TWI631340B - Method for detection of neuraminidase activity - Google Patents

Abstract

A method for detecting neuraminidase activity, comprising the steps of: step (a), providing a biological sample comprising a neuraminidase, and mixing the biological sample with an acetaminophenamine compound of formula (I) The hydrolysis reaction is carried out in the presence of a buffer to produce a hydroxyaromatic compound represented by HO-X ³ , In the formula (I); the step (b), a voltage is applied to cause the hydroxyaromatic compound to undergo a redox reaction, and the current of the hydroxyaromatic compound is detected to confirm the activity of the neuraminidase.

Description

Method for detecting neuraminidase activity

The present invention relates to a method for detecting enzyme activity, and more particularly to a method for detecting neuraminidase activity.

Neuraminidase (NA) is a glycoprotein distributed on the surface of the influenza virus envelope. The neuraminidase is responsible for catalyzing the hydrolysis of glycosidic bonds between sialic acid and glycoproteins, allowing mature influenza viruses to detach from host cells, which in turn infect new cells, causing the spread of influenza virus in a patient. Since the neuraminidase plays an important role in the spread of influenza virus, it is necessary to detect the activity of neuraminidase in order to effectively inhibit the activity of a neuraminidase inhibitor such as zanamivir. To prevent the spread of the flu virus, which in turn reduces the incidence of the disease.

At present, the detection method for the activity of neuraminidase is an enzyme-based immunosorptive assay (Enzyme-Linked ImmunoSorbent Assay, ELISA) using an antibody or an antigen as a matrix and visually reading the coloring result, using a fluorescent substance or coloring Fluorescence analysis using the chemical substance as a substrate and visually reading the color result, and electrochemical analysis using Relisha or Fetuin A.

However, the enzyme immunoassay has the problems of high cost, time consuming, short storage period of the substrate, and poor thermal stability. At the same time, the limit of detection of the concentration of the neuraminidase of the method (LOD) Excessively high (about 7 ng/mL), which is not advantageous as a method for detecting neuraminidase activity in a biological sample containing a low concentration of neuraminidase. This fluorescent analysis method has problems such as being unsuitable for use in biological samples such as blood or urine having color or turbidity. In addition, since the enzyme immunoassay determines the neuraminidase activity by visually observing the color change by the color analysis method, there is a potential problem that a human error is judged. In the electrochemical analysis method using the Relaissance, the activity of the neuraminidase is expressed by the intensity of the current generated by the adsorption between Relisha and a specific electrode, and therefore, the method is limited by the selection of the electrode. It is not convenient to use, and the electrode cost is high. At the same time, the detection limit of the concentration of neuraminidase of the method is too high (about 14.8 ng/mL), which is unfavorable for detecting a low concentration of neuraminidase. A method of neuraminidase activity in a biological sample. In the electrochemical analysis method using the fetuin A, since the fetuin A is first immobilized on the electrode, the fetuin A is easily denatured in a fixed process, which in turn affects the detection efficiency.

Accordingly, it is an object of the present invention to provide a simple, rapid and highly sensitive method for detecting neuraminidase activity.

Thus, the method for detecting neuraminidase activity of the present invention comprises the steps of: step (a), providing a biological sample comprising a neuraminidase, and the biological sample and the acetaminophen acid represented by the formula (I) The compound is mixed and subjected to a hydrolysis reaction in the presence of a buffer to produce a hydroxyaromatic compound represented by HO-X ³ , Formula (I) X ¹ and X ² each represent hydrogen or methyl; X ³ represents , or ; R ¹ , R ³ and R ⁵ each represent hydrogen, -NH ₂ , -OH or -OCH ₃ , but R ¹ , R ³ and R ⁵ may not be hydrogen at the same time; R ² and R ⁴ are hydrogen; R ⁶ and R ⁸ each represents hydrogen, -NH ₂ , -OH or -OCH ₃ , but R ⁶ and R ⁸ may not be hydrogen at the same time; R ⁷ is hydrogen, A is an aromatic ring; and R ⁹ is -NH ₂ , -OH or -OCH ₃ B and C each represent an aromatic ring; in step (b), a voltage is applied to cause a redox reaction of the hydroxyaromatic compound, and a current in the hydroxyaromatic compound is detected to confirm the activity of the neuraminidase.

