5 is a graph showing the degree of activation of phenol oxidase against peptidoglycan as a solution containing plasma fraction B and blood cell hemolytes of honeybee moth larvae with protein contents of 600 μg and 200 μg.

Figure 6 is a graph showing the activation of phenol oxidase according to the concentration of peptidoglycan in a solution containing plasma fraction B and blood cell hemolyte of honeybee moth larvae.

Figure 7 is a standard curve showing the degree of activation of phenol oxidase according to the peptidoglycan concentration 20ng / ml in a solution containing plasma fraction B and blood cell hemolyte of honeybee moth larvae.

FIG. 8 is a graph showing the activation of phenol oxidase using lipopolysaccharide, beta-1,3-glucan, or peptidoglycan as a substrate containing plasma fraction B of a honeybee moth larva and blood cell hemolytes to be.

The present invention relates to a composition capable of detecting peptidoglycan in a sample, a method for producing the same, and a kit for detecting peptidoglycan using the same.

The phenol oxidase system in the present invention means a system that is activated in the phenol oxidase by peptidoglycan, present in the beetle larvae ( Galleria mellonella ) larvae.

The phenol oxidase composition in the present invention means a composition containing all or part of the phenol oxidase system components, and exhibits phenol oxidase activity by peptidoglycan.

In the present invention, the phenol oxidase system of honeybee moth is not required calcium ions to activate phenol oxidase, rather it can be seen that the activity of phenol oxidase is suppressed when calcium ions are added.

The present invention relates to a composition for detecting peptidoglycan comprising a body fluid extract of an insect exhibiting phenol oxidase activity by peptidoglycan without adding calcium. The body fluid extract of the insect may be a plasma solution separated from the body fluid of the insect, and in addition, when the body fluid extract of the insect comprises a plasma solution of the insect body fluid and a hemoglobin lysate, a smaller amount of plasma solution Also, peptidoglycan can be quantified, and even smaller amounts of peptidoglycan contained in the sample can be detected. The solution containing the body fluid plasma solution and blood cell hemolyte of the insect is mixed with the plasma by taking the body fluid of the insect and then separating the plasma and blood cells and hemolyzing the separated blood cells, or part of the blood cell hemolyte or partially purified blood cell hemolyte Can be obtained by addition to the purified plasma. Alternatively, some or all of the blood cells may be hemolyzed as is without separating the blood cells in the body fluid. For example, body fluids or isolated blood cells can be hemolyzed by ultrasonic sonication or high-speed centrifugation. In addition, the plasma solution, blood cell hemolytes, and a mixed solution thereof may be diluted by adding a solution such as a buffer solution, or may be concentrated and used by a conventional concentration method. The hemoglobin hemolytes may be prepared as a lysate obtained by crushing blood cells by adding a solvent to the precipitate except the plasma solution from the insect body fluid extract, preferably, a supernatant separated from the precipitate in the lysate.

In addition, the composition for detecting peptidoglycan of the present invention is that the body fluid extract of the insect is preferably derived from the beetle moth larvae, that is, including all or part of the phenol oxidase system of bee beetle moths, Oxidase. Honey bee moth is shorter than other insects in about 2 months of life and has high fertility, so it can be easily reared in large quantities, so that a large amount of body fluid can be collected.

In addition, the plasma solution in the insect body fluid is obtained by treating the plasma of the honeybee larva moth larvae with a solvent or a buffer to obtain a fraction, and select the fraction showing the phenol oxidase activity by peptidoglycan without adding calcium in the fraction obtained And preferably the solvent or buffer contains sufficient chelating agent to chelate the calcium ions present in the sample and separation process, more preferably the fraction obtained by column chromatography. to be. The column chromatography may be filled with, for example, a resin based on sugar or a resin based on vinyl.

The composition of the present invention that can detect such peptidoglycan can be used to determine whether or not contamination of Gram-positive bacteria, such as staphylococcus, streptococci, pneumococci, and diphtheria bacteria in an individual.

