JPH01142466A - Sampling of insect bodily liquor - Google Patents

Abstract

PURPOSE:To sample an insect bodily liquor handily and efficiently used for assay of beta-1,3-glucan GL or peptide glucan PG, by making a substance for irreversibly blocking serine protease SP coexist in a solution equivalent to the insect liquor used in the sampling of a liquor of an insect. CONSTITUTION:After the 5th instar of a silkworm larva is placed on ice for 10min, a leg of the third abdomen section is cut off and hemolymph exuding is dripped into 10ml of a 0.9% NaCl solution containing 2mM of p-APMSF to sample. About 15ml of hemolymph is obtained from 25 silkworm larvas and about 25ml of a hemolymph diluted liquid is obtained. The hemolymph thus obtained is treated centrifugally at a rate of 1,500Xg at a low temperature to remove blood cells. 37.5ml of an saturated ammonium sulfate is added to 25ml of a supernatant thus obtained to make a 60% saturated ammonium sulfate suspension, which is treated centrifugally at a rate of 10,000Xg for 20min to sample a sediment. This sediment is dissolved in 27ml of a 0.01M tris-malic acid buffer.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an improved method for collecting insect body fluids. More specifically, the present invention relates to β-1,3-glucan (hereinafter abbreviated as GL) or peptidoglycan (
Hereinafter, it will be abbreviated as PG. This relates to an improved method for collecting insect body fluids used for localization of insect body fluids, etc., and is abbreviated as phenol oxidase (hereinafter abbreviated as I) O contained in insect body fluids. This invention relates to a method for easily and efficiently collecting body fluids by suppressing the activation of a cascade reaction (hereinafter, this reaction will be abbreviated as PO cascade reaction) that ultimately activates PO.

[Background of the Invention] In the body fluids of insects, there are molecules activated by GL or PG.
It is known that a cascade reaction of PO exists (
Ashida, M., Shizaki, Y.
Iwahara, H., Bioches+, Bi
ophys, Res, Comn+un,, Lu.

562-568.1983). Using this reaction, G
It is possible to quantify L or PG.

At present, the only method for collecting insect body fluids that can be used for this purpose is the method by Toda (Ashida et al.).

M,, In5ect Blochem,, ■, 57
-65.1981) has been reported. This method involves placing the insect on ice to stop its movement, and then adding sucrose containing cane factor (a polymeric substance consisting of glucose, amino acids, etc. contained in sugarcane) as an impurity, or physiological saline containing cane factor itself. The insect is injected into the body cavity, tied up with a thin thread to prevent the injected liquid from leaking, and left at room temperature for about 20 minutes, then the leg is cut and the exuding body fluid is collected.

As is clear from the above description, this method requires quite complicated operations. Furthermore, depending on the individual, the PO cascade reaction is activated, and the collected body fluid becomes GL or PG.
In many cases, the method cannot be used for quantifying PO, and furthermore, it requires skill to determine whether or not the PO cascade reaction in the collected body fluid is activated.

On the other hand, GL is mainly present in fungi such as molds and yeast, and PG forms the basic structure of the bacterial cell surface, but it is contained more in the cell surface of Durum-positive bacteria than Durum-negative bacteria. It is known that In addition, ribopolysaccharide, known as endotoxin (hereinafter abbreviated as ET), is also a component that constitutes the surface layer of bacterial cells, and it is known that it mainly exists in the cell surface layer of Durham-negative bacteria. Are known.

Therefore, GL, PG, and ET in biological body fluids or drugs, etc.
It is considered that quantifying each content of is particularly useful in the medical and pharmaceutical fields. That is, in the medical field, when diagnosing microbial infections, GL is used as an indicator of fungal infections, PG as an indicator of gram-positive bacterial infections, and ET as an indicator of gram-positive bacterial infections. Ba,
It may be possible to more precisely determine the type of infecting bacteria. Also, in the field of pharmaceutical science, for the same reason,
It is thought that it will be possible to detect what kind of bacteria a drug is contaminated with.

In addition, in the medical and pharmaceutical fields, most of the Limulus tests currently available on the market are not only used for ET but also for GL. Because this measurement method reacts, the amount of E present in the measurement sample is
It is difficult to determine whether it is T, G, or a mixture of both. Therefore, if GL is also measured using a GL measuring reagent prepared from insect body fluid, it becomes possible to confirm whether only ET is reacting to the Limulus test.

For the reasons mentioned above, it has been desired to develop a method that can more easily and efficiently collect insect body fluids that can be used for quantifying GL or PG.