The method of the present invention is that the method for detecting the activity of neuraminidase of the present invention is simple and rapid by passing the acetaminophenamine compound represented by the formula (I) having thermal stability and storage stability together with an electrochemical method. And high sensitivity characteristics. At the same time, it is highly reproducible when applied to the detection of biological samples with color or/and cloudiness.

The contents of the present invention will be described in detail below.

< Biological sample containing neuraminidase>

In this step (a), the biological sample is for example but not limited to a biological fluid. The biological fluid is, for example but not limited to, serum, saliva, urine, or blood. The serum is for example, but not limited to, fetal bovine serum. Preferably, in step (a), the biological sample is selected from the group consisting of serum, saliva, urine, or blood.

The neuraminidase is a pathogen from, for example, an influenza virus or a bacterium. The influenza virus is, for example but not limited to, an influenza A virus, an influenza B virus, or an avian influenza virus. Such bacteria are, for example but not limited to, Cholera toxin, Pseudomonas aeruginosa, or Streptococcus pneumonia.

<Ethylamine neuron acid compound represented by formula (I)>

The aromatic ring is for example but not limited to a benzene ring. The For example but not limited to . The For example but not limited to .

When X ¹ and X ² in the acetaminophen acid compound represented by the formula (I) are both methyl and hydrogen, they can be used for detecting the activity of neuraminidase from influenza virus and bacteria, respectively, based on Thus, the method for detecting neuraminidase activity of the present invention also has the property of identifying the species of the pathogen. When the X ³ in the acetaminophen acid compound represented by the formula (I) is or When the hydroxyaromatic compound produced has a negative oxidation potential, for example, -200 mV to -400 mV, and since the oxidation potential of the biomolecule is positive, the oxidation potential of the hydroxyaromatic compound is designed to be negative, which can be avoided. The problem of background interference caused by the biomolecules to the detection method of the present invention.

In the acetaminophen acid compound represented by the formula (I), when X ³ is or The preparation method of the acetaminophenamine compound represented by the formula (I) is, for example but not limited to, the following method. The method is to or versus Reaction formation Where Ac is expressed as Then, -OAc and -COOCH ₃ are respectively converted into -OH and -COOH under basic conditions to form an acetaminophenamine compound represented by the formula (I).

<buffer>

To reduce the effect of the buffer on the current signal when detecting neuraminidase activity, preferably, in the step (a), the buffer has a pH in the range of 5.0 to 7.0. Preferably, the buffer comprises phosphate and a solvent. The phosphate salt is for example but not limited to sodium phosphate. The solvent is for example but not limited to water.

<hydrolysis reaction>

In the step (a), the operating parameters of the hydrolysis reaction, such as the operating temperature or the operating time, are adjusted depending on the concentration of the acetaminophen acid compound and each component shown in the formula (I) to be used. Preferably, in the step (a), the hydrolysis reaction has an operating temperature in the range of 25 ° C to 40 ° C, and the operating time ranges from 15 minutes to 60 minutes.

<Scan voltammeter>

In this step (b), the voltage is supplied using a scanning voltammeter, and the current of the hydroxyaromatic compound is detected by the scanning voltammeter. The scanning voltammeter is not particularly limited, and a commercially available scanning voltammeter can be used. In the step (b), the applied voltage is adjusted in accordance with the hydroxyaromatic compound to cause the hydroxyaromatic compound to undergo a reversible redox reaction. The reversible redox reaction of the hydroxyaromatic compound, for example, when the hydroxyaromatic compound is 4-aminophenol, the 4-aminophenol can be oxidized to quinoneimine and then reduced to 4-amine Phenolic. The scan voltammeter provides a scan potential interval and the scan potential interval is ±0.8 volts of the voltage. The sweep voltammeter has a potential scan rate ranging from 100 mV/s to 400 mV/s.

The present invention will be further illustrated by the following examples, but it should be understood that this embodiment is intended to be illustrative only and not to be construed as limiting.

Preparation Example 1

Providing an acetaminophenamine compound represented by the formula (I-1), Formula (I-1). In the formula (I-1), the X ³ is And R ¹ , R ² , R ⁴ and R ⁵ are hydrogen and R ³ is —NH ₂ . The method for producing an acetaminophenamine compound represented by the formula (I-1) comprises the steps of: 0.4 mmol of the acetaminophenamine compound represented by the formula (1), and 0.4 mmol of 10 mole. % Pd/C (as a catalyst) and 5 ml of an aqueous methanol solution (a volume ratio of water to methanol of 1/4) were placed in a high pressure reactor in which the acetaminophen acid represented by the formula (1) Compound is . Next, the air in the high pressure reactor is replaced with hydrogen. Then, it was reacted in a hydrogen atmosphere at room temperature for 15 hours. After the reaction, filtration treatment was carried out and the filter cake was washed with methanol to obtain a filtrate. Subsequently, the filtrate was subjected to an evaporation treatment to obtain 0.28 mmol of the acetaminophenamine compound represented by the formula (I-1) in a yield of 69%. Spectroscopic analysis of the acetaminophenamine compound of the formula (I-1): ¹ H NMR (300 MHz, D ₂ O), δ (ppm): 1.86 (t, J = 12.2 Hz, 1H, H- 3 ax), 2.0 (s, 3H, NAc), 2.87 (dd, J = 12.5, 4.7 Hz, 1H, H-3 eq), 3.57~3.87 (m, 9H, H-4, 5, 6, 7 8,9,9',-NH ₂ ), 6.88 (d, J = 8.7 Hz, 2H, Ar-H), 7.04 (d, J = 9Hz, 2H, Ar-H), 3.300 (q, J = 6.6 Hz, 2H, 1×CH ₂ ), 3.434 (t, J = 7.8 Hz, 8H, 4×CH ₂ ), 3.809 (q, J = 7.2 Hz, 6H, 3×CH ₂ ), 4.725 (t, J = 6.0 Hz, 1H, 1 x NH). ¹³ C NMR (125 MHz, D ₂ O), δ (ppm): 22.11, 40.69, 52.23, 62.63, 68.19, 71.98, 73.23, 102.92, 118.84, 123.07, 172.49, 175.13, 178.70. MS: m/z 401.16 (M+H ⁺ ).

Preparation Example 2

Sodium phosphate and water were mixed and placed in a buffer having a pH of 6.0 and a concentration of 0.1 M.

Preparation Example 3

The acetaminophenamine compound of the formula (I-1) of Preparation Example 1 is mixed with the buffer of Preparation Example 2, and is configured as a first stock solution in which the formula is used in the first stock solution. The concentration of the acetaminophen acid compound shown by (I-1) was 10 mM.

Preparation Example 4

1 mg of neuraminidase [label: Sigma-Aldrich; model: N2876; neuraminidase from Clostridium perfringens; activity: 16.8 units/mg (0.0168 mU/ng)], and 1 mL of the buffer of Preparation Example 2 was mixed and configured as a second stock solution.

Example 1

<Steps to provide a relational equation>

An appropriate amount of the first stock solution of Preparation Example 3 and the second stock solution of Preparation Example 4 were mixed, and the buffer solution of Preparation Example 2 was used to configure seven neuraminidase in a total volume of 1 mL and different concentrations. standard solution. In the standard solution of the neuraminidase, the concentration of the neuraminidase was 25 ng/mL, respectively (corresponding to 4.2×10 ^-4 U/mL (25 ng/mL×0.0168 mU/ng)], 30 ng/ mL [equivalent to 5.04×10 ^-4 U/mL (30 ng/mL×0.0168 mU/ng)], 45 ng/mL [equivalent to 7.56×10 ^-4 U/mL (45 ng/mL×0.0168 mU/ng)], 60 ng/mL [equivalent to 10.08×10 ^-4 U/mL (60 ng/mL×0.0168 mU/ng)], 90 ng/mL [equivalent to 15.12×10 ^-4 U/mL (90 ng/mL×0.0168 mU/ng) ], 150 ng/mL [equivalent to 25.2×10 ^-4 U/mL (150 ng/mL×0.0168 mU/ng)], and 300 ng/mL [equivalent to 50.4×10 ^-4 U/mL (300 ng/mL×0.0168 mU) /ng)], and the concentration of the acetaminophenamine compound represented by the formula (I-1) is 1 mM.

The neuraminidase standard solution was subjected to a hydrolysis reaction at 37 ° C for 30 minutes to produce 4-aminophenol, which formed seven standard samples.

A standard range of cyclic potential voltammetry was used to provide a cyclic potential interval for each of the standard samples using a linear voltammetry (label: US CH Instruments; model: 612d). The working electrode of the scanning voltammeter is a glassy carbon electrode (GCE), a reference electrode is Ag/AgCl, and a counter electrode is a platinum wire. The initial potential of the scanning potential interval was -0.1 V, and the end potential was +0.6 V, and the scanning rate was 250 mV/s. The scan voltammeter was used to detect the oxidation current of 4-aminophenol in the standard samples between the oxidation potentials of +0.25 V and +0.35 V. In the present invention, the oxidation current intensity of the 4-aminophenol increases as the concentration of the neuraminidase in the standard samples increases. After the hydrolysis of the neuraminidase standard solution of 150 ng/mL and 300 ng/mL, the oxidation current of 4-aminophenol was -1.59 μA and -2.02 μA, respectively.

The oxidation current of the 4-aminophenol of the standard samples is plotted against the concentration of the neuraminidase of the standard solution of the neuraminidase, and the current (y, unit: μA) is taken as the vertical axis, and the concentration ( x, unit: ng/mL) as the horizontal axis. Through these seven coordinate points, a linear relationship equation between the oxidation current of 4-aminophenol and the concentration of neuraminidase was obtained. The linear relationship equation is y = 0.0025x and R ² is 0.99. In the present invention, the limit of detection (LOD) of the concentration of the neuraminidase is 5.6 ng/mL [equivalent to 0.9408×10 ^-4 U/mL (5.6 ng/mL×0.0168 mU/ng). )].

<Detection of neuraminidase activity in biological samples containing neuraminidase>

The biological sample containing the neuraminidase was mixed with an appropriate amount of the first stock solution of Preparation Example 3, and the buffer solution of Preparation Example 2 was used to prepare a detection solution having a total volume of 1 mL, wherein the detection solution was used in the detection solution. The concentration of the acetaminophenamine compound represented by the formula (I-1) was 1 mM. The test solution was heated to 37 ° C for a hydrolysis reaction for 30 minutes to produce 4-aminophenol to form a test sample.

The test sample was provided with a cyclic potential interval by linear sweep voltammetry using the scanning voltammeter. The initial potential of the scanning potential interval was -0.1 V, and the end potential was +0.6 V, and the scanning rate was 250 mV/s. Using the scanning voltammeter, the oxidation current of the 4-aminophenol in the test sample between the oxidation potentials of +0.25 V and +0.35 V was detected. The oxidation current value is brought into the linear relationship equation to obtain the activity of the neuraminidase in the biological sample.

<Interference experiment and verification reproducibility>

Example 2

An appropriate amount of the first stock solution of Preparation Example 3, the second stock solution of Preparation Example 4, and 245 μL of fetal bovine serum (label: Gibco Life Technologies; model: A31606-01) were mixed, and Preparation Example 2 was used. The buffer solution is configured to have a total volume of 1 mL of the detection solution, wherein the concentration of the neuraminidase in the detection solution is 150 ng/mL and the acetaminophenamine compound represented by the formula (I-1) The concentration is 1 mM. The test solution was heated to 37 ° C for a hydrolysis reaction for 30 minutes to produce 4-aminophenol to form a test sample. The test sample was tested using the scanning voltammeter, and the test conditions were as described in Example 1. The current value of the 4-aminophenol was -1.59 μA.

Examples 3 to 5

Examples 3 to 5 were carried out in the same manner as in Example 2, except that the fetal calf serum was replaced with saliva, urine, and blood, and the amounts used were 500 μL, 495 μL, and 495 μL, as shown in Table 1. Shown. The saliva and the urine are from a healthy human body. The blood was provided by Taipei Medical University.

Comparative Example 1

An appropriate amount of the first stock solution of Preparation Example 3 was mixed with the buffer solution of Preparation Example 2, and configured to have a total volume of 1 mL of the test solution, wherein the test solution had the B shown by the formula (I-1). The concentration of the ruminine compound was 1 mM. The test solution was detected using the scanning voltammeter, and the test conditions were as described in Example 1. The current value obtained after the detection was -0.91 μA.

Comparative Example 2

An appropriate amount of the first stock solution of Preparation Example 3 and 245 μL of fetal bovine serum were mixed, and the buffer solution of Preparation Example 2 was used to prepare a detection solution having a total volume of 1 mL, wherein the detection solution was The concentration of the acetaminophenamine compound shown in I-1) was 1 mM. The test solution was detected using the scanning voltammeter, and the test conditions were as described in Example 1. The current value obtained after the detection was -0.91 μA.

