JP2003325170A - Virus-catching material and virus sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound which can exhibit an excellent anti-viral activity against influenza viruses and the like, and also exhibit a function as a virus sensor. <P>SOLUTION: The sialooligosaccharide chain of YDS-Asn having an influenza virus activity is transferred to a metal complex having fluorescent light by the utilization of the saccharide transfer activity of Endo-M. Since having both the fluorescent property and the influenza virus activity, the obtained compounds (1), (2) can be used as biosensors whose colors are changed, when bound to viruses. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a virus trapping material and a virus sensor.

[0002]

[Prior Art] YDS-Asn (Asn linked Yolk di-sialyl
oligosaccharide) is an asparagine-linked sialo complex type double-chain sugar chain and has two sialyllactose moieties on the non-reducing end side. Since this sialyllactose is known to have an affinity for influenza A virus, YDS-Asn also has a strong affinity.

[Chemical 7]

On the other hand, a metal complex can be regarded as a creator who gives a compound of ever-changing variety depending on a metal species or a coordination species.
Metal complexes are easy to prepare, have various redox potentials and absorption spectra depending on the combination of metal species and coordinating species, and some have fluorescent properties, and are expected as molecular sensors in recent years. It is a functional element. We thought that by combining two compounds, each of which has a specific property, an effect more than the property of each can be expected. The glycosyl transfer activity of Endo-M was used as a means for easily binding the two compounds. Endo-M
Is Endo-β-GlcNA derived from the filamentous fungus Mucor hiemalis
c-ase, which has recently been found to have transglycosylation activity. This enzyme has an extremely broad substrate specificity that acts on any of high-mannose type, complex type, and mixed type sugar chains, and unlike conventional microorganism enzymes, it has sialic acid at the non-reducing end. It also has the specificity of acting on complex-type sugar chains containing That is, when this enzyme acts on a glycopeptide of human serum transferrin having a complex double-chain sugar chain, the sugar chain is released leaving a GlcNAc1 residue on the protein side, while an appropriate receptor containing Glc or Glc is released. When the body is present, the liberated sugar chain is the Gl of its receptor.
It is transferred to part c.

[0004]

An object of the present invention is to provide a compound capable of exhibiting excellent antiviral activity against influenza virus and the like, and capable of exhibiting a function as a virus sensor.

The present invention provides the following means in order to solve the above problems. YDS-Asn having influenza virus activity is used to transfer a sialo-oligosaccharide to a fluorescent metal complex by utilizing the glycosyl transfer activity of Endo-M. Since the compounds (1) to (6) thus produced have both fluorescence and influenza virus activity, they can be biosensors that "change color" when bound to a virus.

Embodiments of the present invention will be described below with reference to examples, but the scope of the present invention is not limited to the following examples.

Example 1 Synthesis of (YDS) -ΔRu conjugate (1-1) Enzymatic reaction in Ru complex First, an enzyme with [Ru (α-Glc-3-bpy) ₃ ] Cl ₂ (ΔRu complex) The reaction was carried out. UV detection was performed at three wavelengths of 214, 254, and 466 nm.・ 214: absorption band such as N-Ac group of YDS-Asn ・ 254: absorption band such as phenyl group of Ru complex ・ 466: absorption band of MLCT of Ru complex Optimal enzyme reaction conditions and HPLC conditions were examined. The following conditions gave the best yields of transglycosylation products. 70 mM YDS-
Incubate 10 ml of a solution containing Asn, 14 mM ΔRu complex, 30 mU / ml Endo M, 20% acetonitrile, 60 mM potassium phosphate buffer (pH 6.25) at 37 ° C for 10 hours. After stopping the reaction, the enzyme solution was analyzed by HPLC (solvent: acetonitrile / water containing 0.1 M ammonium acetate = 15/85). The results are shown in Fig. 1 (a). In addition to the peak (12.5 min) of the raw material ΔRu complex, about three peaks that were considered to be a glycosyl transfer product were confirmed. Peaks 1 and 2 were separated well, but peak 3 had about two peaks overlapping. This peak 3 can be separated by changing the HPLC reaction conditions.