[Object of the invention] The present invention was made in view of the above-mentioned situation, and
The purpose of the present invention is to provide a method for simply and efficiently collecting insect body fluids that can be used for quantifying L or PG.

[Structure of the Invention] In order to achieve the above object, the present invention has the following structure.

``A method for collecting insect body fluids, which is characterized by coexisting a substance that irreversibly inhibits serine protease in a solution that is isotonic with the insect body fluids used when collecting insect body fluids.

That is, the present inventors have conducted intensive research to develop a method for easily and efficiently collecting insect body fluids that can be used for quantifying GL or PG. A substance that irreversibly inhibits serine protease (hereinafter abbreviated as SP), that is, an SP inhibitor (hereinafter abbreviated as spr), is coexisting in a solution that is isotonic with the insect body fluid used for insect production. Collect body fluids and remove excess S
The present invention was completed based on the discovery that insect body fluids retaining reactivity with GL or PG can be easily and efficiently collected by removing PI by dialysis, gel filtration, or the like.

There are no particular restrictions on the insects that can be used in the present invention, but it is preferable to use large insects that have established breeding methods. Examples include, but are not limited to, pterygians, pycnids such as the grasshopper and the beetles, and beetles such as the beetle beetle.

Body fluids include hemolymph obtained from body cavities.
lymph) is the easiest to obtain and is more common.

Solutions used in the present invention that are isotonic with insect body fluids include:
Any material can be used without particular limitation as long as it is isotonic with insect body fluids, but usually yA! J
Physiological saline or the like is preferably used because of its simplicity.

The SPT used in the present invention is not particularly limited as long as it irreversibly inhibits SP and can be separated from components that react with GL or PG present in insect body fluids. However, it is preferable from the viewpoint of operation that the molecular weight is relatively small, for example, 2,000 or less, and that it can be removed by dialysis. Examples of such SPI include (p-amidinophenyl)methanesulfonyl fluoride (p-APMSF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), p-nitrophenyl-p'-guanidino Examples include benzoic acid (NPGB) and dihydroxychloromethylcoumarin (DCC'). These SPs
The use of I is for S, which becomes activated during the collection of body fluids.
It is not particularly limited as long as it is added to the above isotonic solution in an amount equal to or more than the amount necessary to inactivate P, but preferably 0.1-1 On+M, more preferably 0.5
It is added in the range of ~5a+M.

The present invention may be carried out, for example, as follows.

That is, the body fluid exuded by injuring a part of the insect's body is directly dropped into a solution containing SPI that is isotonic with the insect's body fluid, or the solution containing SPi is isotonic with the insect's body fluid. After injecting a solution into an insect, the body fluid exuded by touching a part of the insect's body is collected. Next, the collected liquid is centrifuged to remove blood cells and the like. Furthermore, SP1 present in excess in the solution containing the obtained body fluid is removed by a dialysis method.

Among the separation and purification methods commonly used in the field of biochemistry, such as gel filtration, ion exchange, and high-performance liquid chromatography, one or more of the most suitable methods are selected depending on the nature of the SPI used. By selecting and removing two or more types, a solution containing the desired insect body fluid can be obtained. This SPI removal operation can also be performed after centrifugation and after concentrating and purifying the insect body fluid components to some extent by conventional methods such as ammonium sulfate fractionation.

In the solution containing the plasma component of the insect body fluid thus obtained, there is a substance that does not react with ET but reacts specifically with GL to express enzymatic activity (or a substance that induces expression).
and a substance that reacts specifically with PG to express enzyme activity (
or a substance that induces expression), which specifically reacts with GL and expresses enzyme activity (
If the substance (or inducing expression) is removed, it can be used as a reagent that specifically reacts with PC, or among these, a substance that specifically reacts with PG to express (or induce expression) enzyme activity. If removed, it can be used as a reagent that specifically reacts with GL.

From this solution, a substance that specifically reacts with GL to express (or induces expression) enzyme activity, or a substance that specifically reacts with PG to express (or induces expression) enzyme activity. Methods for removing substances include gel filtration method, electrophoresis method,
Separation and purification methods commonly used in the field of biochemistry such as high-performance liquid chromatography and affinity chromatography can be used, but this method can be achieved by affinity chromatography using a carrier bound to PG or GL. It is particularly preferable to carry out this procedure because it can be carried out extremely easily and efficiently. Furthermore, when performing this by affinity chromatography, a chelating agent is usually used to prevent unnecessary activation of the PO cascade reaction, but the SPI according to the present invention
may be used instead.