Comparative Examples 3 to 5

Comparative Examples 3 to 5 were carried out in the same manner as in Comparative Example 2, except that the fetal calf serum was replaced with saliva, urine, and blood, and the amounts used were 500 μL, 495 μL, and 495 μL, as shown in Table 1. Shown.

Table 1  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> "--" means not added</td><td> fetal calf serum (μL) < /td><td> Saliva (μL) </td><td> Urine (μL) </td><td> Blood (μL) </td><td> Neuraminidase concentration (ng/mL) </td><td> Current value of detection solution (μA) </td><td> Current value (μA) of 4-aminophenol in standard sample or test sample </td></tr><tr ><td> The neuraminidase standard solution of Example 1</td><td> -- </td><td> -- </td><td> -- </td><td> -- </td><td> 150 </td><td> — </td><td> -1.59 </td></tr><tr><td> Test solution of Comparative Example 1</td>< Td> -- </td><td> -- </td><td> -- </td><td> -- </td><td> -- </td><td> -0.91 < /td><td> — </td></tr><tr><td> The detection solution of Example 2</td><td> 245 </td><td> -- </td><td > -- </td><td> -- </td><td> 150 </td><td> — </td><td> -1.59 </td></tr><tr><td > Test solution of Comparative Example 2</td><td> 245 </td><td> -- </td><td> -- </td><td> -- </td><td> - - </td><td> -0.91 </td><td> — </td></tr><tr><td> Detection solution of Example 3</td><td> -- </td ><td> 500 </t d><td> -- </td><td> -- </td><td> 150 </td><td> — </td><td> -1.59 </td></tr>< Tr><td> Test solution of Comparative Example 3</td><td> -- </td><td> 500 </td><td> -- </td><td> -- </td> <td> -- </td><td> -0.89 </td><td> — </td></tr><tr><td> The detection solution of Example 4</td><td> - - </td><td> -- </td><td> 495 </td><td> -- </td><td> 150 </td><td> — </td><td> -1.59 </td></tr><tr><td> Test solution of Comparative Example 4</td><td> -- </td><td> -- </td><td> 495 </ Td><td> -- </td><td> -- </td><td> -0.90 </td><td> — </td></tr><tr><td> Example 5 Detection solution</td><td> -- </td><td> -- </td><td> -- </td><td> 495 </td><td> 150 </td> <td> — </td><td> -1.59 </td></tr><tr><td> Test solution of Comparative Example 5</td><td> -- </td><td> - - </td><td> -- </td><td> 495 </td><td> -- </td><td> -0.89 </td><td> — </td></ Tr></TBODY></TABLE>

It can be seen from the experimental results in Table 1 that the method for detecting the activity of the neuraminidase of the present invention is not easily disturbed by the complexity of the biological sample, resulting in a large deviation in the detection, so the method for detecting the activity of the neuraminidase of the present invention is Good accuracy.

<Storage Stability>

Example 6

The acetaminophenamine compound represented by the formula (I-1) of Preparation Example 1 was allowed to stand at room temperature for 30 days. Next, it was mixed with the buffer of Preparation Example 2 to prepare a third stock solution in which the concentration of the acetaminophenamine compound represented by the formula (I-1) was 10 mM. An appropriate amount of the third stock solution and the second stock solution of Preparation Example 4 were mixed, and the buffer solution of Preparation Example 2 was used to prepare a detection solution having a total volume of 1 mL, wherein the neuraminidase was detected in the test solution. The concentration was 150 ng/mL and the concentration of the acetaminophenamine compound represented by the formula (I-1) was 1 mM.

The test solution was heated to 37 ° C for a hydrolysis reaction for 30 minutes to produce 4-aminophenol to form a test sample. The test sample was tested using the scanning voltammeter, and the test conditions were as described in Example 1. The current value of the 4-aminophenol was -1.59 μA.

Examples 7 to 8

Examples 7 to 8 were carried out in the same manner as in Example 6, except that the acetaminophenamine compound of the formula (I-1) of Preparation Example 1 was allowed to stand at room temperature for 60 days and 90 hours. Days, and the test results are shown in Table 2.

Table 2  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> The concentration of neuraminidase from Example 1 was 150 ng/ Standard sample of mL neuraminidase standard solution</td><td> Test sample of the example</td></tr><tr><td> 6 </td><td> 7 </td> <td> 8 </td></tr><tr><td> Current value of 4-aminophenol (μA) </td><td> -1.59 </td><td> -1.59 </td ><td> -1.59 </td><td> -1.59 </td></tr></TBODY></TABLE>

As is apparent from the experimental results in Table 2, the acetaminophenamine compound represented by the formula (I) has stability and is not easily deteriorated, so that the method for detecting the activity of the neuraminidase of the present invention does not have a large deviation in detection.

In summary, the method for detecting neuraminidase activity of the present invention is simple and rapid by passing the acetaminophenamine compound represented by formula (I) having thermal stability and storage stability in combination with an electrochemical method. High sensitivity characteristics. At the same time, it is highly reproducible when applied to the detection of biological samples such as blood or serum, and thus it is indeed possible to achieve the object of the invention.

However, the above is only the embodiment of the present invention, and the scope of the invention is not limited thereto, and all the equivalent equivalent changes and modifications according to the scope of the patent application and the patent specification of the present invention are still The scope of the invention is covered.

Claims (10)

A method for detecting neuraminidase activity, comprising the steps of: (a), providing a biological sample comprising a neuraminidase, and mixing the biological sample with an acetaminophenamine compound of formula (I) The hydrolysis reaction is carried out in the presence of a buffer to produce a hydroxyaromatic compound represented by HO-X ³ , Formula (I) X ¹ and X ² each represent hydrogen or methyl; X ³ represents , or ; R ¹ , R ³ and R ⁵ each represent hydrogen, -NH ₂ , -OH or -OCH ₃ , but R ¹ , R ³ and R ⁵ may not be hydrogen at the same time; R ² and R ⁴ are hydrogen; R ⁶ and R ⁸ each represents hydrogen, -NH ₂ , -OH or -OCH ₃ , but R ⁶ and R ⁸ may not be hydrogen at the same time; R ⁷ is hydrogen, A is an aromatic ring; and R ⁹ is -NH ₂ , -OH or -OCH ₃ B and C each represent an aromatic ring; in step (b), a voltage is applied to cause a redox reaction of the hydroxyaromatic compound, and a current of the hydroxyaromatic compound is detected to confirm the activity of the neuraminidase. A method for detecting neuraminidase activity according to claim 1, wherein in the step (b), the voltage is supplied using a scanning voltammeter and the current of the hydroxyaromatic compound is detected. The method of detecting neuraminidase activity according to claim 2, wherein in the step (b), the scanning voltammeter provides a scanning potential interval, and the scanning potential interval is ±0.8 volts of the voltage. The method for detecting neuraminidase activity according to claim 3, wherein in the step (b), the scanning voltammeter has a potential scanning rate ranging from 100 mV/s to 400 mV/s. The method for detecting neuraminidase activity according to claim 1, wherein in the step (a), the temperature of the hydrolysis reaction ranges from 25 ° C to 40 ° C, and the time ranges from 15 minutes to 60 minutes. The method for detecting neuraminidase activity according to claim 1, wherein the pH of the buffer ranges from 5.0 to 7.0. A method for detecting neuraminidase activity according to claim 6, wherein the buffer comprises phosphate. The method for detecting neuraminidase activity according to claim 1, wherein in the step (a), the biological sample is selected from the group consisting of serum, saliva, urine, or blood. The method for detecting neuraminidase activity according to claim 1, further comprising a step (c) of providing a relationship equation between the current of the hydroxyaromatic compound represented by HO-X ³ and the concentration of neuraminidase, and Using the relationship equation and the current of the hydroxyaromatic compound obtained in the step (b), the concentration of the neuraminidase in the biological sample is calculated. The method for detecting neuraminidase activity according to claim 9, wherein in the step (c), a step of obtaining the relationship equation includes providing a plurality of neuraminidase standard solutions, and the like The neuraminidase standard solution comprises a neuraminidase, an acetaminophenamine compound represented by the formula (I), and a buffer, and the concentration of the neuraminidase in the standard solution of the neuraminidase is known and not The same; each of the neuraminidase standard solutions is subjected to a hydrolysis reaction to produce a hydroxyaromatic compound represented by HO-X ³ ; a voltage is applied to cause the hydroxyaromatic compound to undergo a redox reaction, and the hydroxyaromatic compound is detected Current; the current is plotted against the concentration of neuraminidase of the neuraminidase standard solution to obtain the equation of interest.