Further, the peak area ratios of the respective peaks over time under these reaction conditions are shown in FIG. As the reaction time became longer, the yield of the raw material ΔRu complex decreased, and the yields of peaks 1, 2, and 3, which are considered to be transglycosylation products, increased. In addition, the reaction reached equilibrium in about 10 hours, and the yield hardly changed thereafter. Since this reaction is an equilibrium reaction between the transglycosylation reaction and the hydrolysis reaction, the transglycosylation reaction proceeds as the reaction time increases,
After that, it is considered that the glycosyl transfer product was decomposed by the hydrolysis reaction.

Therefore, when the scale-up was carried out under the above conditions (the reaction was charged at 100 ml), the transglycosylation reaction hardly proceeded. Prolonged reaction time (24 hours) did not change much, so add 0.
After adding 5 volumes and incubating, 6 hours later,
The transfer reaction occurred in good yield. The conditions for mass synthesis were changed and examined, but this condition was the optimum. Mass synthesis under these conditions, collecting each peak,
^It was identified by ¹ H-NMR. The results are shown in Figure 2. Measured ¹ H-NMR
When the chart was compared with the ¹ H-NMR chart of the raw material ΔRu complex, YDS-Asn, it was found that YDS-Asn was transformed to the ΔRu complex. Also, from the integration ratio of 1H, the peak
1 has one sialo-oligo 10 sugar, peak 2 has sialo-oligo 10
It was possible to identify it as a product in which two sugars were transferred. However, the configuration of the two transferred products is unknown.

(1-2) UV, CD and fluorescence spectra of (YDS) -ΔRu conjugate ΔRu complex as a raw material, UV of the product of transfer of one sialo-oligo decasaccharide, and transfer of two sialo-oligo decasaccharide UV, CD And fluorescence spectra were measured at a concentration of 10 ⁻⁵ M in water at 25 ° C. The results are shown in Fig. 3. In the UV and CD spectra, no significant difference was observed between the raw material ΔRu complex and the transglycosylation product. In the fluorescence spectrum, a very strong fluorescence with a fluorescence quantum yield of 0.2 or more was emitted in the visible region of about 605 nm with an excitation wavelength of 450 nm. (The fluorescence quantum yield is a value indicating how much light is emitted as fluorescence when the light of 1 is applied, and the larger this value, the stronger the fluorescence. Fluorescence quantum yield of the Bpy-Ru complex. Since the rate is 0.042, the fluorescence was measured under exactly the same conditions, and the fluorescence quantum yield was determined from the magnitude of the fluorescence intensity and the molar extinction coefficient.) The increase in fluorescence intensity was due to the addition of sugar to water. It is thought that this is because the molecules are less accessible to the complex and the release of heat energy is reduced, but this is also unclear because there are various factors.

Example 2 Hemagglutination Inhibition Test of Glycosyl Transfer Products Using Lectins In order to evaluate the molecular recognition ability of glycosyl transfer products, hemagglutination inhibition tests using lectins were performed. For lectins,
An elder collectin was used. This method is an immunochemical research tool that has been advanced to analyze the antigen-antibody reaction. In particular, by replacing the lectin with the transglycosylation product, the interaction between them can be analyzed semi-quantitatively. On the surface of red blood cells, glycoproteins and glycolipids having various sugar chains attached are present. When lectins that specifically recognize these sugar chains are mixed with erythrocytes, a three-dimensional cross-linking structure is formed between a protein called hemagglutinin on the surface of erythrocytes and the lectins to form aggregates. However, erythrocyte aggregation can be inhibited by treating the lectin binding site with a sugar that is specifically recognized. Hemagglutination is YDS-Asn
Or YDS-ΔRu complex, and the smaller the minimum concentration of the inhibitor at that time, the higher the inhibitory capacity. Since a 2-fold dilution series of inhibitor solution was used for the measurement, a difference of approximately 2-fold is within the error range.

