Reagents for determining peptidoglycan and beta-1,3-glucan

Abstract: A reagent comprising a fraction obtained from plasma of an insect such as silkworm larvae and capable of reacting specifically with β-1,3-glucan or peptidoglycan can be used for determining β-1,3-glucan or peptidoglycan.

Description: BACKGROUND OF THE INVENTION This invention relates to reagents for determining β-1,3-glucan or peptidoglycan and a method for determining β-1,3-glucan or peptidoglycan using the same. β-1,3-Glucan (hereinafter referred to as "β-G") is present in nature as a cell wall component of true fungi such as yeast and molds and as a major polysaccharide component in the fruit bodies of many basidiomycota. It is well known that β-G is positive to the so-called Limulus test which is a method for detecting endotoxin using a reagent purified from horseshoe crab hemocyte lysate. By applying this property of β-G, a method has been proposed for determining β-G specifically which is not influenced by endotoxin, in which a reagent purified from horseshoe crab hemocyte lysate is used [Rinsho Byori (Clinical Pathology) 33 , 639-644, 1985]. However such a method has not yet been used in practice, because of difficulties such as instability of the reagent. There has also been proposed a method for detecting endotoxins or β-G by using a reagent obtained by purifying blood cell lysate from a crustacean or an insect [PCT, International Publication No. WO 83/02123]. But the reagent used for this process reacts with both the endotoxins and β-G. Therefore, in order to use this reagent for determing specifically β-G, it is necessary to add anti-endotoxin factors purified from crayfish blood cells. On the other hand, peptidoglycan (hereinafter referred to as "PG") is a kind of glycopeptide polymer which is a constituent of cell walls of bacteria and which is contained more in cell walls of Gram-positive bacteria than in cell walls of Gram-negative bacteria. In general, PG is characterized by containing N-acetyl- or N-glycolylmuramic acid and D-amino acid. It has been known that PG has many biological activities such as causing attacks of fever; lowering liver function and kidney function; enhancing endotoxin activity; and increasing the effect of immune function (adjuvant activity). Thus studies of PG have been carried out in the fields of medical science, pharmacology, and microbiology, but a specific determination method has not been found. Some of the present inventors previously found that a body fluid obtained from silkworm does not react with endotoxin but reacts with PG or β-G so as to activate at least three kinds of enzymes, i.e. an esterase hydrolyzing N-α-benzoyl-L-arginine ethyl ester (BAEEase), prophenoloxidase activating enzyme (PPAE), and phenoloxidase (PO) [Insect Biochem, vol 16, No. 3, pp. 539-545, 1986]. But since there was a problem with specificity, this reaction was not applied to the determination of β-G or PG. As mentioned above, since suitable determination methods have not been established, the effects of β-G or PG on human bodies is not yet fully known. But recently, there has been a question whether β-G is one cause of shock when hemodialysis films of cellulose derivatives are used. There has also been a suggestion that β-G is present in the body fluid of patients suffering from mycotic infection. Therefore, appropriate determination of β-G or PG is becoming more and more important in the fields of medical science, such as pharmacology and microbiology. SUMMARY OF THE INVENTION The present inventors have succeeded in removing a component, which specifically binds to β-G or PG, from insect plasma, and found that it is possible to determine β-G or PG by reacting the resultant plasma fractions with a sample to be tested containing β-G or PG and measuring expressed enzymatic activities of for example BAEEase, PPAE, PO or measuring the time required for these enzymatic activities to be expressed. Thus, the present invention has been accomplished. In one aspect this invention provides a reagent for determining β-1,3-glucan. The reagent comprises a fraction obtained from the plasma of an insect which is capable of reacting specifically with β-1,3-glucan. This invention also provides a process for determining β-1,3-glucan which comprises reacting a sample with a reagent comprising a fraction obtained from the plasma of an insect which is capable of reacting specifically with β-1,3-glucan, and measuring the enzymatic activity generated. The enzymatic activity generated can be quantified by measuring the time required for an enzymatic activity to be expressed. In another aspect this invention provides a reagent for determining peptidoglycan. The reagent comprises a fraction obtained from the plasma of an insect which is capable of reacting specifically with peptidoglycan. This invention further provides a process for determining peptidoglycan which comprises reacting a sample with a reagent comprising a fraction obtained from the plasma of an insect which is capable of reacting specifically with peptidoglycan, and measuring the enzymatic activity generated. The enzymatic activity generated can be quantified by measuring the time required for an enzymatic activity to be expressed. This invention still further provides a process for producing the mentioned reagent as defined in present claims 8 and 9. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing change of absorbance at 520 nm depending on reaction time when various samples obtained in Example 3 are reacted with substrate solutions. Fig. 2 shows a calibration curve obtained in Example 4. Fig. 3 shows a calibration curve obtained in Example 5. Fig. 4 is a graph showing change of absorbance at 520 nm depending on reaction time when various samples obtained in Example 6 are reacted with substrate solutions. Fig. 5 shows a calibration curve obtained in Example 7. Fig. 6 shows a calibration curve obtained in Example 8. DESCRIPTION OF THE PREFERRED EMBODIMENTS There is no limitation to the insects which can give body fluids usable in this invention, but larger ones with known rearing methods therefor are preferable. Examples of such insects are Manduca serta, Gelleria melonella, Hyalphoma ceropia, and Bombyx mori (silk-moth), belonging to the Lepidoptera order; Sarcophaga peregrina, and musca, belonging to the Diptera order; Locusta, and migratoria Teleogryllus, belonging to the Orthoptera order; and Cerambyx belonging to the Coleoptera order. Hemolymph which is the body fluid obtained from the body cavity is most easily obtained and thus usually used. The body fluid of insects can be collected, for example, by the method disclosed by M. Ashida [Insect Biochem., 11 , 57-65 (1981)]. That is, insects, for example, silkworm larvae, are anesthetized on ice, and physiological saline containing sugar including as impurities cane sugar factor (polymeric substances included in sugar cane and comprising for example glucose, and amino acids), or containing cane sugar factor itself is injected into the heamocoel (body cavity) of the silkworm larvae. Then the larvae body is tied with a fine thread so as to prevent leakage of the injected saline. After being allowed to stand at room temperature for about 20 minutes, a leg is cut and the body fluid is collected. The collected fluid is centrifuged to remove blood cells, then dialysed and insect plasma is obtained. Alternatively, the body fluid of insects (comprising the above mentioned reagents) can be collected by a process newly devised by some of the present inventors. That is, to a solution isotonic to an insect body fluid to be collected and containing serine protease (hereinafter referred to as "SP") inhibitor (hereinafter referred to as "SPI") which is a substance irreversely inhibiting SP, an insect body fluid is added and then excess SPI is removed. The insect body fluid without free SPI holds reactivity with β-G or PG. Further, the body fluid of insects (comprising the above mentioned reagents) can also be collected by a process comprising injecting an isotonic solution for the insect to be used which contains a substance irreversely inhibiting serine protease to the insect, cutting a part of the body, collecting a body fluid leaking out and removing an excess amount of the inhibiting substance. The insect body fluid without free SPI holds reactivity with β-G or PG. As the SPI, there is no particular limitation thereto so long as it can irreversely inhibit SP and it can be separated from the fraction of an insect body fluid which react with β-G or PG. Among the SPIs, preferable ones from the viewpoint or handling are those having relatively small molecular weights, e.g., 2000 or less, and being able to be removed by dialysis. Preferable examples of the SPI are (p-amizinophenyl)methanesulfonyl fluoride (p-APMSF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosporic acid (DFP), p-nitrophenyl-p'-guanidinobenzoic acid (NPGB), and dihydroxychloromethylcoumalin. It is sufficient to add to the above-mentioned isotonic solution the SPI in an amount at least necessary to inactivate SP which is to be inactivated during the collection of body fluid. Preferable amount of SPI in the isotonic solution is 0.1 to 10 mM, more preferably 0.5 to 5 mM. This method can be carried out, for example, as follows: To a solution containing SPI and isotonic to an insect body fluid to be collected, a body fluid leaked out of an injured portion of an insect is directly dropped. Alternatively, after injecting a solution containing SPI and isotonic to an insect body fluid to be collect into an insect, a body fluid leaked out of an injured portion of the insect is collected. Then, the collected fluid is centrifuged to remove blood cells, and the like. Further, excess SPI present in the resulting body fluid-containing solution is removed by one or more most