The present invention also relates to a method for preparing a composition comprising an insect body fluid extract exhibiting phenol oxidase activity by peptidoglycan under conditions in which calcium is not added using a plasma of honeybee moth larva as a sample. In the method of the present invention, after obtaining a plasma from the body fluid of honeybee larva moth larvae, the obtained sample is treated with a solvent or a buffer to obtain a fraction, and phenol oxidase activity is induced by peptidoglycan without adding calcium in the obtained fraction. Consisting of selecting the fractions indicated.

In the method of the present invention, an anticoagulant buffer which can suppress the coagulation of body fluids is preferably used when taking samples from honeybee larvae. The anticoagulant buffer may be used as long as it is a buffer solution capable of inhibiting body fluid coagulation of insects, and citric acid buffer is particularly preferable. A serine protease inhibitor that can irreversibly inhibit serine protease can be added to the anticoagulant buffer. The serine protease inhibitor is a phenol oxidase composition fraction from honey bee moth larva fluid that irreversibly inhibits the serine protease. Anything that can be used can be used. Preferably, p-APMSF (para-amijinophenyl) -methanesulfonyl fluoride), PMSF (phenylmethanesulfonyl fluoride), DFP (diisopropylfluorophosphoric acid) and the like can be used and the concentration is 0.2 mM It can be used to be abnormal. In addition, chelating agents may be added to the anticoagulant buffer to prevent intercellular adhesion and to prevent the phenol oxidase system from being activated during bodily fluid collection.

The process of obtaining a fraction by treating the sample obtained in the method of the present invention with a solvent or buffer may be carried out, for example, by column chromatography.

The kind of solvent or buffer used in the separation step in the method of the present invention is not particularly limited. An anticoagulant buffer used for sampling from larvae can be used, and preferably, a chelating agent can be added to the anticoagulant buffer to inhibit a reaction related to protein aggregation to obtain a desired phenol oxidase composition.

As a chelating agent sufficient to chelate the calcium ions present in the sampling and separation process, conventionally known chelating agents can be used without particular limitation, and for example, EDTA, EGTA, citric acid and the like can be used. . The amount of the chelating agent may vary depending on the separation process conditions such as the target insect sample or column type, the solvent used, and may be an amount sufficient to chelate calcium ions present in the insect sample and the separation process. Thus, ordinary experts in the art will be able to determine the amount of chelating agent without undue experimentation.

In the present invention, an example of a method of treating the plasma of honeybee moth larvae with a solvent or a buffer is column chromatography, and after loading the plasma in a resin-filled column, eluting with a solvent or a buffer such as an anticoagulant buffer. Can be obtained. In the present invention, by performing column chromatography without a separate difficult purification process such as affinity chromatography, a composition capable of specifically detecting peptidoglycan can be purified.

In the present invention, the resin that can be used in column chromatography is a carrier of sugars such as monosaccharides and polysaccharides, and a resin of agarose or dextran as a raw material or a resin of vinyl is preferable. For example, Sephadex or Toyoperal may be used.

The composition according to the present invention can specifically detect peptidoglycan and thus can be used for diagnosing microbial infections having peptidoglycan as a cell wall component.

Accordingly, the present invention relates to a method for detecting the presence of peptidoglycan in a sample, wherein a sample is taken from the sample, and a composition exhibiting phenol oxidase activity by peptidoglycan in the absence of calcium is added to the sample. And phenol oxidase activity in the sample. The composition exhibiting phenol oxidase activity by the peptidoglycan is intended to include the composition for detecting the peptidoglycan, and in one embodiment of the present invention, sufficient chelating to chelate calcium ions present in the sample and separation process. No calcium ions were added to the fraction obtained by treating the plasma of the honeybee moth larvae recovered in the presence of the agent with a solvent containing a chelating agent sufficient to chelate the calcium ions present in the sample and the separation process. It may be a composition prepared by selecting a fraction for activating phenol oxidase by peptidoglycan in.

In the peptidoglycan detection method according to the present invention, the specimen may be an animal including a human or an environment in which an organism lives. For example, blood may be collected from a sample to diagnose Gram-positive bacteria infection. In another example, aquaculture can collect water from aquaculture farms and diagnose microbial infections that contain peptidoglycans as cell wall components, such as Gram-positive bacteria.

In order to improve the specificity of diagnosing microbial infections having peptidoglycan as a cell wall component, pretreatment may be performed to remove lipopolysaccharides that may be present in the sample sample if necessary. For example, the effects of lipopolysaccharide can be removed by treating the sample sample with a substance that specifically binds to lipopolysaccharide, such as polymyxin, or can precipitate lipopolysaccharide .

The method for measuring phenol oxidase activity in the present invention can be used as it is or by modifying a known phenol oxidase measuring method. As an example, phenol oxidase activity can be measured by measuring absorbance using a color reaction using 4-methylcatechol / 4-hyperiproline ethyl ester (4-MC / 4-HP) or a melanin-forming reaction using dopamine as described below. It can be determined from this, it is easy to know the presence of peptidoglycan.

The present invention also relates to a kit for detecting peptidoglycan. The peptidoglycan detection kit of the present invention contains a composition exhibiting phenol oxidase activity by peptidoglycan without adding calcium. In one example of the present invention, the composition for detecting peptidoglycan is a composition exhibiting phenol oxidase activity by peptidoglycan under the condition that calcium ions are not added, and preferably kills calcium ions present in the sample and separation process. In a fraction obtained by treating a sample, which is a plasma of honeybee moth larvae prepared in the presence of a chelating agent sufficient for rating, with a solvent containing a chelating agent sufficient for chelating the sample and calcium ions present in the separation process. The composition may be prepared by selecting a fraction for activating phenol oxidase by peptidoglycan without adding calcium ions.

The present invention will be described in more detail with reference to the following illustrative examples, but the protection scope of the present invention is not intended to be limited to the following examples.

Example 1 Preparation of Plasma and Blood Cells in Honeybee Larva Moth Larvae

Among the honey bee larvae ( Galleria mellonella ) larvae, 2.5 to 3 cm in length were selected for anesthesia on ice for 10 to 30 minutes, and then anti-coagulant buffer containing 0.2 mM p-APMSF (Wako, Japan) A needle of a 5 ml syringe with a 23G needle containing 4.6) was stabbed into the second section of the larva's head. After cutting half of the second node at the tail, the buffer solution was pushed into the syringe and received 4 to 5 drops to extract the body fluid of the honeybee larvae. The anticoagulant buffer comprises NaCl 15 mM, trisodium citrate 30 mM, citric acid 26 mM, and EDTA 20 mM.

50 ml of the collected body fluid was centrifuged at 4 ° C. and 371 × g for 20 minutes, and the supernatant was named plasma, and the precipitate was named hemocyte.

Example 2: Preparation of Plasma Solution

2-1: Pretreatment of Extracted Plasma Solution

Concentrate the plasma solution to the final level of 2ml using an ultrafiltration kit (membrane cut off. 3000) and measure the phenol oxidase activity under the condition that 5mM of calcium is added or not at the final concentration. The stability was confirmed and shown in FIG. 1 (1: plasma solution + no Ca ²⁺ , 2: plasma solution + 1 μg lipopolysaccharide (LPS) + Ca ²⁺ no addition, 3: plasma solution + 1 μg beta −) 1.3-glucan (BG) + Ca ²⁺ free, 4: plasma + 1 μg peptidoglycan (PG) + Ca ²⁺ free, 5: plasma + Ca ²⁺ added, 6: plasma + 1 μg LPS + Ca ²⁺ addition, 7: plasma solution + 1 μg BG + Ca ²⁺ addition, 8: plasma solution + 1 μg PG + Ca ²⁺ addition). The phenol oxidase activity of 1 μg lipopolysaccharide, peptidoglycan and beta-1,3-glucan was measured using plasma solution. We observed low phenol oxidase activity (bar 2) on saccharides, but strong phenol oxidase activity on beta-1,3-glucan (bar 3) and peptidoglycan (bar 4). In addition, the bee-flying moth larva system can be activated with phenol oxidase without calcium ions. Rather, the addition of calcium ions inhibited the activity of phenol oxidase.

2-2: Separation of Fractions Specific to Peptidoglycans

Sephadex G-100 resin was charged to a column of 1.0 × 45 cm and equilibrated with anticoagulant buffer (pH 5.0). The concentrated sample (500 mg protein) was loaded into an equilibrated column, and then the anticoagulant buffer was flowed at a rate of 3.0 ml / tube, and the eluate was fractionated 3 ml up to 1 to 30 times.

The proteins were separated into groups A (fractions 1-6), B (fractions 7-10), and C (fractions 11-16). Groups B and C were concentrated using ultrafiltration kits to a protein concentration comparable to group A, and pre-loading samples, samples of groups A, B and C, β-1,3-glucan, peptidoglycan, lipopolysaka Phenolic oxidase activity by the ride was measured and the results are shown in FIG. 2 (1: plasma solution, 2: plasma solution + 1 ug lipopolysaccharide (LPS). 3: plasma solution + 1 μg beta-1,3-glucan) (BG), 4: plasma solution + 1 μg peptidoglucan (PG), 5: group A, 6: group A + 1 μg LPS, 7: group A + 1 μg BG, 8: group A + 1 μg PG, 9: group B, 10: group B + 1 μg LPS, 11: group B + 1 μg BG, 12: group B + 1 μg PG, 13: group C, 14: group C + 1 μg LPS, 15: group C + 1 μg BG, 16: group C + 1 μg PG). In group B it can be seen that the phenol oxidase activity against peptidoglycan only.

Example 3: Phenoloxidase Activity of the Plasma Fraction

This example was performed to investigate the sensitivity to peptidoglycan to group B, which exhibits phenol oxidase activity specifically for peptidoglycan.

The peptidoglycan solution was first suspended in 1 mg of insoluble peptidoglycan (Fluka) in a 20 mM Tris (pH8.0) solution, 100 μl was added to 900 μl Tris buffer, and then ultrasonically treated for several seconds. 10 μl was taken and used to measure phenol oxidase activity.

Phenoloxidase activity was measured by modifying the spectrometry of Pye using 4-methylcatechol (4-MC) and 4-hydroxyproline ethyl ester (4-HP) as substrates. 30㎕ sample (protein amount of 600 u g) in 20mM Tris-cost peptidoglycan suspended in a buffer solution (pH8.0) 1 u g / a mixture of 10㎕ then reacted at 30 ℃ 5 bungan ago. Negative control was added with 10 µl of 20 mM Tris buffer (pH 8.0) only. The protein content in the sample is obtained by measuring the absorbance at 280 nm, and refers to the total amount of the protein contained in the sample obtained by separation in the above method. Then, 442 μl of 20 mM Tris buffer solution (pH 8.0) was dispensed into the test tube, and 4-MC 1 mM, 4-HP, which was used as a substrate, was added at a final concentration of 2 mM and then adjusted to a volume of 500 μl. After reacting at 30 ° C for 20 minutes, the samples were removed from the test tubes by 100 μl and diluted twice in 100 μl of anticoagulant buffer (pH4.6) to measure the absorbance at 520 nm of the spectrometer. Multiply by to determine the actual phenol oxidase activity. When calcium was added when phenol oxidase activity was measured, phenol oxidase activity was measured by the above method using 20 mM Tris buffer (pH8.0) to which CaCl ₂ 5.65 mM was added.

30 μl (protein content 600 μg) of a Group B solution exhibiting phenol oxidase activity specifically for the peptidoglycan obtained in Example 2, in which phenol was not added at each concentration of the water-soluble peptidoglycan. Oxidase activity was measured and the results are shown in FIG. 3 (1: group B, 2: group B + 3 ng peptidoglycan (PG), 3: group B + 5 ng PG, 4: group B + 10 ng PG, 5 : Group B + 20 ng PG, 6: group B + 30 ng PG). Group B solution was able to quantify peptidoglycan up to 3ng / ml.

Example 4 hemocyte lysate preparation of honeybee moth

In Example 1, the blood cells separated from the body fluid of honeybee moths were added 50 mM Tris, and a pH 6.5 Tris buffer containing 1 mM EDTA to 1/2 of the volume of the blood cells. Broke the cell. Thereafter, centrifugation was performed at 3,586 × g for 20 minutes at 4 ° C to name this supernatant as the first sample. The supernatant from which the supernatant was removed was re-added by 1/2 of the volume of blood cells and subjected to one more ultrasound and centrifugation in the same manner as above, and the supernatant obtained at this time was referred to as a second sample. The first sample and the second sample thus prepared were called a hemolyte solution, and stored in a freezer at -80 ° C until use.

Example 5: Phenyloxidase Activity and Specificity for Peptidoglycans of Blood Cell Lysate Solution

In order to determine whether blood cell hemolytes specifically respond to peptidoglycan, phenol oxidase activity against beta 1,3-glucan and peptidoglycan was measured and shown in FIG. 4. The experimental solution is as follows:

(1: plasma fraction B solution of Example 2 (protein content: 200 µg), 2: plasma fraction B solution (200 µg) + beta 1,3-glucan (1 µg), 3: plasma fraction B solution (200 µg) ) + Peptidoglycan (1 μg), 4: blood cell hemolyte (protein content: 100 μg), 5: blood cell hemolyte (100 μg) + beta 1,3-glucan (1 μg), 6: blood cell hemolyte (100 μg) + peptidoglycan (1 μg), 7: plasma fraction B solution (200 μg) + blood cell hemolyte (100 μg), 8: plasma fraction B solution (200 μg) + blood cell hemolyte (100 μg) ) + Beta 1,3-glucan (1 μg) 9: plasma fraction B solution (200 μg) + hemocytic hemolysate (100 μg) + peptidoglycan (1 μg))

As a result, when 1 µg of beta 1,3-glucan and 1 µg of peptidoglycan were added to the solution in which the anticoagulant buffer was added to the plasma fraction B obtained in Example 2 (protein content: 200 µg) (2 and 3 times) Bar) showed no phenol oxidase activity, and phenol oxidase was also observed when hemoglobin hemolyte solution (protein content of 100 μg) was added with 1 μg of beta 1,3-glucan and 1 μg of peptidoglycan (bars 5 and 6). No activity was shown. However, when the plasma fraction B solution (protein content: 200 µg) was added to the cell hemolyte solution 100 µg and 1 µg peptidoglycan (bar 9), phenol oxidase activity was increased. In addition, it was found that phenol oxidase activity was not observed when 100 μg of hemolytic hemolyte solution and 1 μg of beta 1,3 glucan were added to the plasma fraction B solution (protein content: 200 μg). Therefore, the mixture of the plasma fraction B solution and the blood cell hemolyte solution specifically recognizes peptidoglycan and exhibits phenol oxidase activity, and by adding the blood cell hemolyte solution less than the plasma fraction B solution alone, It can be seen that the phenol oxidase activity can be measured using the plasma fraction B solution.

Example 6 Effect of Blood Cell Hemolyte on Phenoloxidase Activity

Plasma fraction B solution (protein content 600 μg), plasma fraction B solution (protein content 600 μg) + peptidoglycan (1 μg), plasma fraction B solution (protein content 600 μg) + blood cell hemolyte according to Example 2 (100 μg of protein content), Plasma Fraction B solution (600 μg of protein content) + hemoglobin hemolyte (protein content of 100 μg) + peptidoglycan (1 μg) for each 30 μl solution of Example 3 after 15 minutes The phenol oxidase activity was measured in substantially the same manner as the phenol oxidase activity assay.

In addition, phenol oxidase activity was measured in substantially the same manner except for diluting by adding anticoagulant buffer solution to the plasma fraction B solution according to Example 2 (protein content of 200 µg). Shown in 5, each histogram is as follows: 1: Fraction B solution (600 μg), 2: Fraction B solution (600 μg) + peptidoglycan (1 μg), 3: Fraction B solution (600 μg) + Hemocytic hemolysate (100 μg), 4: fraction B solution (600 μg) + hemocytic hemolyte (100 μg) + peptidoglycan (1 μg), 5: fraction B solution (200 μg), 6: fraction B Solution (200 μg) + peptidoglycan (1 μg), 7: fraction B solution (200 μg) + hemolyte (100 μg), 8: fraction B solution (200 μg) + hemolyte (100 μg) + pepti Doglycan (1 μg)).

As a result, the plasma fraction B solution with a protein content of 600 µg showed high phenol oxidase activity against peptidoglycan (bar 2), whereas the peptide fraction glycoprotein with a protein content of 200 µg showed a peptidoglycan activity. Weak phenol oxidase activity against rods (bar 6) was observed

Therefore, in order to further enhance the activity of phenol oxidase, hemoglobin hemolyte solution (protein content: 100 μg) was added to the plasma fraction B solution (protein content of 200 μg). In the case of 200 μg, the phenol oxidase activity was all very high (rod 4, rod 8). Therefore, it can be seen that a component that activates phenol oxidase specifically to peptidoglycan exists among components of blood cell hemolytes.

Example 7: Phenoloxidase Activity of a Composition Comprising a Plasma Fraction B Solution and Blood Cell Lysate

30 μl (fraction) containing a solution prepared by diluting by adding anticoagulant buffer to the plasma fraction B prepared in Example 2 (protein content of plasma fraction B 200 μg) and the blood cell hemolyte solution prepared in Example 4 The phenol oxidase activity according to the concentration of peptidoglycan was measured for 200 μg of protein content and 100 μg of blood cell hemolysis. In order to obtain the standard curve, the phenol oxidase activity was measured in the same manner as the above method except that the peptidoglycan concentration was used at 2, 5, 10, and 20 ng / ml. Decided.

Each peptidoglycan solution was prepared at concentrations of 2 ng / ml, 20 ng / ml and 200 ng / ml of peptidoglycan in substantially the same manner as prepared in Example 3. The peptidoglycan solution was reacted for 1 hour at 30 ° C by 4-MC / 4-HP color reaction, and the absorbance was measured at 520 nm. The results are shown in FIG. 6, and the standard curves of the experimental results according to the peptidoglycan concentration are shown in FIG. 7. As shown in FIG. 7, the correlation coefficient between peptidoglycan concentration and phenol oxidase activity was 0.98, indicating that a trace amount of peptidoglycan could be detected.

Example 8: Specificity for Peptidoglycan

To determine the specificity for peptidoglycan for the plasma fraction B solution (200 μg protein content) and 30 μl blood cell hemolyte (protein content 100 μg) according to Example 2, lipopolysaccharide and beta Phenolic oxidase activity against -1,3-glucan was measured.

Substrates at concentrations of 2 ng / ml, 20 ng / ml and 200 ng / ml were prepared by suspending beta-1,3-glucan in 20 mM Tris buffer (pH 7.6). After suspending lipopolysaccharide (Sigma) in 20 mM Tris buffer (pH 7.6), substrates were prepared at 2ng / ml, 20ng / ml and 200ng / ml concentrations by sonication for 2-3 minutes. Using the lipopolysaccharide solution and the beta-1,3-glucan solution as a substrate, 4-MC / 4-HP color reaction was carried out in the same manner as in Example 3 using the phenol oxidase composition, and 20ng of peptidoglycan was used. / ml is used as a substrate and the results are shown in FIG. 8.

As a result, even if the reaction time was increased even at high concentrations of lipopolysaccharide and beta-1,3-glucan, that is, 200 ng / ml, the phenol oxidase composition of the present invention was peptidoglycan specific. It can be seen that the detection can be done by.

The composition of the present invention can quantify trace amounts of peptidoglycans in human blood, tissues, body fluids or water, and foods, and diagnose microbial contamination with peptidoglycans as cell wall components. It is expected to be useful for the prevention or treatment of food poisoning, pneumonia, as a diagnostic reagent that can diagnose pathogenic Gram-positive bacteria infection in animals or humans in advance.

Claims (18)

A composition for detecting peptidoglycan comprising a body fluid extract of an insect that exhibits phenol oxidase activity by peptidoglycan without adding calcium. The composition of claim 1, wherein the body fluid extract of the insect is a plasma solution separated from the body fluid. 2. The composition of claim 1, wherein said body fluid extract of said insect comprises a plasma solution of an insect body fluid and a blood cell lysate. The composition of claim 1, wherein the body fluid extract of the insect is derived from a honeybee moth larva. The composition according to claim 2, wherein the bodily fluid extract of the insect is a fraction showing phenol oxidase activity by peptidoglycan without adding calcium in the fraction obtained by treating the insect bodily fluid plasma with a solvent or a buffer solution. According to claim 3, wherein the extract of the body fluid of the insect is phenol by peptidoglycan without hemolysis of blood cells in the insect body fluid including plasma solution and blood cells, and without adding calcium in the fraction obtained by treatment with a solvent or a buffer solution. A composition that is a fraction that exhibits oxidase activity. According to claim 3, wherein the body fluid of the insect is obtained by treating the plasma of the honey bee moth larva with a solvent or buffer solution, hemocyt hemolysin or partially purified hemocyt hemolysin is added, and without the addition of calcium peptide A composition which is a fraction which exhibits phenol oxidase activity by doglycan. 8. The composition of any one of claims 5, 6 and 7, wherein the solvent or buffer contains sufficient chelating agent to chelate the calcium ions present in the sample and separation process. 8. A composition according to any one of claims 5, 6 and 7, wherein said fraction is a fraction by column chromatography. 10. The composition according to claim 9, wherein the column chromatography is filled with a resin based on sugar or a resin based on vinyl. A method for detecting peptidoglycan, comprising collecting a sample from a sample, adding a composition for detecting peptidoglycan according to any one of claims 1 to 4, and measuring phenol oxidase activity. The method for detecting peptidoglycan according to claim 11, wherein the body fluid of the insect is a fraction showing phenol oxidase activity by peptidoglycan without adding calcium in the fraction obtained by treating the insect body fluid plasma with a solvent or a buffer solution. . 12. The phenol oxidase according to claim 11, wherein the body fluid extract of the insect is a peptidoglycan by means of peptidoglycan without the addition of calcium in the fraction obtained by hemolyzing blood cells in the insect body fluid including plasma solution and blood cells and treating with a solvent or a buffer solution. Peptidoglycan detection method that is a fraction showing the activity. 12. The method according to claim 11, wherein the bodily fluid of the insect is added to the peptidoglycan without adding blood cell hemolyte or partially purified blood cell hemolyte to the fraction obtained by treating the insect body fluid plasma with a solvent or buffer solution. Peptidoglycan detection method which is a fraction showing phenol oxidase activity. Peptidoglycan detection kit containing a composition for detecting peptidoglycan according to any one of claims 1 to 4. 16. The peptidoglycan detection method according to claim 15, wherein the body fluid extract of the insect is a fraction showing phenol oxidase activity by peptidoglycan without adding calcium in the fraction obtained by treating the insect body fluid plasma with a solvent or a buffer solution. Kit. 16. The phenol oxidase according to claim 15, wherein the body fluid extract of the insect is a peptidoglycan by means of peptidoglycan without the addition of calcium in the fraction obtained by hemolyzing blood cells in the insect body fluid including plasma solution and blood cells and treating with a solvent or a buffer solution. Peptidoglycan detection kit that is a fraction showing the activity. 16. The method according to claim 15, wherein the bodily fluid of the insect is added to the peptidoglycan without the addition of blood cell hemolyte or partially purified blood cell hemolyte to the fraction obtained by treating the insect body fluid plasma with a solvent or buffer solution. Peptidoglycan detection kit which is a fraction showing phenol oxidase activity.