(2-1) Preparation of erythrocyte suspension treated with actinase E After 2 ml of human blood (A type) was sampled, citrate buffer solution (pH 3.8)
Mixed into 2 ml. Then centrifuge (4 ℃, 1000rp
m, 10 min) and the supernatant (plasma, white blood cells) was removed to leave only red blood cells. About 10 times the amount of 10 mM phosphate buffered saline (PBS) having a pH of 7.2 was added to the erythrocytes, mixed well, centrifuged, and repeated until the supernatant became transparent. Next, the supernatant was removed and 10 mg of actinase E was added to PBS 18
The solution dissolved in ml was added and incubated at 45 ° C for 30 minutes. This was centrifuged to remove the supernatant, PBS was added, and the supernatant was washed until it became transparent. This red blood cell 0.9m
1 was placed in a 30 ml Erlenmeyer flask and diluted with 30 ml of PBS.
((Actinase E-treated erythrocytes) -PBS suspension) Also, add 1.1 mg of chicken collectin (SS
A) was added and diluted with 1 ml of PBS.

(2-2) Measurement of erythrocyte agglutination titer of chicken collectin By determining the minimum concentration of lectin necessary for agglutinating erythrocytes, the agglutination titer of lectin is measured to determine the lectin concentration. Were determined. 96 holes-microtiter U
Make a 2-fold dilution series of 20 μl of lectin in PBS on a letter plate, and then add 20 μl of red blood cells ((Actinase E-treated red blood cells) -PBS suspension) to each well and shake the plate gently to homogenize. After the reaction, the mixture was allowed to stand for 1 hour, and the hemagglutination titer was visually evaluated. The hemagglutination titer was determined at the maximum dilution where agglutination was observed (the lectin concentration at that time was 5.2 × 10 ^-11 M). In the actual inhibition test, lectin at a concentration 8 times this aggregation titer was used (4.2 × 10 ^-10 M).

(2-3) Hemagglutination inhibition test of lectin by glycosyl transfer product NeuAc, YDS-Asn, (YDS) ₁ -ΔR
u complex, (YDS) ₂ -ΔRu complex, ΔRu complex solution (1mg / ml)
A 2-fold dilution series with PBS was prepared with a microtiter plate, and 20 μl of a lectin solution (4.2 × 10 ⁻¹⁰ M) at a concentration of 8 times the maximum dilution that causes aggregation was added to each well.
Red blood cells ((Actinase E treated red blood cells) -P
(BS suspension) (40 ml) was added, and the mixture was allowed to stand for 1 hour, and then the minimum inhibitory concentration was determined from the dilution ratio of the sample solution. The results are shown in Fig. 4. NeuAc without Gal moiety and ΔRu complex without sialogooligosaccharide were not recognized by lectins.
On the other hand, YDS-Asn with two NeuAcα2-6Gal was 2.2 × 10 ^-7
M was strongly recognized by the lectin. The (YDS) ₁ -ΔRu complex was 2.5 × 10 ^-8 M, and the (YDS) ₂ -ΔRu complex was 3.5 × 10 ^-8 M. (The minimum inhibitory concentration per sialic acid was 4.4 × 10 ^-7 M and 4.9 × 10 respectively.
^-8 M and 1.4 × 10 ^-7 M. From these results, it can be said that the molecular recognition ability of the sialo-oligosaccharide is about 10 times stronger on the complex. This is because, by using an aromatic ring as the hydrophobic group of the aglycone part of the glycolipid, π-π * stacking occurs between the amino acid residue at the sugar chain binding site of the lectin and the binding ability is increased. Number 10
The ~ 100-fold improvement was almost in agreement with that reported by Soaks et al. Also, (YDS) ₁ -ΔRu
The complex and the (YDS) ₂ -ΔRu complex had almost the same molecular recognition ability for lectins. (In the hemagglutination inhibition test, the difference of about 2 times is within the error range) Since the X-ray crystal structure analysis of the lectin has not been performed, it is not known where the binding site is, but the spatial arrangement is different. I think that can be said from this result. Certainly, it is thought that the molecules are cross-linked and the recognition ability per molecule increases, but it can be said that the binding ability does not change.

Example 3 Synthesis of (YDS) -ΛRu conjugate (3-1) The reaction was carried out under the same conditions as the transglycosylation reaction (1-1) in the ΛRu complex. The Δ form was also reacted in the same manner, and the transglycosylation yields were compared over time. Results are shown in FIG. From these results, it can be seen that the reaction reached equilibrium in about 2 hours, and that the Δ form had a higher transglycosylation yield than the Λ form. In the Δ type, the yield was lower than that of the previous time (FIG. 1), and the equilibrium was reached immediately, so it can be expected that the enzyme activity was reduced. The higher transglycosylation yield in the Δ form than in the Λ form may be due to the difference in conformation. Dr. Hasegawa's [Ru (α-Glc-3-bp
Studies of y) ₃ ] Cl ₂ show that the Λ type has a more compact conformation than the Δ type. Therefore, it seems that the Glc at the terminal of the Δ type was more flexible, and thus it was easier to transfer sugars than the Λ type.

Next, the optimum conditions for the enzymatic reaction in Λ type were examined.

[Table 1] From Table 1, it can be seen that the higher the amount of enzyme, the higher the transglycosylation yield. Here, the yield was shown after 4 hours, but the reaction had already reached equilibrium. In No.3, the best yield was obtained after 1 hour (about the same as No.2).
After the lapse of time, the transglycosylation product was slightly hydrolyzed. From the above results, the No. 2 condition was the most excellent. Next, scale up is performed, and (YDS) -Λ
A large amount of Ru conjugate was synthesized. Each peak was collected by HPLC and identified by ¹ H-NMR. Results are shown in FIG. The measured ¹ H-NMR chart is based on the raw ΛRu complex, YDS-As
Comparison with the ¹ H-NMR chart of n revealed that YDS-Asn was transformed into a ΛRu complex. Moreover, from the integration ratio of 1H, it was possible to identify that peak 1 was a product with one sialo-oligo decasaccharide transferred, and peak 2 was a product with two sialo-oligo decasaccharide transferred. However, the configuration of the two transferred products is unknown.

(3-2) UV, CD of (YDS) -ΛRu conjugate
And Fluorescence spectra The UV, CD and fluorescence spectra of the raw material ΛRu complex, one sialo-oligo decasaccharide transfer and two sialo-oligo decasaccharide transfer were measured at 10 ^-5 M concentration in water at 25 ° C. did. The results are shown in Fig. 7. In the UV and CD spectra, no significant difference was observed between the raw material ΛRu complex and the transglycosylation product. There was a weak crown difference in the fluorescence spectra, but such a difference can be regarded as an error. (Fluorescence is very sensitive, and even slight differences in concentration can make a difference.)

Example 4 Inhibitory Activity Experiment with Influenza Virus In order to examine the molecular recognition ability of the glycosyl transfer product, an inhibitory activity experiment with a target influenza A virus was conducted. This experiment was performed under the guidance of Tadanobu Takahashi, Takashi Suzuki, and Yasuo Suzuki (Department of Biochemistry, Faculty of Pharmaceutical Sciences, University of Shizuoka). The virus used this time is A / Memphis / 1/71 (H3N2)
This virus strain has a receptor binding property of 2-
It is known to be type 6 and is used frequently because it has the most stock because it is used frequently in biochemistry classes.
In addition, in this experiment, rather than measuring LDH activity as has been done so far, a method of counting the number of infected cells by staining the secondary antibody with the primary antibody / secondary antibody is used. I was there. This is because the sensitivity is better and the substrate specificity by the antibody allows the infection inhibition activity to be measured more accurately and quantitatively.

(4-1) Cell culture 24-well plate with MDCK (Madin-Darby canine kidney)
Put cells a) and MEM (containing 5% FBS) b) into cells to make them confluent. (1-2 days)

(4-2) Preparation of sample and virus In an Eppendorf tube, 100 μl of a virus diluted in MEM (without FBS) c) was prepared so that HA was 2 ^-2 . Similarly, 100 μl of a sample diluted in PBS was prepared so that the maximum concentration was as follows (when mixed with virus), and then diluted twice.

[Table 2]

(4-3) The solutions prepared in the infection inhibition experiment (4-2) were mixed (200 μl in total in one Eppen) and incubated at 4 ° C. for 1 hour. (4-1)
Wash MDCK cells on 24-well plate once with PBS (200 μl / w
ell), a mixed solution of the sample and the virus was added on MDCK cells in the well (200 μl / well) and incubated at 34 ° C. for 1 hour. Then, wash the well once with PBS, MEM (without FBS)
500 μl / well was added at 17 ° C at 34 ° C in the presence of 5% CO _2.
Incubated for hours. Wash once with PBS and MeOH (500 μl /
well) was added and shaken for 5 minutes to immobilize the cells on the plate. After washing once with PBS, 200 μl / well of anti-HA monoclonal antibody (2E10, IgG1) diluted in PBS was added and shaken at room temperature for 30 minutes. HRP diluted with PBS after washing once with PBS
200 μl / well of labeled anti-mouse IgG + M antibody was added, and the mixture was shaken at room temperature for 30 minutes. Wash once with PBS, 100 mM citric acid b
uffer (pH 6.0) 10 ml, 30% H ₂ O ₂ 1 μl, 200 μl each of 0.06 M DEPDA and 0.1 M 4-CN diluted in acetonitrile
It was added to well and shaken at room temperature for 10 minutes. Then PBS
Replaced with.

(4-4) Results The number of stained cells was counted by a microscope, and the results of plotting the sample concentration and the cell number (%) are shown in FIG. Further, the IC ₅₀ obtained from this graph is shown in FIG. YDS-Asn and (YDS) -R
Comparing u, the binding inhibitory activity of the virus was increased about 100 times only by the binding of YDS to the complex. In addition, when one YDS bound and two YDS bound, the inhibitory activity increased about two-fold when two YDS bound. Since the inhibitory activity is the same at the molar concentration per one YDS, it can be seen that the cluster effect due to the increase in sugar chains is not working.

[Example 5] Fluorescence change by addition of virus In order to evaluate the function as a virus sensor, (YD
Influenza virus was added to (S) -ΔRu conjugate and it was investigated whether the fluorescence changed (Ex = 450nm, Em = 500-700
nm). Because we observe the change in fluorescence due to virus binding,
(YDS) -ΔRu conjugate was performed at a low concentration (2.5 × 10 ^-7 M). In addition, it was measured at 4 ° C so that it would not be decomposed by sialidase after it was bound once. As a reference
Inactivated virus by heat treatment at 100 ℃ for 3 minutes (n.
a. virus) and that with YDS-Asn added to prevent competition.

(5-1) Experimental method ΔRu complex, (YDS) ₁ -ΔRu conjugate and (YDS) _2-
Dilute ΔRu conjugate in PBS buffer (pH 7.4) and add 1
A 0 ^-6 M solution was prepared (). Influenza virus and navirus (1.5 × 10 ^-4 pfu / ml) in PBS buffer
A solution (1.5 × 10 ⁻⁷ pfu / ml) diluted 1000 times with (pH 7.4) was prepared (). In addition, YDS-Asn was prepared to be 10 ^-2 M (). These prepared solutions were thoroughly degassed before use. Add 50 μl of any volume and PBS buffer to an Eppendorf tube and bring the total volume to 200.
μl. The solution was incubated at 4 ° C. for 1.5 hours, and the fluorescence intensity was measured at Ex = 450 nm and Em = 500-700 nm.

(5-2) Result 1 (virus (A / Memphis / 1/7
Changes in fluorescence due to addition of 1) etc.) Changes in fluorescence spectrum are shown in FIG. ΔRu complex that does not bind to virus (all terminals are α-Glc) showed no change in fluorescence even when virus was added (Fig. 10 (a)). However, it has a sialo-oligosaccharide chain site to which the virus binds
(YDS) ₁ -ΔRu conjugate and (YDS) ₂ -ΔRu conjugate
Clearly decreased the fluorescence intensity when virus was added ((Fig. 10 (b), (c)).), And YDS-Asn was added to (YDS) -ΔR
When the competition with u conjugate was inhibited, the fluorescence intensity was almost unchanged. This is because YDS-Asn was added at a high concentration, so that the virus was not YDS-ΔRu conjugate but YDS-Asn.
-It is thought that it is because it has combined with Asn. In addition, even when navirus was added, the fluorescence increased only in the weak crown, but did not decrease. We believe that the weak crown increase is due to non-specific binding to the virus (when the virus is heated, the hydrophobic surface is exposed and hydrophobic interaction occurs). From the above, it was found that the fluorescence decreased when (YDS) -ΔRu conjugate was bound to a virus.