suitable methods generally used in the field of biochemistry for separation and purificattion depending on the nature of SPI used, e.g. a dialysis method, a gel filtration method, an ion exchange method, high performance liquid chromatography. As a result, the desired solution containing insect plasma is obtained. Further, the SPI removal operation can be conducted after subjecting the centrifuged solution to concentration and purification to some extent by a conventional method such as fractionation with ammonium sulfate. The thus obtained plasma contains a substance which does not react with endotoxin but specifically binds to and reacts with β-G to express an enzymatic activity (or to induce expression of enzymatic activity) and another substance which does not react with endotoxin but specifically binds to and reacts with PG to express an enzymatic activity (or to induce expression of enzymatic activity). When the substance which reacts with β-G to express the enzymatic activity (or to induce expression of enzymatic activity) is removed, a fraction which specifically reacts with PG is obtained. In contrast, when the substance(s) which reacts with PG to express the enzymatic activity (or to induce expression of enzymatic activity) is removed, the fraction which specifically reacts with β-G is obtained. As the method for removing substance(s) which reacts with β-G or PG to express the enzymatic activity (or to induce expression of enzymatic activity), there can be used any separation and purification methods conventionally used in the field of biochemistry such as gel filtration, electrophoresis, high performance liquid chromatography, affinity chromatography. Among these methods, affinity chromatography using a carrier bonded to β-G or PG is particularly preferable due to its remarkably easy and effective operation. Such affinity chromatography is explained below in detail. As the carrier for bonding β-G or PG, there can be used cellulose, agarose, dextran, or polyacrylamide, porous glass. These carriers are usually used as carriers in affinity chromatography. Among them, agarose is particularly preferable. Commercially available carriers are agarose type carriers such as Sepharose (Pharmacia Fine Chemicals), Biogel A (Bio-Rad Laboratries); dextran type carriers such as Sephadex (Pharmacia Fine Chemicals), P-L DEX (P-L Biochemicals); polyacrylamide type carriers such as Sephacryl (Pharmacia Fine Chemicals), and Biogel P (Bio-Rad Laboratories). In order to bond β-G or PG to such a carrier, it is necessary to activate the carrier. The carrier can be activated, for example, by activation with CNBr in the case of bonding PG to an agarose type carrier. It is also possible to use epoxy activated (epichlorohydrin activated) agarose as activated agarose for both β-G and PG. As the β-G bonded to a carrier, there can be used a natural one obtained from various bacteria (e.g. Alcaligenes genus, and Agrobacterium genus), yeasts (e.g. Saccharomyces genus) , and mushrooms (e.g. Cortinellus shiitake, Schizophyrum commune, Coriolus versicolor); or storage polysaccharides of algae, e.g. brown algae, Laminaria genus, Euglena, diatomeae, In the case of using a material such as curdlan which can be processed to an insoluble carrier (e.g. beads) by itself in place of using the carrier bonded to β-G mentioned above, it is possible to use such a material as a carrier without bonding to other carriers to carry out the affinity chromatography. The curdlan beads used for such a purpose can be prepared, for example, by processes disclosed in U.S. Patent Nos. 4,143,201 and 4,493,894. On the other hand, as the PG bonded to a carrier, there can be used a natural one obtained from cell walls of various bacteria (e.g. Micrococcus genus, Streptococcus genus, Mycobacterium genus, Bacillus genus, and Staphylococcus genus) ; or a decomposed (digested) PG which is decomposed (digested) to some extent by a suitable enzyme such as egg white lysozyme. It is also possible to use PG itself as a carrier in place of the carrier bonded to PG for carrying out affinity chromatography. In order to carry out affinity chromatography more effectively, it is desirable to remove the influence of cations such as Ca²⁺, and Mg²⁺, present in the body fluid by adding a chelating agent to plasma and eluate for affinity chromatography. As the chelating agent, examples are sodium ethylenediaminetetraacetate (EDTA), and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetate sodium salt (EGTA). The chelating agent is preferably used in a concentration of 1 to 10 mM in plasma or eluate. It is also possible to use the above-mentioned SPI in place of the chelating agent. Preferable amount of SPI in plasma or eluate is 0.1 to 10 mM, more preferably 0.5 to 5 mM. The procedure of affinity chromatography can be carried out according to the conventional procedure. By treating the insect plasma by the affinity chromatography using the carrier bonded to β-G or PG mentioned above, the fraction which specifically reacts with PG or β-G can easily be obtained. By using the thus obtained fraction, the determination of β-G or PG can be carried out as follows. A sample containing β-G or PG is well mixed with a reagent comprising a fraction which specifically reacts with β-G or PG (hereinafter referred to as "β-G reagent" or "PG reagent") to prepare a reaction solution. After a certain period of time, an enzymatic activity, e.g. activity of BAEEase, PPAE, PO, in the reaction solution is measured by a conventional method and compared with calibration curves previously obtained by using β-G or PG standard solutions with known concentrations to determine the amount of β-G or PG (hereinafter this method is referred to as "end method"). Alternatively, it is possible to apply a phenomenon that a time required for the activation of PO depends on the concentration of β-G or PG in the sample. That is, after mixing the β-G reagent or PG reagent with a sample in the presence of the substrate of PO, a time required for reaching a certain value of the amount of reaction product generated by PO is measured. This method was devised by the present inventors (hereinafter referred to as "time method"). Prior to the determination of β-G or PG by using either the end method or the time method, it is necessary to add divalent metal ions such as Ca²⁺, Mg²⁺, which are removed from the reagent solution during the preparation of the fraction which specifically reacts with β-G or PG, to the reaction solution. Final concentration of these divalent metal ions in the reaction solution is preferably about 4 to 10 mM. In this invention, it is possible to use additives or reagents which are necessary for conventional determination of enzymatic activities, for example, substrates, buffers, coupling enzymes, coenzymes, as well as color developing agents, enzyme activity imparting agents, stabilizers for enzymes and dyes, and surface active agents, depending on enzymes of which activities are to be measured. These additives or reagents may be dissolved in the β-G or PG reagent previously, or in the case of end method, it is possible to react a part of the reaction solution not containing these additives or reagents which is taken out as a sample with a reagent for measuring enzymatic activity. In the determination of β-G or PG by using these methods, the reaction temperature is not particularly limited so long as the reaction between a sample containing β-G or PG and the β-G or PG reagent can proceed, and is preferably 20° to 40°C. The reaction pH changes depending on enzymes to be measured. In order to maintain the reaction pH, a buffer is usually used so long as it does not influence the reaction. Examples of the buffers are phosphate buffer, borate buffer, acetate buffer, and Tris-HCl buffer, Good's buffer. As β-G measurable by the methods mentioned above, are so-called β-G such as zymosan, curdlan, and pachyman ; and glucose polymers having β-1,3-linkage and derivatives thereof such as Sclerotan, lentinan, schizophyllan, coriolan, laminaran, lichenan. This invention is illustrated by way of the following Examples, but not limited thereto. Reference Example 1 Preparation of Silkworm Plasma Silkworm plasma was prepared according to the Ashida method (Insect Biochem., 11 , 57-65, 1981) as follows. Silkworm larvae of fifth instar were anesthetized on ice for 10 min. An amount of half the body weight of physiological saline containing 20 mM of cane sugar or 6 µg/ml of cane sugar factor purified from sugar cane was injected into haemocoel through a hypodermic needle inserted at the junction of the fifth and sixth abdominal segments. After the injection, the larvae body was tied with a fine thread at the posterior part of the fifth abdominal segment to prevent leakage of the injected saline. After allowing to stand at room temperature for 20 minutes, a leg was cut for bleeding at the third abdominal segment. The collected hemolymph was centrifuged at 1500 g for 5 minutes at a low temperature (about 4°C) to remove hemocytes. The supernatant in an amount of about 100 ml was subjected to dialysis at a low temperature for 2 days against 3 liters of 0.01 M-tris-malate buffer (pH 6.5 containing 0.15 M KCl) to give the desired silkworm plasma. Reference Example 2 Preparation of Peptidoglycan Cell walls of Micrococcus luteus ATCC 4698 was suspended in 150 ml of cold water and 0.6 g/ml of glass beads of 0.1 mm in diameter were added thereto. Ultrasonic treatment was carried out at 0°C to break the cell walls. After removing the glass beads, centrifugation was conducted at 2200 g for 10 minutes to remove a precipitation, followed by centrifugation of the supernatant at 20000 g for 45 minutes. The obtained precipitate was suspended in 150 ml of a 1 M NaCl solution, followed by collection of fractions by centrifugation at 2200 g to 