JPH08166388A - Method and sensor for immunoassay of enzyme - Google Patents

Abstract

PURPOSE: To provide the simple enzyme immunity sensor and the measuring method thereof, which do not require cleaning and substrate adding operation at the time of measurement. CONSTITUTION: Together with antigen to be measured 3, enzyme marker antigen 4 and an enzyme substrate 5 for the antigen 4 and pH buffer material 8 are dissolved into a sample. A pH sensor, wherein antibody 2 is fixed on a pH sensitive part 1, is immersed into the solution. The antigen to be measured 3 and the enzyme marker antigen 4 are made to compete and bonded to the antibody 2. In a product 6, which is formed by the reaction of the enzyme marker antigen 4 bonded to the antibody 2 and the substrate, the antibody 2 indicates the action of a limited transmission film and blocks the pH buffer material 8. Therefore, the product 6 contributes to the pH change of the pH sensor part. The product 6, which is formed by the enzyme marker antigen 4, which is not bonded to the antibody 2, receives the pH buffering of the pH buffering material 8 and does not contribute to the pH change. Thus, the concentration of the antigen 3 in the sample can be obtained by the amount of the pH change.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the concentration of a substance contained in a sample solution in a trace amount, and more particularly to an enzyme immunoassay method and an enzyme immunosensor.

[0002]

2. Description of the Related Art An immunoassay is known as a method for measuring a trace amount of a component by utilizing a specific reaction between an antigen and an antibody. Among immunoassays, there is a method using an enzyme-labeled antigen or antibody, which is called enzyme immunoassay. The enzyme immunoassay method includes a competitive method and a non-competitive method (sandwich method), and the principle is shown below.

In the competitive reaction method, a sample (including an antigen to be measured) is first added with a fixed amount of an enzyme-labeled antigen in which the same antigen as that to be measured is labeled with an enzyme in advance, and both antigens and a fixed amount of antibody are added. React. Next, the antigen-antibody complex is separated from unreacted antigen or antibody by an appropriate method. Finally, the activity of the labeling enzyme in the antigen-antibody complex is measured to obtain the concentration of the antigen to be measured.

In the non-competitive reaction method, a two-step antigen-antibody reaction is carried out. First, the antigen to be measured is reacted with the primary antibody. Then, the enzyme-labeled secondary antibody is reacted. Then, unreacted substances are removed. Finally, the activity of the labeling enzyme of the immune complex is quantified to determine the concentration of the antigen to be measured.

Since the enzyme immunoassay is based on the measurement of enzyme activity as described above, it is simpler to use and more sensitive than other methods (for example, radioisotope), and is widely applied in the field of clinical examination and the like. There is.

In the enzyme immunoassay, it is important to measure the enzyme activity. Conventionally, the enzyme activity can be measured by measuring the absorbance and fluorescence intensity of a substrate or product which is affected by the enzyme, and by measuring pH and oxygen concentration. The measurements have taken place. Devices based on these methods were large in size, and the amount of sample required for measurement was not small. For this reason, JP-A 64-59
Japanese Patent Application Laid-Open No. 057 and Japanese Patent Application Laid-Open No. 64-59058 propose a small enzyme immunoassay method and sensor capable of measuring a small amount of sample. In order to clarify the prior art in more detail, a specific description will be given with reference to the drawings. FIG. 3 schematically shows a case where the measuring method disclosed in JP-A 64-59057 is a competitive reaction. The antibody 2 is immobilized on the pH sensor 1 which detects the pH change of the solution (1). The sensor 1 is reacted with a sample (including the antigen 3 to be measured) to which the enzyme urease-labeled antigen 4 is added in a fixed amount (2). Next, when the sensor 1 is washed and the sample is removed, the labeled antigen 4 and the antigen 3 to be measured are bound to the antibody 2 in accordance with the concentration ratio when the sample was contained (3). Next, when the sensor 1 is immersed in a solution containing urea 5 which is a substrate of the enzyme urease, ammonia 6 is produced by the enzyme urease (4). By measuring the pH change due to ammonia with a pH sensor, the concentration of the antigen to be measured can be determined. By immobilizing the antibody on the minute pH sensor as described above, a small-sized enzyme immunosensor capable of measuring a small amount of sample has been realized. However, the sensor needs to be washed and replaced with a solution containing the substrate.