(5-3) Result 2 (virus (A / Memphis / 1/71
Strain) Fluorescence Change According to Addition Amount) Next, it was examined how the fluorescence intensity changes by changing the addition amount of virus. In Figure 10, the amount of virus added is 10
Since it was 0 μl, the fluorescence change was examined by shifting the concentration from 0 to 100 μl at about 5 points. The results are shown in Fig. 11. as a result,
The fluorescence intensity decreased as the amount of virus added increased. With (YDS) ₁ -ΔRu conjugate, the fluorescence intensity gradually decreased with the addition of virus, and decreased to about 1/3 (Fig. 11 (b)). On the other hand, the fluorescence of (YDS) ₂ -ΔRu conjugate changed significantly at low concentration of virus, but did not change at some concentration (50 μl). And the reduction amount is also 3/5
The fluorescence change was smaller than that of (YDS) ₁ -ΔRu conjugate (Fig. 11 (c)). This is (YDS) ₂ -ΔRu conjugate
Probably possesses two virus-binding sites and can bind to the virus at a low concentration. Also, (YDS) ₁ -ΔRu con
Since the jugate has only one virus binding site (YDS site), it is considered that the conformational change and the fluorescence change when bound to the virus were larger than those of the (YDS) ₂ -ΔRuconjugate. Also from this, (YD
This result suggests that neither of the S) ₂ -ΔRu conjugate YDS is bound to the virus.
This is because it is considered that both of them have a larger conformational change and a larger fluorescence change when bound to a virus. In addition, it is considered that the reason for the decrease in fluorescence was that the YDS site that had been rounded in the solution was extended due to the binding with the virus.

(5-4) Result 3 (Fluorescence Change by Addition of A / PR / 8/34 Strain) Until now, a virus strain A / Memphis / 1/71 which specifically binds to NeuAcα2-6Gal was used. I have been experimenting. (YDS)-
ΔRu conjugate has NeuAcα2-6Gal at the end, so A / Me
It was bound to the mphis / 1/71 strain and a change in fluorescence was observed. This time refe
Experiments were performed using other virus strains as rence. In particular, the A / PR / 8/34 strain that specifically binds to NeuAcα2-3Gal was used. Since this virus strain does not bind to the (YDS) -ΔRu conjugate, it can be expected that the fluorescence will not change. As a result, even if A / PR / 8/34 strain was added, (YDS) ₁ -ΔRu conjugate
No change in fluorescence was observed in both (YDS) ₂ -ΔRuconjugate. The result is shown in FIG. From this
(YDS) -ΔRu conjugate does not detect all influenza viruses, but its receptor specificity is NeuA
It can be said that it can specifically recognize only cα2-6Gal. Moreover, the detection sensitivity was 1 × 10 ⁵ to 1 × 10 ⁶ pfu / ml, which was about the same as that of the virus diagnostic agents currently on the market. The product of the present invention is bound to a virus via a sugar chain. Influenza virus is easy to mutate, but no matter how much it mutates, the basic property of binding to sugar chains remains, so
It can be said that the product of the present invention is effective as a sensor. By the way,
It is known that infection with one virus increases to 100 after 1 hour, 1 million after 24 hours, and 10 million after 36 hours. In addition, since 1 million cases develop the disease and 10 million cases develop a high fever, it can be said that the present invention can be used for diagnosis before the onset of the disease, and the combination use with zanamivir may save the suffering from influenza.

The present invention is not limited to the description of the embodiments and examples of the invention. Various modifications are also included in the present invention within a range that can be easily conceived by those skilled in the art without departing from the scope of the claims.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of an enzymatic reaction between a ΔRu complex and YDS-Asn. (a) shows HPLC after 10 hours of reaction, and (b) is a diagram showing the relationship between enzymatic reaction time and yield of transglycosylation product. ◆ indicates ΔRu complex, ■ indicates peakl, ● indicates peak
2 and ▲ indicate peak3.

FIG. 2 is a diagram showing a ¹ H-NMR spectrum of (YDS) -ΔRu conjugate. (a) is the spectrum of (YDS) ₁ -ΔRu. (b) is the spectrum of (YDS) ₂ -ΔRu.

FIG. 3 is (a) UV, (b) C of (YDS) -ΔRu conjugate.
It is a figure which shows D and (c) fluorescence spectrum.

FIG. 4 is a diagram showing the results of a lectin (SSA) and hemagglutination inhibition test.

FIG. 5 is a diagram showing a time course of a sugar transfer reaction by an enzymatic reaction of (YDS) -Asn with (a) ΔRu and (b) ΛRu complex.

FIG. 6 is a diagram showing a ¹ H-NMR spectrum of (YDS) -ΛRu conjugate. (a) is the spectrum of (YDS) ₁ -ΛRu. (b) is the spectrum of (YDS) ₂ -ΛRu.

FIG. 7 is (a) UV, (b) CD of (YDS) -ΛRu conjugate.
And (c) is a diagram showing a fluorescence spectrum.

[Fig. 8] Fig. 8 is a view showing the results of an infection inhibition experiment of (YDS) -Ru conjugate with influenza virus (A / Memphis / 1/71).

[Fig. 9] Fig. 9 is an IC in the infection inhibition experiment with the influenza virus (A / Memphis / 1/71) of (YDS) -Ru conjugate.
It is a figure which shows ₅₀ value.

FIG. 10 shows fluorescence of (YDS) -ΔRu conjugate solution added with influenza virus (A / Memphis / 1/71 strain), inactivated virus (navirus), and influenza virus and YDS-Asn. It is a figure which shows a spectrum. (a) is ΔRu complex, (b) is (YDS) ₁ -ΔRu conjug
ate and (c) are fluorescence spectra in (YDS) ₂ -ΔRu conjugate.

FIG. 11 is a diagram showing how fluorescence changes when the amount of influenza virus (A / Memphis / 1/71) added to a solution of (YDS) -ΔRu conjugate is changed. is there. The horizontal axis represents the amount of virus added, and the vertical axis represents the fluorescence change rate. (b) is (YDS) ₁ -ΔRu conjugate, (c) is (YDS) ₂ -ΔRu
It is a fluorescence spectrum in conjugate.

FIG. 12 is a diagram showing a fluorescence spectrum and an amount of change when an influenza virus (A / PR / 8/34 strain) was added to a solution of (YDS) -ΔRu conjugate. (b) is (YD
S) ₁ -ΔRu conjugate, (c) is the spectrum of (YDS) ₂ -ΔRu conjugate.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/483 G01N 33/483 C 4C090 // C08B 37/00 C08B 37/00 Z (72) Inventor Hasegawa Teruaki 909-1 Minamigomizuka, Kusu-cho, Mie-gun, Mie Prefecture (72) Inventor Toshihiro Yonemura 30-7 Wakakusa-cho, Minami-ku, Nagoya-shi, Aichi (72) Takashi Suzuki 519-1 Orito, Shimizu-shi, Shizuoka Prefecture 104 F-term (reference) 2G045 AA28 BA11 FB12 2G054 AA06 BB13 CA21 CE02 EA03 GA04 4B063 QA01 QQ08 QQ10 QR54 QR66 QS36 QX02 4B065 AA95X BD50 CA46 4C0BB ABB BBBB36BB32 BB36BB32 BB36BB32A14 BB32BB34A16 BB13BB14ABB BB32BB34A13 A04 ZB33 4A09 A04 ZB33 4A06 AA02 CA42 DA23 DA25

Claims (7)

[Claims] 1. A virus trapping material represented by any of the following formulas (1) to (6). [Chemical 1] [Chemical 2] [Chemical 3] [Chemical 4] [Chemical 5] [Chemical 6] 2. A virus sensor represented by any of the following formulas (1) to (6). [Chemical 1] [Chemical 2] [Chemical 3] [Chemical 4] [Chemical 5] [Chemical 6] 3. A method for detecting an influenza virus, which comprises capturing the influenza virus with the virus sensor according to claim 2, irradiating excitation light and observing fluorescence. 4. The detection method according to claim 3, wherein the excitation light is approximately 450 nm. 5. Having affinity with influenza virus
An influenza virus trapping material obtained by transferring a sialo-oligosaccharide chain of YDS-Asn to a metal complex by utilizing the glycosyl transfer activity of Endo-M. 6. Affinity with influenza virus
An influenza virus sensor obtained by transferring a sialo-oligosaccharide chain of YDS-Asn to a metal complex having a fluorescent property by utilizing the glycosyl transfer activity of Endo-M. 7. The metal complex is a Ru complex and has a content of about 45.
The influenza virus sensor according to claim 6, wherein the influenza virus sensor is excited by 0 nm excitation light.