Furthermore, the factors that make up the cascade reaction of PO present in the body fluids of insects have not yet been fully elucidated, and even in the relatively well-researched reaction system of silkworms, only a small amount of PO exists in the body fluids of insects.
O, prophenol oxidase activating enzyme (PPAE)
), benzoyl arginine ethyl ester degrading enzyme (
BAE Ease) has only been discovered, and it has not yet been elucidated what other factors are involved in the overall reaction system. That is, among these, P
It is known that PAE and BAEEase are SPs, but SPI suppresses the cascade reaction of PO in insect body fluids, which is easily activated by conventional collection methods, and moreover, is GL or P
It was surprising that the ability to be activated by G was retained.

The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.

[Example] Example 1゜Fifth instar silkworm larvae were placed on ice for 10 minutes and stopped moving,
Cut the leg of the third abdominal segment and remove the hemolymph that oozes out.
It was collected by dropping it into a 0.9% sodium chloride solution 101 containing 11M p-APMSF. Approximately 15 ml of hemolymph was obtained from 25 silkworm larvae, and approximately 251 hemolymph dilutions were obtained. This was centrifuged at 1,500×g for 5 minutes at low temperature to remove blood cells. Obtained supernatant 25
Add 37.5ml of saturated ammonium sulfate solution to -1 to achieve 60% saturated ammonium sulfate! 3. After making a suspension, 10. The precipitate was collected by centrifugation at OOOXg for 20 minutes. This precipitate was dissolved in 271 0.01M Tris-malate buffer (containing 0.15M NaCl,
After adding O, 1 ml of 200 m p-APMSF solution, OIM) Lys-malate buffer (containing 0,15 M NaCl, pH 6,5)
It was dialyzed four times at 11. 10. The obtained dialyzed fluid was OO
The target silkworm plasma was obtained by centrifugation in OXg for 20 minutes at low temperature.

Note that similar results were obtained when NPGB was used instead of p-APMSF.

Example 2 After placing the fifth instar silkworm larva on ice for 10 minutes and stopping its movement,
A 0.9% sodium chloride solution containing 2mM p-APMSF was injected into the silkworm between the 5th and 6th abdominal segments. To prevent the injected fluid from leaking, the front of the fifth abdominal segment was tied with a thin thread, the leg of the third abdominal segment was cut, and the exuding body fluid (hemorymph) was collected.

The collected body fluid was centrifuged at 1,500×3 for 5 minutes at low temperature to remove blood cells. Add 200ml to 10ml of the supernatant obtained.
Add 0.05 ml of p-APMSF solution of M, and add 10 ml of p-APMSF solution.
．． After centrifugation at low temperature for 20 minutes at OOOXg, remove the supernatant.
．． 01M) Lis-malate buffer (0,15M Na
Sephadex G-25 (trade name, Pharmacia) column (2.5X) equilibrated with
15c++1), and the protein peaks eluted in the void volume of the column were collected to obtain the desired silkworm plasma.

Reference Example 1゜(1) Preparation of PG-immobilized Sepharose 4B column Micrococcus luteus (Micrococc luteus)
Purified PG 153 obtained from
+sg was suspended in 8011IM ammonium acetate 1531, 1.5s+g of egg white lysozyme was added, and the mixture was stirred and heated at 45°C for 4 minutes, and then digested at 37°C for 2 hours. It was filtered through a Millipore filter (HAWPo 4700) and the filtrate was lyophilized. 5. Dissolve the freeze-dried product in distilled water 61.
51 to a column of Sephadex G-50SF (trade name of Pharmacia) (eluent: 50mM ammonium carbonate solution, 2.5X90cim, elution rate: 15a+l/
Gel filtration was performed at hr).

The core of the active fraction of reducing sugars resulting from the digestion was collected and lyophilized, and the lyophilized product was dissolved in 6.3 ml of O9 group sodium carbonate buffer (pH 0) to prepare PG.
It was made into a solution. This PGN? a and CNBr-activated Sepharose 4B (trade name of Pharmacia) 2.73 were reacted according to a conventional method to obtain PG-immobilized Sepharose 4B.

The PG-immobilized Sepharose 4B obtained in this way was packed into a column (0.6 x 1.7 cm), and 0.01 M
) Lys-malate buffer [0,15M KCI and 1
Contains mM EDTA (ethylenediaminetetraacetic acid). pH below 6.5°, abbreviated as TMB. ] to equilibrate and prepare a PG column.