FIG. 4 is a schematic sectional view of the immunosensor disclosed in Japanese Patent Laid-Open No. 64-59058. N on the substrate 16
Type silicon 9 and p type silicon 10, gate oxide film 1
1. Two pH-sensitive semiconductor sensors composed of the silicon nitride film 12 are formed. Further, the pseudo reference electrode 13 is formed on the substrate 16. The antibody 2 is immobilized on the pH-sensitive portion of one of the pH-sensitive semiconductor sensors. A protein cross-linking film 14 is formed on the other pH sensing portion and the pseudo reference electrode 13. The flat plate 15 is arranged so as to face the sensor substrate 16 with a minute gap therebetween. The urease labeled antigen 4 is coated on the flat plate 15.
The method for measuring the antigen by this sensor is described in the above-mentioned JP-A-64-5.
This is almost the same as that of the 9057 publication. First, when the sensor is dipped in the sample, a fixed amount of the sample is generated on the substrate 16 by the capillary phenomenon.
Is sucked up into the space between the flat plate 15. At this time, the urease-labeled antigen 4 dissolves in the sample and competes with the antigen to be measured in the sample to bind to the antibody 2. Next, the sensor is washed and immersed in a solution containing urea to cause an enzymatic reaction. At this time, the pH change due to ammonia generated by the reaction between the enzyme (urease) and the substrate (urea) is measured by the pH-sensitive reaction sensor on which the antibody 2 is immobilized. By providing the plate 15 coated with the enzyme-labeled antigen having a known enzyme and antigen concentration in advance, it became unnecessary to quantify the sample, but this enzyme immunosensor also has the above-mentioned JP-A-64-5.
As in Japanese Patent No. 9057, it is necessary to clean the sensor and replace it with a urea solution.

[0008]

As described above, in the conventional enzyme immunosensor, the operation of washing the sensor and the operation of adding the substrate solution of the enzyme are required. Therefore, there is a problem that not only the user himself is troublesome but also an error occurs.

Therefore, an object of the present invention is to provide an enzyme immunoassay method and an enzyme immunosensor, which does not require the user to perform any operation other than immersing the sensor in a sample.

[0010]

To solve the above problems, the enzyme immunoassay method of the present invention achieves the above object by dissolving a pH buffer substance and an enzyme substrate in a sample together with an enzyme-labeled antigen. There is.

The first aspect of the present invention is to provide a sample containing an antigen to be measured with an enzyme-labeled antigen in which an enzyme for converting a substrate into an acid or a basic substance is pre-labeled with the antigen, and a substrate for the enzyme. And a pH buffer substance is added, and the pH sensor in which the antibody specifically reacting with the antigen is immobilized on the pH sensing unit is immersed in the added sample,
An enzyme immunoassay method characterized by measuring an antigen concentration in a sample by detecting a pH change due to an enzyme of an enzyme-labeled antigen bound by an antibody-antigen reaction with an antibody immobilized on a pH sensing unit. is there.