20000 g to give a crude cell wall preparation. The obtained crude cell wall preparation was suspended in 80 ml of water, heated at 100°C for 20 minutes, cooled, added with 140 ml of 2M acetic acid-sodium acetate buffer (pH 5.9) and 10 mg of ribonuclease and incubated at 37°C for 3 hours. Then, centrifugation was conducted at 20000 g for 1 hour and the obtained precipitate was suspended in a 50 mM Tris-HCl buffer [pH 7.5, containing 20 mM MgCl₂, 1 mM CaCl₂ and 7 mg of DNase I (mfd. by Sigma Chem. Co., Ltd.) and incubated at 37°C for 3 hours. Subsequently, centrifugation was conducted at 20000 g for 1 hour, and the obtained precipitate was suspended in 100 ml of a 0.4% sodium dodecylsulfate solution and allowed to stand at room temperature for 1 hour. Then the precipitate was washed with distilled water 6 times, followed by freeze-drying to give a purified cell wall preparation. The obtained pure cell wall preparation was suspended in 0.1N HCl and allowed to stand at 60°C for 24 hours. Then, centrifugation was conducted at 20000 g for 1 hour, and the obtained precipitate was washed with distilled water and freeze-dried to give peptidoglycan. Reference Example 3 Preparation of Curdlan Beads Column To 9 g of curdlan powder (mfd. by Wako Pure Chemical Industries, Ltd.), 270 ml of pure water was added and stirred to give a slurry. When 30 ml of 1N NaOH was add to the slurry, the curdlan was dissolved to give a sodium hydroxide aqueous solution of curdlan. In a 8-liter beaker, 1200 ml of toluene and 6 g of surface active agent Emalex HC-80 (a trade name, polyoxyethylene cured castor oil derivative, mfd. by Nihon Emulsion Co., Ltd.) were placed and added thereto dropwise the sodium hydroxide aqueous solution of curdlan at room temperature with stirring at a speed of 800 r.p.m. using a screw-type stirrer. The thus obtained curdlan dispersion was added to a liquid comprising 2000 ml of toluene and 1000 ml of acetic acid with stirring at a rate of 800 r.p.m. After stirring for 1 hour, the resulting curdlan dispersion was allowed to stand about 8 hours to precipitate produced beads. The solvent was removed by decantation. The obtained precipitate was washed with 8 liters of pure water 5 times to make the pH neutral, to remove the organic solvent completely to give 240 ml of curdlan beads. The beads were classified and those of particle size of 50 to 100 µm (average particle size: about 80 µm) were equilibrated with 0.01 M tris-malate buffer (pH 6.5, containing 0.15 M KCl and 1 mM EDTA; hereinafter referred to as "TMB") and packed in a column (1.3 cm in diameter and 2.3 cm long) to give a curdlan beads column. Example 1 Silkworm larvae (Bombyx mori) of fifth instar were anesthetized on ice for 10 min. A leg was cut at the third abdominal segment and hemolymph leaked out was dropped into 10 ml of a 0.9% NaCl solution containing 2 mM of p-APMSF and collected. About 15 ml of hemolymph was obtained from 25 silkworm larvae. From this, about 25 ml of a hemolymph diluted solution was obtained. The hemolymph diluted solution was centrifuged at 1500 g for 5 minutes to remove hemocytes. To 25 ml of the resulting supernatant, 37.5 ml of saturated ammonium sulfate solution was added to give a 60% saturated ammonium sulfate suspension, which was centrifuged at 10000 g for 20 minutes to give a precipitate. This precipitate was dissolved in 27 ml of 0.01 M Tris-malate buffer (pH 6.5, containing 0.15 M NaCl), added with 0.1 ml of a 200 mM p-APMSF solution and dialyzed 4 times against 1 liter of 0.01 M Tris-malate buffer (pH 6.5, containing 0.15 M NaCl). The dialyzed solution was centrifuged at 10000 g for 20 minutes at low temperatures to give the desired silkworm plasma. The same results were also obtained when NPGB was used in place of p-APMSF. Example 2 Silkworm larvae (Bombyx mori) of fifth instar were anesthetized on ice for 10 minutes. A 0.9% NaCl solution containing 2 mM of p-APMSF was injected into haemocoel at the junction of the fifth and sixth abdominal segments. After the injection, the larvae body was tied with a fine thread at the posterior part of the fifth abdominal segment to prevent leakage of the injected saline. Then, a leg was cut at the third abdominal segment to collect a body fluid (hemolymph) leaked out. The collected hemolymph was centrifuged at 1500 g for 5 minutes at low temperatures to remove hemocytes. To 10 ml of the obtained supernatant, 0.05 ml of a 200 mM p-APMSF solution was added and centrifuged at 10000 g for 20 minutes at low temperatures. The supernatant was passed through a Sephadex® G-25 column (2.5 cm in diameter and 25 cm long) equilibrated with 0.01 M Tris-malate buffer (pH 6.5, containing 0.15 M NaCl). After collecting peaks of protein eluted into void volume of the column, the desired silkworm plasma was obtained. Example 3 (1) Preparation of Sepharose 4B Column Coupled with Peptidoglycan The purified peptidoglycan obtained in Reference Example 2 in an amount of 153 mg was suspended in 153 ml of 80 mM ammonium acetate and 1.5 mg of egg white lysozyme was added thereto. After heating at 45°C for 4 minutes with stirring, digestion was carried out at 37°C for 2 hours. Then, the reaction solution was filtered with a millipore filter (HAWPO 4700) and the filtrate was freeze-dried. The freeze-dried product was dissolved in 6 ml of distilled water. The resulting solution in an amount of 5.5 ml was subjected to gel filtration using a Sephadex® G-50 SF column (eluate: a 50 mM ammonium carbonate solution; column 2.5 cm in diameter and 90 cm long; elution rate; 15 ml/hr). Central portions of active fractions of reducing sugar produced by the digestion were collected and freeze-dried. The freeze-dried product was dissolved in 6.3 ml of 0.1 M sodium carbonate buffer (pH 10) to give a peptidoglycan solution. The peptidoglycan solution was reacted with 2.7 g of CNBr activated Sepharose 4B® (mfd. by Pharmacia Fine Chemicals) according to a conventional method to give Sepharose 4B coupled with peptidoglycan. The thus obtained Sepharose 4B coupled with peptidoglycan was packed in a column (0.6 cm in diameter and 1.7 cm long) and equilibrated with TMB to give a PG column. (2) Preparation of β-G Reagent. To the silkworm plasma obtained in Reference Example 1, a 100 mM EDTA solution (pH 6.5) was added so as to make the final concentration of EDTA 1 mM. 15 Ml of the resulting solution was treated with the PG column obtained in above (1) (eluate: TMB; elution rate: 6 ml/hr). The eluate in an amount of 12 ml obtained immediately after pouring the sample was collected to give a β-G reagent. (3) Measurement of Activation of Zymogens (inactive forms of BAEEase, PPAE and PO) by Zymosan (β-1,3-glucan) or PG in β-G Reagent or Silkworm Plasma. [Measuring procedure] To 200 µl of the silkworm plasma obtained in Reference Example 1 or the β-G reagent obtained in (2), 20 µl of a 80 mM CaCl₂ solution was added and further 1 mg/ml of Zymosan solution or 1 mg/ml of PG solution was added thereto with well mixing to react at 25°C. The reaction solution was sampled at intervals to measure the degree of activation of PO or BAEEase activity value. 1 ○ Measurement of PO Activity To 1 ml of a substrate solution (a 0.1 M phosphate buffer containing 4 mM 4-methylcatechol and 8 mM 4-hydroxyproline ethyl ester, pH 6.0), 10 µl of a sample (the above-mentioned reaction solution) was added and reacted at 30°C for 10 minutes. Then, absorbance of the produced quinone dye stuff at 520 nm was measured to obtain the degree of activity of PO. Fig. 1 shows changes of absorbance at 520 nm depending on the reaction time when various samples were reacted with the substrate solution. In Fig. 1, the changes of absorbance are shown by the curve ―― in the case of reacting zymosan with the β-G reagent, ―▴― in the case of reacting PG with the β-G reagent, ―o― in the case of reacting zymosan with the silkworm plasma, and ―▵― in the case of reacting PG with the silkworm plasma. 2 ○ Measurement of BAEEase Activity To 1 ml of a substrate solution [containing 2 mM N-α-benzoyl-L-arginine ethyl ester, 1 mM NAD (nicotinamide adenine dinucleotide), 0.1 mg/ml alcohol dehydrogenase, 0.25 M tris(hydroxymethyl)aminomethane, and 0.2 M semicarbazide, pH 8.5] previously heated at 25°C, 30 µl of a sample (the above-mentioned reaction solution) was added and well mixed to react at 25°C. An increase of absorbances at 340 nm of NADH (nicotinamide adenine dinucleotide, reduced form) produced by the reaction was measured. One unit (U) of BAEEase is defined as an amount which can produce 1 nmole of ethanol in one minute under the above-mentioned conditions. The results are shown in Table 1. Reagent Sample solution BAEEase activity (U/ml) Silkworm plasma Zymosan solution 93.7 PG solution 91.0 β-G reagent Zymosan solution 84.0 PG solution 0.0 As is clear from Table 1, the enzymes in the silkworm plasma are activated by zymosan and PG, but the enzymes in the β-G reagent are only activated by zymosan and not activated by PG. The same results as mentioned above were obtained when the β-G reagents prepared by using the silkworm plasma obtained in Examples 1 and 2 were used. Example 4 Formation of Calibration Curve for β-1,3-glucan using Curdlan [Measuring procedure] To 2 ml of the β-G reagent obtained in Example 3(2), 200 µl of a 80 mM CaCl₂ solution was added and mixed well. To 10 µl of the resulting mixture, 10 µl of a curdlan solution with a predetermined concentration was added and incubated at 30°C for 60 minutes. Then, 1 ml of the same substrate solution for measuring PO activity as used in Example 3(3) was added and reacted at 30°C for 10 minutes, followed by measurement of absorbance at 520 nm (measured value: Es). Blank value (E Bℓ ) was obtained in the same manner as mentioned above by using purified water in place of the curdlan solution. [Results] Fig. 2 shows the relationship between the curdlan concentration and (E S -E Bℓ ) values by using logarithmic scales at both ordinate and abscissa axes. As is clear from Fig. 2, good linearity is obtained. Example 5 Formation of Calibration Curve for β-1,3-glucan using Curdlan [Measuring procedure] To 2 ml of the β-G reagent obtained in Example 3(2), 200 µl of a 80 mM CaCl₂ solution was added and mixed well. To 70 µl of the resulting mixture, 70 µl of 0.1 M phosphate buffer (containing 20 mM L-dopa, pH 6.0) and 70 µl of a curdlan solution with a predetermined concentration were added and mixed well. Then, by using a Toxinometer ET-201 (mfd. by Wako Pure Chemical Industries, Ltd.), a time (Δt) required for reducing the transmitted light amount by 5% at 30°C was measured. [Results] Fig. 3 shows the relationship between the Δt and the curdlan concentration by using logarithmic scales at both ordinate and abscissa axes. As is clear from Fig. 3, good linearity is obtained. As mentioned above, by using the reagent containing the component which specifically reacts with β-G, β-G can be determined. By using such a determination process, the detection of contamination with true fungi, examinations of blood dialysis films of cellulose derivatives, examinations of reactive substances reactive to Limulus test other than endotoxin, etc., can be carried out with easy and precisely. Example 6 (1) Preparation of PG Reagent To 5 ml of the silkworm plasma obtained in Reference Example 1, 20 ml of TMB was added and mixed well to give a sample, which was treated with the curdlan beans column obtained in Reference Example 3. The protein fraction passed through in an amount of about 25 ml was dropped in 225 ml of a saturated ammonium sulfate solution, followed by stirring overnight. A precipitate was collected by centrifugation (at 16000 g for 20 minutes) and then dissolved in 4 ml of TMB, followed by dialysis against 500 ml of TMB. The resulting solution was centrifuged again (at 16000 g for 20 minutes) and the supernatant was made 5 ml as a total with TMB to give a PG reagent. (2) Measurement of Activation of Zymogens (inactive forms of BAEEase, PPAE and PO) by Zymosan (β-1,3-glucan) or PG in PG Reagent or Silkworm Plasma. [Measuring procedure] To 200 µl of the silkworm plasma obtained in Reference Example 1 or the PG reagent obtained in above (1), 20 µl of a 80 mM CaCl₂ solution was added and further 20 µl of a 1 mg/ml zymosan solution or a 1 mg/ml PG solution (prepared by using that obtained in Reference Example 2) was added and mixed well to react at 25°C. The reaction solution was sampled at intervals to measure the degree of activation of PO or BAEEase activity value. 1 ○ Measurement of PO Activity To 1 ml of a substrate solution (a 0.1 M phosphate buffer containing 4 mM 4-methylcatechol and 8 mM 4-hydroxyproline ethyl ester, pH 6.0), 10 µl of a sample (the above-mentioned reaction solution) was added and reacted at 30°C for 10 minutes. Then, absorbance of the produced quinone dye stuff at 520 nm was measured to obtain the degree of activity of PO. Fig. 4 shows changes of absorbance at 520 nm depending on the reaction time when various samples were reacted with the substrate solution. In Fig. 4, the changes of absorbance are shown by the curve ―― in the case of reacting zymosan with the PG reagent, ―▴― in the case of reacting PG with the PG reagent, ―o― in the case of reacting zymosan with the silkworm plasma, and ―▵― in the case of reacting PG with the silkworm plasma. 2 ○ Measurement of BAEEase Activity. To 1 ml of a substrate solution [containing 2 mM N-α-benzoyl-L-arginine ethyl ester, 1 mM NAD (nicotinamide adenine dinucleotide), 0.1 mg/ml alcohol dehydrogenase, 0.25 M tris(hydroxymethyl)aminomethane and 0.2 M semicarbazide, pH 8.5] previously heated at 25°C, 30 µl of a sample (the above-mentioned reaction solution) was added and well mixed to react at 25°C. An increase of absorbances at 340 nm of NADH (nicotinamide adenine dinucleotide, reduced form) produced by the reaction was measured. One unit (U) of BAEEase is defined as an amount which can produce 1 nmole of ethanol in one minute under the above-mentioned conditions. The results are shown in Table 2. Reagent Sample solution BAEEase activity (U/ml) Silkworm plasma Zymosan solution 93.7 PG solution 91.0 PG reagent Zymosan solution 0.0 PG solution 89.3 As is clear from Table 2, the enzymes in the silkworm plasma are activated by zymosan and PG, but the enzymes in the PG reagent are only activated by PG and not activated by zymosan. The same results as mentioned above were obtained when the PG reagents prepared by using the silkworm plasma obtained in Examples 1 and 2 were used. Example 7 Formation of Caliburation Curve for PG [Measuring procedure] To 2 ml of the PG reagent obtained in Example 6(1), 200 µl of a 80 mM CaCl₂ solution and mixed well. To 10 µl of the resulting mixture, 10 µl of the PG solution having a predetermined concentration was added and incubated at 30°C for 60 minutes. Then, 1 ml of the same substrate solution for measuring PO activity as used in Example 6(2) was added thereto and further reacted at 30°C for 10 minutes, followed by measurement of absorbance at 520 nm (measured value: E S ) Blank value (E Bℓ ) was obtained in the same manner as mentioned above by using purified water in place of the PG solution. [Results] Fig. 5 shows the relationship between the PG concentration and (E S -E Bℓ ) values by using logarithmic scales at both ordinate and abscissa axes. As is clear from Fig. 5, good linearity is obtained. Example 8 Formation of Calibration Curve for PG [Measuring procedure] To 2 ml of the PG reagent obtained in Example 6(1), 200 µl of a 80 mM CaCl₂ solution was added and mixed well. To 70 µl of the resulting mixture, 70 µl of 0.1 M phosphate buffer (containing 20 mM L-dopa, pH 6.0) and 70 µl of PG solution with a predetermined concentration were added and mixed well. Then, by using Toxinometer ET-201 (mfd. by Wako Pure Chemical Industries, Ltd.), a time (Δt) required for reducing the transmitted light amount by 5% at 30°C was measured. [Results] Fig. 6 shows the relationship between the Δt and the PG concentration by using logarithmic scales at both ordinate and abscissa axes. As is clear from Fig. 6, good linearity is obtained. As mentioned above, by using the reagent containing the component which specifically reacts with PG, PG can be determined. By using such a determination process, PG can be determined easily and precisely.

Claims: A reagent for the determination of either β-1,3-glucan or peptidoglycan comprising a fraction obtainable from plasma of an insect from which has been removed the substance which binds to and reacts with the other compound to leave a fraction capable of reacting specifically with the compound for which determination is desired. A reagent according to claim 1, wherein the insect is one member selected from orders of Lepidoptera, Diptera, Orthoptera, and Coleoptera. A reagent according to claim 1, wherein the insect is silkworm larvae. A reagent for the determination of peptidoglycan according to any preceding claim, wherein the substance which binds to and reacts with β-1,3-glucan has been removed by affinity chromatography using a carrier bonded to β-1,3-glucan A reagent for the determination of β-1,3-glucan according to any one of preceding claims 1-3, wherein the substance which binds to and reacts with peptidoglycan has been removed by affinity chromatography using a carrier bonded to peptidoglycan. A process for using the reagent of any one of preceding claims 1-4 for the determination of peptidoglycan, said reagent comprising a fraction capable of reacting specifically with peptidoglycan, wherein a sample comprising peptidoglycan is reacted with said reagent and either a measurement is taken of enzymatic activity generated or a measurement is taken of a time required for an enzymatic activity to be expressed wherein the enzyme used in the measurement is one which is activated by the presence of peptidoglycan. A process for using the reagent of any one of preceding claims 1-3 and 5 for the determination of β-1,3-glucan, said reagent comprising a fraction capable of reacting specifically with β-1,3-glucan, wherein a sample comprising β-1,3-glucan is reacted with said reagent and either a measurement is taken of enzymatic activity generated or a measurement is taken of a time required for an enzymatic activity to be expressed, wherein the enzyme used in the measurement is one which is activated by the presence of β-1,3-glucan. A process for producing the reagent according to any one of claims 1-5 which comprises the steps of adding an insect body fluid to a solution which is isotonic to the body fluid of the insect and contains a substance irreversely inhibiting serine protease, and removing an excess amount of the inhibiting substance, removing the substance which binds to and reacts with the other compound to leave a fraction capable of reacting specifically with the compound for which determination is desired. A process for producing the reagent according to any one of claims 1-5 which comprises the steps of injecting into the insect an isotonic solution for the insect to be used which contains a substance irreversely inhibiting serine protease, cutting a part of the body, collecting a body fluid leaking out and removing an excess amount of the inhibiting substance, removing the substance which binds to and reacts with the other compound to leave a fraction capable of reacting specifically with the compound for which determination is desired.

Inventors: ASHIDA MASAAKI, MATSUURA SHUJI, SAKATA YOSHITSUGU, TSUCHIYA MASAKAZU, ASHIDA, MASAAKI, MATSUURA, SHUJI, SAKATA, YOSHITSUGU, TSUCHIYA, MASAKAZU
Applicants: WAKO PURE CHEM IND LTD, WAKO PURE CHEMICAL INDUSTRIES LTD
Application year: 1987
Main classification: C12Q1
IPC codes: C12Q1