(2) Preparation of GL reagent A 100mM EDTA solution (p) 16.5) was added to the silkworm plasma obtained in Example 1 so that the final concentration of EDTA was 111M, and 15ml of the solution obtained in (1) was added. (Eluent: TMB, Elution rate:
6ml/hr). Immediately after injecting the sample, 12 ml of eluate was collected and used as a GL reagent.

(3) Measurement of the degree of activation of inactive enzymes in GL reagent and silkworm body fluid by zymosan (β-1,3-glucan) or PG (measurement procedure) Whole plasma obtained in Example 1 or (2) Add 20 μm of 80+*M CaCl2 solution to 200 μl of the GL reagent obtained above, and add I B/ml zymosan solution or 111
Add 20μm of g/l+I PG warm solution and mix well.
The reaction was carried out at 5°C. A predetermined amount of the reaction solution was collected at a predetermined time, and the degree of PO activation or BAEEase activity value was measured.

■Measurement of PO activation degree Substrate solution (4mM 4-methylcatechol and 8wM4-
0.1M phosphate buffer containing hydroxyproline ethyl ester, pH 6.0) Add sample (above reaction solution) to 1 ml
After adding 10 μm and causing a lθ partial reaction at 30° C., the degree of PO activation was determined by measuring the absorbance of 520 na+ of the quinone dye produced.

FIG. 1 shows changes in absorbance at 520 ns depending on the reaction time when various samples were reacted with a substrate solution. However, ...- means zymosan and GL reagent, and -mu- means P.
G and GL reagent, -〇- means zymosan and whole plasma,
Moreover, -Δ- indicates the absorbance change when using samples obtained by reacting PG and whole plasma, respectively.

■Measurement of BAEEa, se activity Substrate solution kept at 25°C in advance [2mM N-α-benzoyl-arginine ethyl ester, 1mM N.A.
D is cotinamide adenine dinucleotide), 0.11
8/ml alcohol dehydrogenase, 0.25M)
Contains lis(hydroxymethyl)aminomethane and 0.2M semicarbazide, 1) H8.5, at 25°C] 1m
307 L of sample was added to 1, mixed well, and reacted at 25° C., and the increase in absorbance of the resulting NA (reduced nicotinamide adenine dinucleotide) at 340 was measured.

In addition, one unit (U) of BAEEase was defined as a mold that produces lnmol of ethanol per park under the above reaction conditions.

The results are shown in Table 1. In addition, in the table, sample A is whole plasma and zymosan solution, sample B is whole plasma and PG warm solution, sample C is GL reagent and zymosan solution, and sample is G
Samples obtained by reacting the L reagent and the PG warm solution are shown.

Table 1 As is clear from these results, the enzyme in the whole plasma is activated by zymosan and PG, but the enzyme in the GL reagent is activated only by zymosan and not by PG.

Incidentally, when the same operation was performed using the whole plasma obtained in Example 2, similar results were obtained.

Reference example 2. Creation of a calibration curve using curdlan (measurement operation) Add 80 wMC to the GL reagent 21 obtained in (2) of Reference Example 1.
200 μm of aCl 2 solution was added and mixed well. Add 10 μl of curdlan solution of a specified concentration to this 10 μm.
After tactile heating at 0°C for 60 minutes, the substrate solution 11 for PO activity measurement used in Reference Example 1 was added, and after further reaction at 30°C, the absorbance at 520 nm was measured (measured value; E, )
. Separately, a blind value (Ear) was obtained by performing the same operation using purified water instead of the curdlan solution.

(Results) FIG. 2 shows the relationship between the curdlan concentration and the (E-Ellll) value using logarithmic axes for both the horizontal and vertical axes.

As is clear from this result, the calibration curve showed good linearity.

Reference example 3. Creation of a calibration curve using curdlan (measurement operation) Add 80mMC to the GL reagent 21 obtained in (2) of Reference Example 1.
200 μm of aCl 2 solution was added and mixed well. Add 0.1M phosphate buffer (20w+M L-
Dopa-containing, pH 6,0) 70 μm and 70 μm of curdlan solution of the specified concentration were added, mixed well, and heated at 25°C.
The time (Δt) until the amount of transmitted light decreased by 5% was measured using a toxinometer (manufactured by Wako Pure Chemical Industries, Ltd.).

(Results) FIG. 3 shows the relationship between Δt and curdlan concentration using logarithmic axes for both the horizontal and vertical axes.

As is clear from this result, the calibration curve in this case also showed good linearity.

Reference example 4. Preparation of Curdlan Bead Column Curdlan (
Wako Pure Chemical Industries, Ltd.! 270++ liters of pure water was added to 9 g of powder and stirred to obtain a slurry. To this, lN-NaOH3
When 0a+l was added, curdlan was dissolved, and an aqueous sodium hydroxide solution of curdlan was obtained.

To an 81-volume beaker, 1.21 g of toluene and 6 g of surfactant Emarex IC-80 (product name of Nippon Emulsion Co., Ltd., polyoxyethylene hardened castor oil derivative) were added, and the mixture was stirred at a speed of 800 rpm+m using a screw-type stirring blade. The above sodium hydroxide aqueous solution of curdlan was added dropwise at room temperature. The thus obtained curdlan dispersion was added to a solution consisting of 21 parts of toluene and 11 parts of acetic acid at 800 rpm.
and stirring was continued for about 1 hour. By allowing the mixture to stand for about 8 hours, the generated beads were precipitated. The solvent was removed by decantation, and the resulting precipitate was washed five times with pure water 81 to make the pH neutral, and the organic solvent was completely removed.
I got 1.

This was classified, and the particles with a particle size of 50 to 100 μm (average particle size of about 80 μa1) were equilibrated with TMB column (1, 3
x2, 3 cm) to form a curdlan bead column.

Reference Example 5゜(1) Preparation of PG Reagent 820 ml of 7M was added to the whole plasma 51 obtained in Example 2 and mixed well, which was used as a sample and treated with the curdlan beads column obtained in Reference Example 4.

Approximately 251 of the protein fraction passed through was dropped into a saturated ammonium sulfate solution of 2251, followed by stirring.

Collect the precipitate by centrifugation (16,000 x g, 20 min), dissolve the precipitate in TMB 50
0111 was dialyzed as an external solution. Centrifuge this again (
16,000×g for 20 minutes), and the supernatant was made up to a total volume of 51 with TMB to use as a PG reagent.

(2) PG reagent and measurement of the degree of activation of inactive enzymes in silkworm plasma by zymosan (β-1,3-glucan) or PG (measurement procedure) Whole plasma obtained in Example 1 or (2) Add 20μm of 80+mM CaCl2 solution to 200μ+ of the PG reagent obtained in , and add I nag/ml zymosan solution or I
Add 20 μm of B/ml PG warm solution and mix well.
The reaction was carried out at 5°C. A predetermined amount of the reaction solution was collected at a predetermined time, and the degree of PO activation or BAEEase activity value was measured.

■Measurement of po activation degree Substrate solution (4mM 4-methylcatechol and 8mM 4-methylcatechol
After adding 108 m of the sample (above reaction solution) to hydroxyproline ethyl ester-containing 0.1 M phosphate buffer, pH 6,0) 1 + was measured to determine the degree of PO activation.

FIG. 4 shows changes in absorbance at 520 nm depending on reaction time when various samples were reacted with a substrate solution. However, -・- means zymosan and PG reagent, and -mu- means PG reagent.
The graph shows the change in absorbance when using samples obtained by reacting zymosan with the PG reagent, -0- with zymosan and whole plasma, and -Δ- with PG and whole plasma, respectively.

■Measurement of BAEEase activity Substrate solution [2n+M N-α-benzoyl-thi-arginine ethyl ester, 1mM N
AD.

0.1mg/ml alcohol dehydrogenase, 0.2
5M) lis(hydroxymethyl)aminomethane and 0
．． Contains 2M semicarbazide, pH 8.5, at 2S°C
30 μl of the sample was added to Co. 11, mixed well, and reacted at 25° C., and the increase in absorbance of the resulting NADH at 340 rv was measured.

Incidentally, the l unit (U) of B A E Ease was defined as the amount that would produce 1 rvol of ethanol per minute under the above reaction conditions.

The results are shown in Table 2. In the table, sample A' contains zymosan and whole plasma, sample B' contains PG and whole plasma, sample C9 contains zymosan and PG reagent, and sample D' contains PG and PG reagent. The samples obtained by each reaction are shown.

Table 2 As is clear from these results, the enzyme in silkworm plasma is activated by zymosan and PG, but the enzyme in the PG reagent is activated only by PG and not by zymosan.

Incidentally, when the same operation was performed using the whole plasma obtained in Example 2, similar results were obtained.

Reference example 6. Creation of a calibration curve using PG (measurement operation) Add 80+wM to the PG reagent 21 obtained in Reference Example 5 (1)
200 μl of CaCI 2 solution was added and mixed well. To this lOμl, add lOμm of a PG solution with a predetermined concentration and hold at 30°C.
After tactile heating for 60 minutes, substrate solution 11 for PO activity measurement used in Reference Example 5 was added, and after further reaction at 30° C., the absorbance at 520 n+s was measured (measured value; E).

Separately, a blind value (Eel) was obtained by performing the same operation using purified water instead of the PG solution.

(Results) FIG. 5 shows the relationship between the PCl1 degree and the (E--Eel) value using logarithmic axes for both the horizontal and vertical axes.

As is clear from this result, the calibration curve showed good linearity.

Reference example 7. Creation of a calibration curve using PG (measurement operation) Add 80w+M to the PG reagent 21 obtained in Reference Example 5 (1)
200 μm of CaCI 2 solution was added and mixed well. Add 0.1M phosphate buffer (20mM L-
Add 70 μl of dopa-containing, pH 6,0) and 70 μm of curdlan solution of a specified concentration, mix well, and heat at 25°C.
The time (Δt) until the amount of transmitted light decreased by 5% was measured using a toxinometer (manufactured by Wako Pure Chemical Industries, Ltd.).

(Results) FIG. 6 shows the relationship between Δt and PCl3 degrees using logarithmic axes for both the horizontal and vertical axes.

As is clear from this result, the calibration curve in this case also showed good linearity.

[Effects of the Invention] As described above, the present invention provides a simple and efficient method for collecting insect body fluid, which is a raw material for GL or PG measuring reagents, and makes a major contribution to this industry. This is an invention.

[Brief explanation of the drawing]

Figure 1 shows the reaction time of 520 rv (
It shows the change in absorbance at this point, and connects the points obtained by plotting the absorbance at 520n+s obtained for each time (minute) on the horizontal axis along the vertical axis. However, -・- is a reaction solution of GL reagent and zymosan as a sample, -mu- is a reaction solution of GL reagent and PG, -〇-
1 shows the results when the reaction solution of whole plasma and zymosan was used, and -Δ- shows the result when the reaction solution of whole plasma and PG was used, respectively. Figure 2 shows the calibration curve obtained in Reference Example 2, where the horizontal axis shows the curdlan concentration (ng/+*I), and the vertical axis shows the blinded value at 520% subtracted. The absorbance is shown respectively. Figure 3 shows the calibration curve obtained in Reference Example 3, with the horizontal axis representing the curdlan concentration (ng/ml) and the vertical axis representing the time required for the amount of transmitted light to decrease by 5% (minutes). are shown respectively. 4th [! l is 520n depending on the reaction time when the various samples obtained in Reference Example 5 were reacted with the substrate solution.
s (indicates the change in absorbance at this point, each time (minute) on the horizontal axis)
It connects the points obtained by plotting the absorbance at 520n- obtained for 520n along the vertical axis. However, -・- is a reaction solution of PG reagent and zymosan as a sample, -mu is a reaction solution of PG reagent and PG, -〇-
1 shows the results when the reaction solution of whole plasma and zymosan was used, and -Δ- shows the result when the reaction solution of whole plasma and PG was used, respectively. FIG. 5 shows the calibration curve obtained in Reference Example 6, where the horizontal axis represents PCl3 degrees (ng/w+l), and the vertical axis represents 52 degrees PCl (ng/w+l).
The absorbance at 0 rv after subtracting the blind value is shown. Figure 6 shows the calibration curve obtained in Reference Example 7, where the horizontal axis represents the PG concentration (ng/ml) and the vertical axis represents the time (minutes) until the amount of transmitted light decreases by 5%. Show each. Patent applicant Wako Pure Chemical Industries, Ltd. Figure 1 Time (minutes) 2al Curdlan concentration (ng/ml) Figure 3 0.1 1,0 10.0 Curdlan concentration (ng/ml) 411 II Figure 5 PO stage (n9/ml)

Claims (3)

[Claims] (1) A method for collecting insect body fluids, which comprises collecting insect body fluids using a solution that is isotonic with insect body fluids and in which a substance that irreversibly inhibits serine protease is present. (2) The method for collecting insect body fluid according to claim 1, which is carried out by dropping the insect body fluid into a solution that is isotonic with the insect body fluid and in which a substance that irreversibly inhibits serine protease is present. . (3) The insect body fluid according to claim 1, wherein the body fluid is collected after injecting into the body of the insect a solution that is isotonic with the insect body fluid and in which a substance that irreversibly inhibits serine protease is present. Collection method.