A second aspect of the present invention is a pH sensor in which an antibody that specifically reacts with an antigen to be measured is immobilized on a pH sensing unit, and an enzyme that converts a substrate into an acid or a basic substance. An enzyme-labeled antigen pre-labeled with an antigen, a substrate for the enzyme, and a pH buffer substance are immobilized on a plate surface, and the immobilization surface is installed facing the surface of the pH sensing unit with a minute gap. It is an enzyme immunosensor characterized in that it is composed of a flat plate.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing an enzyme immunoassay method using a pH sensor according to an embodiment of the present invention. The case where urease is used as the enzyme is shown. The antibody 2 is immobilized on the pH sensing unit 1 of the pH sensor. The measurement target antigen 3 and the added urease-labeled antigen 4 in the sample compete with each other,
It binds to the antibody 2 according to the concentration ratio of the measurement target antigen 3 and the urease-labeled antigen 4 when added and mixed. The labeled enzyme urease converts the added substrate (urea) 5 into a basic product (ammonia) 6 by the action of the enzyme, regardless of whether it binds to the antibody 2. The product 6 (ammonia) produced by urease of the urease-labeled antigen 7 that is not bound to the antibody is consumed by the action of the pH buffer substance 8 in the sample, and therefore does not contribute to the pH change of the pH sensor surface 1. Can not. On the other hand, the product 6 (ammonia) produced by the urease of the urease-labeled antigen 4 bound to the antibody 2 is located near the pH sensor surface 1 and the immobilized antibody 2 exhibits the function of a limiting permeable membrane.
Since it impedes the permeation of the H buffer substance 8, it does not need to undergo pH buffering and changes the pH of the pH sensor surface 1. Therefore, only the urease-labeled antigen bound to antibody 2 is
Since it can be measured from the change, the concentration of the antigen 3 to be measured can be obtained from the value.

Further, FIG. 2 shows a structure (cross-sectional view) of an enzyme immunosensor which is an embodiment of the present invention.

A total of two pH-sensitive semiconductor sensors consisting of n-type silicon 9 (source and drain) and p-type silicon 10 (channel), gate oxide film 11 and silicon nitride film 12 are formed on the substrate 16. There is. Further, the pseudo reference electrode 13 is formed on the substrate 16. The antibody 2 is immobilized on the pH-sensitive portion of one of the pH-sensitive semiconductor sensors. PH of the other pH-sensitive semiconductor sensor
A protein cross-linking film 14 is formed on the sensing section and the pseudo reference electrode. The flat plate 15 is opposed to the sensor substrate with a minute gap therebetween. A urease-labeled antigen 4, a substrate (urea) 5, and a pH buffer substance 8 are coated on the plate 15. The operating method of the pH-sensitive semiconductor sensor is to apply a constant voltage between the source and the drain and change the voltage between the source and the pseudo reference electrode 13 so that a constant current flows. Since this voltage value is proportional to the pH value of the solution, the pH value of the solution can be detected. In practice, in order to eliminate the influence of the background output due to the sample, the value obtained by subtracting the output of the pH sensor without the antibody immobilized from the output of the pH sensor with the antibody immobilized is used as the response.

The enzyme immunoassay method using the enzyme immunosensor is as follows. First, when the sensor is dipped in the sample, a fixed amount of the sample is sucked up into the space between the substrate 16 and the flat plate 15 by the capillary phenomenon. At this time, the urease-labeled antigen 4, the substrate 5, and the pH buffer substance 8 on the plate 15 are dissolved in the sample.
The urease-labeled antigen 4 competes with the antigen to be measured in the sample and binds to the antibody 2. The urease of the urease-labeled antigen 4 bound to the antibody 2 is located in the vicinity of the pH-sensing part, and the immobilized antibody 2 acts like a limiting permeation membrane to prevent the pH buffer substance 8 from permeating, so that The enzyme activity produces a product (ammonia), which changes the pH of the pH sensing surface. On the other hand, the product produced by the urease of the urease-labeled antigen 4 that is not bound to the antibody 2 is consumed by the action of the pH buffer substance 8 in the sample, and therefore cannot contribute to the pH change of the pH sensitive part. Therefore, only the urease-labeled antigen 4 bound to the antibody 2 can be detected as a pH change, and the concentration of the measurement target antigen can be determined from the value.

Next, the results of actually measuring the concentration of human immunoglobulin G (hereinafter abbreviated as human IgG) antigen in the sample using the enzyme immunosensor according to the present invention will be described. The enzyme immunosensor was prepared as follows.
First, 15 on the pH sensing part of one sensitive semiconductor sensor.
A protein cross-linked film was formed by spin coating a mixed solution of 1% bovine serum albumin and 1% glutaraldehyde. Next, a 15% bovine serum albumin containing 10% anti-human IgG antibody and a 1% glutaraldehyde mixed solution were spin-coated to immobilize the antibody on the pH sensitive part. In addition, 1% urease (enzyme) -labeled human IgG antigen solution 4 μl, 0.5% HEPES-NaOH pH buffer solution 4 μ
1 and 4 μl of 0.1% urea (substrate) were individually applied and attached, and the flat plate was arranged and mounted facing the pH sensor substrate at intervals of 0.1 mm. About 2 μl of the serum sample was sucked between the pH sensor substrate and the plate, and the response output was measured for 5 minutes. The resulting sensor response output and human I in the sample
FIG. 5 shows the relationship between the gG concentrations. Although the response output of the sensor according to the present invention is slightly smaller than that of the conventional sensor, it shows a substantially equivalent measurable range.

Although human IgG has been described as an example of the antigen to be measured in this example, other antigens can be measured by the enzyme immunosensor of the present invention in exactly the same manner by using corresponding antibodies. Is possible. For example, proteins corresponding to proteins such as immunoglobulins and enzymes, peptides such as human chorionic gonadotropin and α-fetoprotein, drugs such as zicoxin and theopherin, viruses such as hepatitis, pesticides such as atrazine and triazine, and corresponding antibodies to pH sensing parts. If the above is made, the measurement according to the present invention can be applied. Also, the enzyme used for the measurement may be any one as long as it converts a substrate into an acid or a basic substance.

Although an example based on the competitive reaction method has been described in the present embodiment, the measurement can be performed by the method of the present invention in the same manner even in the non-competitive reaction method. That is, it can be applied by immobilizing the primary antibody on the surface of the pH sensor and using the urease-labeled secondary antibody instead of the urease-labeled antigen.

[0020]

INDUSTRIAL APPLICABILITY As described above, the enzyme immunosensor and the method for measuring the same according to the present invention, by dissolving the substrate and the pH buffer substance together with the enzyme-labeled antigen in the sample,
There is an effect that the measurement can be performed without any operation such as washing or addition of a reagent at the time of measurement other than immersing the sensor in the sample, and the operation is simple.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a measuring method of an enzyme immunosensor according to the present invention.

FIG. 2 is a schematic view showing a cross section of an enzyme immunosensor according to the present invention.

FIG. 3 is an explanatory view showing a measuring method of a conventional enzyme immunosensor.

FIG. 4 is a schematic diagram showing a cross section of a conventional enzyme immunosensor.

FIG. 5 is a diagram showing the results of measuring human immunoglobulin G by an enzyme immunosensor according to the present invention and a conventional sensor.

[Explanation of symbols]

 1 pH sensor 2 Antibody 3 Antigen to be measured 4 Enzyme (urease) -labeled antigen 5 Substrate (urea) 6 Product (ammonia) 7 Enzyme (urease) -labeled antigen that did not bind to antibody 8 pH buffer substance 9 n-type silicon 10 p Type silicon 11 gate oxide film 12 silicon nitride film 13 pseudo reference electrode 14 protein cross-linking film 15 flat plate 16 substrate

Claims (2)

[Claims] 1. A sample containing an antigen to be measured, an enzyme-labeled antigen in which an enzyme for converting a substrate into an acid or a basic substance is pre-labeled with the antigen, and a substrate for the enzyme.
A pH buffer substance was added, and a pH sensor in which an antibody specifically reacting with the antigen was immobilized on the pH sensing unit was immersed in the added sample, and immobilized on the pH sensing unit. An enzyme immunoassay method, which comprises measuring an antigen concentration in the sample by detecting a pH change due to an enzyme of the enzyme-labeled antigen bound to the antibody by an antigen-antibody reaction. 2. A pH sensor in which an antibody that specifically reacts with an antigen to be measured is immobilized on a pH sensing unit, and an enzyme that converts a substrate into an acid or a basic substance is pre-labeled on the antigen. The enzyme-labeled antigen, the substrate for the enzyme, and the pH buffer substance are immobilized on a plate surface, and the immobilization surface is placed at a minute interval so as to face the pH sensing unit surface. An enzyme immunosensor comprising: