JPH1010065A - Immunosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To inexpensively and simply detect an antibodyantigen reaction by measuring the electric change between antibody immobilized electrodes or between antigen immobilized electrodes accompanying the antibody-antigen reaction by use of a general measuring instrument. SOLUTION: The electrodes of a pair of antibody immobilized electrodes or antigen immobilized electrodes are electrically connected to each other by a protein layer containing antibody or antigen to constitute an electrode part. In the electrode part, two antibody immobilized electrodes are used when a subject is antigen, and two antigen immobilized electrodes when the subject is antibody. As the antibody of the antibody immobilized electrode, each of monoclonal antibody and polyclonal antibody, if it has a property of specifically recognizing antigen, can be used. As the method of immobilizing the antibody and antigen to the electrode surface, immobilization by adsorption and chemical bonding to electrode surface are given. The protein layer containing the antibody or antigen between the electrodes preferably has a conductivity of 10<-5> -105<-6> S/cm. For measurement of electric change of the electrode part, a general conductimeter can be used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunosensor for detecting the presence or absence of an antibody-antigen reaction or the degree of progress thereof.

[0002]

BACKGROUND ART As a method for detecting a substance using an antibody, a method of immobilizing an antibody or antigen on a solid phase such as plastics and labeling the antigen or antibody with a radioisotope or an enzyme is widely known. For example, the details are described in a review by Kuniyo Inoue (Chemistry and Biology, vol. 34, p240, 19).
96). Further, for example, JP-A-6-261784,
JP-A-7-159403, Forensics
science international, vol.
27, p49, 1985 and Journalof a
analytic toxicology, vol.
16, p211, 1992, etc., have proposed specific methods.

A method using a piezo element as a method for measuring a mass change accompanying an antibody-antigen reaction (for example, Japanese Patent Application Laid-Open No.
No. 95443 and Sensors and Actu
ators B, vol. 13-14, p188, 19
93, etc.) and a method using photoacoustic (for example, JP-A-3-29
No. 836) or a method using a surface acoustic wave (for example, JP-A-6-133759) has been proposed.

As a method for optically measuring an antibody-antigen reaction, a method using surface plasmon resonance is disclosed in, for example,
JP-A-224647, JP-A-2-103469,
It has been proposed in Japanese Patent Application Laid-Open No. 2-297053.

As a method for measuring an antibody-antigen reaction as an electrical change, a method for measuring a membrane potential difference of a membrane on which an antibody or an antigen is immobilized (for example, Journal of Membrane).
Brane Science, vol. 7, p1, 19
80 and Analytica Chimica Acta,
vol. 274, p59, 1993, etc.) and a method for measuring the electrode potential difference of an electrode on which an antibody or an antigen is immobilized (for example, JP-B-59-37462, Clin. Chem.,
26, p1569, 1980), a method using an ion-sensitive field-effect transistor having an antibody or antigen immobilized on a gate portion (for example, Japanese Patent Application Laid-Open No. 1-119753 and Japanese Patent Publication No. 3-26345) has been proposed. I have.

A method for measuring the change in the electric conductivity of pure water filled between the electrodes on which the antibody or the antigen is immobilized using the electrode on which the antibody or the antigen is immobilized is disclosed in Japanese Patent Application Laid-Open No. 6-82.
No. 3287, 1993 Annual Meeting of the Electrochemical Society of Japan, vol. 60th, p. 214, 1993.

The method of using an antigen or an antibody labeled with a radioisotope or an enzyme measures the radioactivity and the enzyme activity after washing after the antibody-antigen reaction, so the operation is complicated, and the measurement is time-consuming. Take it. Since the measurement of an antigen (hapten) having a smaller molecular weight is performed by a competitive method, high sensitivity measurement cannot be expected.

A method using a piezo element, photoacoustic, surface acoustic wave or surface plasmon resonance cannot be a general-purpose method because the measuring device is large and expensive. Further, conventional methods for measuring an antibody-antigen reaction as an electrical change have been proposed, although many methods have been proposed, but have problems in measurement sensitivity and operability, and have not yet been put to practical use.

A detailed examination of the contents of Japanese Patent Publication No. 6-823287, which has been proposed as a method for measuring the change in electric conductivity, shows that the electrode area is 0.785 cm ² , the gap between electrodes is 0.
The electric conductivity of pure water measured with a 05 cm electrode cell is 65.
μS / cm, which is 1 μS / c which is a general concept of pure water.
m or less.

Further, a pole area of 0.785 described in this publication
In order to measure 1 μS / cm or less, which is the concept of pure water, using an electrode cell having a cm ² of 0.05 cm between the electrodes, it is necessary to make the distance between the opposed antibody-immobilized electrodes about 8 μm. Although it is calculated, it can be easily estimated that it is extremely difficult to prepare an electrode cell having a pole area of 0.785 cm ² and a distance between the electrodes of about 8 μm. Thus, Japanese Patent Application Laid-Open No. Hei 6-8232
The method proposed in the 87th Gazette has to be said to be incorrect or impractical.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to cause an antibody-antigen reaction to be performed on an antibody-immobilized electrode or on an antigen-immobilized electrode, and to determine the presence or absence of the reaction or the extent of the reaction in accordance with the antibody-antigen reaction. It is an object of the present invention to provide an inexpensive and easy-to-operate immunosensor that can be measured as a change in electrical properties between immobilized electrodes or between antigen-immobilized electrodes.

[0012]

Means for Solving the Problems The present inventors measure the antibody-antigen reaction by measuring the change in the electrical properties between the antibody or the antigen-immobilized electrode accompanying the antibody-antigen reaction using a general-purpose measuring instrument. Many experiments have been performed with the aim of providing an inexpensive and simple method.

As a result, the electrical properties between the antibody or antigen-immobilized electrode due to the antibody-antigen reaction using two antibody-immobilized electrodes or antigen-immobilized electrodes on which the antibody or antigen is immobilized on the surface. Changes were measurable. Further, in that case, by changing the area of the antibody-immobilized electrode or the antigen-immobilized electrode from each other, the change in the electrical properties between the antibody-immobilized electrodes or between the antigen-immobilized electrodes is increased, and It has been found that the change requires an antibody or antigen-containing layer for electrically coupling between the antibody-immobilized electrodes or between the antigen-immobilized electrodes, and the present invention has been accomplished.

According to the present invention, there is provided (A) a pair of an antibody-immobilized electrode or an antigen-immobilized electrode having an antibody or an antigen immobilized on the surface thereof, and the electrode immobilized electrode or the antigen-immobilized electrode is provided between the electrodes. The present invention relates to an immunosensor comprising an electrode portion electrically connected by a protein layer containing an antibody or an antigen, and (B) a measuring device for measuring electric properties between these electrodes.

In using the immunosensor according to the present invention, the electrode portion comprises two antibody-immobilized electrodes when the object to be measured is an antigen, and the electrodes when the object to be measured is an antibody. The section uses two antigen-immobilized electrodes, respectively.

The object to be measured is an antigen, and the electrode part is 2
In the case of a single antibody-immobilized electrode, any of a monoclonal antibody and a polyclonal antibody can be used as long as the antibody has a property of specifically recognizing an antigen to be measured. However, it is needless to say that the monoclonal antibody is superior to the polyclonal antibody in consideration of the detection sensitivity and stability of the properties of the antibody.

As the material of the electrode for immobilizing the antibody or the antigen, any material can be used as long as a change in the electrical properties of the electrode portion can be detected. For example, gold, silver, and the like can be used.
Examples thereof include metals such as copper, platinum, nickel and tin, metal oxides such as zinc oxide, and various kinds of conductive synthetic resins.

As a method for immobilizing an antibody or an antigen on an electrode surface, an electrochemical method-applied measurement manual (edited by Osaka and Koyama, Kodansha Scientific, 1991)
As described in (1), for example, immobilization by adsorption, chemical bonding to the electrode surface, and the like can be mentioned. Further, specific methods of the chemical bonding method to the electrode surface are described in Japanese Patent Application Laid-Open No. Hei 6-823287 and 1993 Annual Meeting of the Electrochemical Society of Japan (vol. 60th, p2).
14, 1993), using a silane coupling agent such as δ-aminopropyltriethoxysilane to bond an antibody or antigen to a functional group such as an amino group introduced on the electrode surface with a cross-linking agent such as glutaraldehyde. Methods can be mentioned.

As a crosslinking agent for binding an antibody or an antigen to a functional group such as an amino group or a carboxyl group introduced on the electrode surface, besides glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is used. Dimethylformamide, maleimidobenzoyloxysuccinimide and the like.

The pair of antibody-immobilized electrodes or antigen-immobilized electrodes constituting the electrode portion used in the present invention has a property that the measurement sensitivity is improved due to the different areas. In the method proposed in Japanese Patent Application Laid-Open No. Hei 6-823287, various measures have been proposed to increase the area of the antibody-immobilized electrode in order to increase the sensitivity. There is no technical idea to increase or decrease one of the electrodes.

The present inventors use a comb-shaped electrode portion in which thin electrodes are arranged in parallel on a single substrate, and detect the area ratio of two electrodes that form a pair of an antibody-immobilized electrode or an antigen-immobilized electrode. The sensitivity was studied. As a result, it has been found that the detection sensitivity is improved as the area ratio of two pairs of antibodies or antigen-immobilized electrodes is different from one.

Of course, in the present invention, it is preferable that one of the pair of electrodes has a large electrode area. For this purpose, the concept of a comb-shaped electrode or a spiral-shaped electrode described in Japanese Patent Application Laid-Open No. 6-823287 can be applied.

In the electrode portion used in the present invention, it is important that the antibody-immobilized electrode or the antigen-immobilized electrode is electrically connected by a protein containing an antibody or an antigen. Japanese Patent Application Laid-Open No. 6-823287 describes that an antibody is immobilized on an electrode, but the technical idea of interposing a protein layer containing the antibody or antigen between the electrodes is described. Has no suggestion. The conductivity of the protein layer containing the antibody or the antigen is 10 ⁻⁴ S / cm or less, and preferably 10 ⁻⁷ S / cm or more. A particularly preferred range of the conductivity is 10 ^-5 to 10 ^-6 S / cm.

In the present invention, when an antibody-antigen reaction is detected using an electrode portion having only one pair of an antibody-immobilized electrode or an antigen-immobilized electrode, the electric potential between the electrodes is measured before and after the antibody-antigen reaction. Each change must be measured,
Two measurement operations are essential. However, if a reference electrode on which no antibody or antigen is immobilized is incorporated in the electrode part, the measurement of the antibody-antigen reaction can be performed once.

The preparation of the reference electrode without immobilizing the antibody or antigen is performed by physically masking the surface of the reference electrode with a Teflon tape or the like so that the immobilization reaction of the antibody or antigen does not reach. This can be easily performed.

Further, by incorporating a guard electrode into the electrode portion used in the present invention, electrical noise can be eliminated and sensitivity can be further improved.

As a specific example of the measuring device for measuring the electrical change of the electrode portion of the present invention, a general-purpose electric conductivity meter can be mentioned. However, since the electrical conductivity is the reciprocal of the resistance value of the solution, and the resistance value is a function of the current value and the voltage value, the measuring instrument can measure the resistance value, the current value or the voltage value between the electrodes. It goes without saying that any measuring instrument may be used.

The immunosensor of the present invention is extremely useful for a method for detecting methamphetamine.

With the increase in stimulant related cases,
There is an increasing need to simply, quickly and accurately recognize the user of methamphetamine, and a method and a measurement kit for measuring methamphetamine in a simple, highly sensitive and highly selective manner are desired.

At present, a measurement system using a monoclonal antibody that specifically recognizes methamphetamine is an enzyme immunoassay (Enzyme Linked Immuno).
A competitive method based on the sorbent assay method (ELISA method) has been proposed. However, in this method, a special scientific instrument such as a spectrophotometer is required to determine the result, and the user is required to have a special technique due to the complicated operation.

In addition, the currently proposed assay kit using a monoclonal antibody is subject to laws and regulations in production and sales because the immobilized antigen which is a component of the kit is methamphetamine. JP-A-7-63755 and JP-A-7-159403 propose a method using N-methylphenethylamine or the like as a pseudoantigen instead of methamphetamine as a component of a kit. The immunosensor of the present invention can be effectively used for detecting such methamphetamine and N-methylphenethylamine.

As the antibody used in the present invention, any antibody can be used as long as it is an anti-methamphetamine antibody. Specifically, for example, described in JP-A-1-96198 and JP-A-6-261784,
Mention may be made of monoclonal antibodies which show very high specific reactivity to methamphetamine obtained by culturing cell lines obtained by immunizing animals.

[0033]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1-1: Preparation of antigen for immunization (1) Synthesis of N-methyl-N- (4-aminobutyl) methamphetamine In order to bind methamphetamine to an appropriate protein, for example, the method of Cheng et al. [FEBS LETTE
RS 36 , 339 (1973)], and an amino group was introduced into N-methylphenethylamine according to the method of Iwasaki et al. [Nichiho-Medical Journal 41 (3), 217 (1987)]. That is, 400 mg of methamphetamine is added to 4
Dissolved in 0 ml of dehydrated benzene and 2.3 g of N-
(4-Bromobutyl) phthalimide and 0.9 g of sodium carbonate were added, and the mixture was refluxed at 80 ° C. for 40 hours in the presence of nitrogen gas. Next, the sodium carbonate was removed by filtration, an equal volume of 1M HCl was added, and the mixture was extracted three times with an equal volume of benzene. An equal volume of chloroform was added to the aqueous layer, and extracted three times. The obtained chloroform layers were combined, dehydrated and concentrated. By this operation, N-methyl-N-butylphthalimide-methamphetamine was obtained.

To the obtained N-methyl-N-butylphthalimide-methamphetamine were added 10 ml of ethanol and 0.1 ml of ethanol.
One ml of 90% hydrazine hydrate was added and reacted at 80 ° C. for 2 hours in the presence of nitrogen gas. After completion of the reaction, ethanol was distilled off, and 20 ml of 1N hydrochloric acid was added. The aqueous solution was extracted three times with an equal volume of chloroform, and the pH of the aqueous layer was adjusted to 10 by dropwise addition of 2N sodium hydroxide. This aqueous solution was extracted three times with an equal volume of chloroform, the chloroform layers were combined, dehydrated, and concentrated. Thus, 1.27 mg of N-methyl-N- (4-aminobutyl) methamphetamine was obtained. Further, using a mass spectrum and a nuclear magnetic resonance analyzer, this N-methyl-N-
The structure of (4-aminobutyl) methamphetamine was confirmed.

(2) Preparation of N-metal-N- (4-aminobutyl) methamphetamine-bovine serum albumin complex An antigen for immunization was synthesized using the above-mentioned N-methyl-N- (4-aminobutyl) methamphetamine. I went. That is, 6 mg of N-methyl-N- (4-aminobutyl) methamphetamine was dissolved in 1 ml of deionized water, 6 mg of bovine serum albumin was added, and 20 mg of 1-ethyl-3- (3-dimethylaminopropyl) was further added. ) Carbodiimide was added, and the pH of the reaction solution was adjusted to 8 with HCl. The reaction was carried out at room temperature with stirring for 16 hours. After the reaction,
The whole reaction solution was treated with a phosphate buffer solution containing physiological saline (pH 7.0).
Dialyzed against 4) and unreacted 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide and N-methyl-N- (4-aminobutyl) methamphetamine,
An N-methyl-N- (4-aminobutyl) methamphetamine-bovine serum albumin complex was obtained. This N-methyl-N- (4-aminobutyl) methamphetamine-bovine serum albumin complex was used as a mouse immunizing antigen.

Example 1-2: Screening for monoclonal antibody specifically reacting with methamphetamine N-methyl-N- (4-aminobutyl) methamphetamine-bovine serum albumin complex synthesized in Example 1-1 was immunized. Mice were inoculated as antigens for 6 months. Mouse spleen cells were taken out according to a standard method and hybridized with myeloma cells (SP2 / 0-Ag14 strain) to obtain hybridoma cells. After cloning,
Using the culture supernatant of each cell, a monoclonal antibody specifically reacting with methamphetamine was searched.

A search for a monoclonal antibody which specifically reacts with methamphetamine was carried out using 96
Using a well microplate, methamphetamine,
Competition inhibition by ephedrine, methylephedrine, etc. was used as an index. An antibody produced by the hybridoma cell line 8Cl was selected as an antibody strongly inhibited only by methamphetamine. This point is described in the specification of Japanese Patent Application No. 6-232314 filed by the present inventors.

Example 1-3: Hybridoma cell 8Cl
Purification of Monoclonal Antibody Produced by Strain (Deposit No. FERM P-13148) Ammonium sulfate was added to 400 ml of the culture supernatant of the 8Cl strain so as to be 45% saturated, and a protein fraction containing the antibody was obtained as an insoluble substance. The protein fraction containing the antibody was separated from the culture supernatant by centrifugation at 15000 rpm for 30 minutes. The protein fraction containing the antibody was redissolved in a phosphate buffer at pH 8, and subjected to affinity chromatography using protein G as a ligand. 26 mg of a monoclonal antibody was obtained by a series of operations.

Example 1-4 Hybridoma Cell 8Cl
Examination of Reaction Specificity of Monoclonal Antibody Produced by Strain (Deposit No. FERM P-13148) Example 1 was added to each well of a carrier (96-well plate for enzyme immunoassay) carrying a methamphetamine-bovine serum albumin complex. 26 mg / ml of the monoclonal antibody produced by the 8Cl strain obtained in Step 3 and 0.1 mg / m of various amines
1 ml of the mixture was dispensed, and allowed to stand at room temperature for 30 minutes. Next, the content of each well was discarded, and the well was washed once with deionized water. A horseradish peroxidase-labeled anti-mouse immunoglobulin G antibody solution was dispensed at 0.1 ml per well, and left still at room temperature for 30 minutes. Thirty minutes later, the contents of each well were discarded, and the well was washed once with deionized water. Then, 0.1 ml of a coloring substrate solution for horseradish peroxidase (ABTS solution) was dispensed per well, and reacted at room temperature for 2 minutes. Two minutes later, the reaction was stopped by adding 10 ml of 1N sulfuric acid per well, and the absorbance at 415 nm was measured using a microplate reader.

The results are shown in FIG. Methamphetamine and N-methylphenethylamine were selected as the amines that gave only 1/3 or less of the color development when the value of the well to which the amines were not added was 1, and the monoclonal antibody specifically reacting with methamphetamine was N-methylphenethylamine. It has been found that can also be recognized.

Example 1-5: Anti-methamphetamine antibody 8
Preparation of Antibody-Immobilized Electrode (Comb Shape) Using Cl (1) Introduction of Amino Group to Electrode Surface As shown in FIG. 2, glass epoxy of 50 mm length and 40 mm width (made by impregnating glass fiber with epoxy resin) FR
P) After copper plating and then gold plating on the substrate, 20 gold-plated copper foil electrodes each having a pole width of 1 mm were vertically formed by etching to prepare an electrode substrate (see FIG. 3A). The electrode substrate was cleaned of fat-soluble stains on the surface with acetone, chloroform and tetrahydrofuran, dried and immersed in 0.1N HCl for 5 minutes to oxidize the surface. The electrode whose surface was oxidized was immersed in a 2 wt% acetone solution of δ-aminopropyltriethoxysilane,
Heat treatment was carried out at 0 ° C. for 18 hours to introduce amino groups on the surface (see FIG. 3B).

(2) FR on electrode surface and between electrodes
P surface Monoclonal antibody 8Cl purified in Example 1-3
It was adjusted to be 00 mg / ml. Glutaraldehyde was added to this solution to a concentration of 0.1 mg / ml, and the electrode (amino group-introduced) substrate prepared in (1) of Example 1-5 was immersed in the solution. After reacting for 4 hours, the antibody was placed on the electrode surface and FRP.
It was immobilized on the surface (see FIG. 3 (c)).

(3) Blocking treatment In order to prevent non-specific adsorption of proteins and the like to the electrode surface,
The FRP substrate treated in the above paragraph (2) was immersed in a 1 wt% bovine serum albumin solution for 18 hours, and bovine serum albumin was fixed between the monoclonal antibodies 8Cl fixed on the FRP surface between the electrodes. Thus, an antibody-containing protein layer was formed between the electrodes (see FIG. 3D).

Example 1-6: Examination of Area Ratio of Two Antibody-Immobilized Electrodes as a Pair That Affects Sensitivity Using the comb-shaped antibody-immobilized electrode produced in Example 1-5,
The effect of pole area on sensitivity was studied. First, an electrode part which is not subjected to any antibody immobilization treatment of the same type as the above-mentioned comb-shaped antibody immobilization electrode (one which has not been subjected to amino group introduction, monoclonal antibody treatment, or blocking treatment)
Was used to measure the conductivity (background value) of pure water. After confirming that the 20 electrodes are electrically connected, the conductivity of pure water between two adjacent electrodes of each of the 20 electrodes is measured using a comb-shaped antibody-immobilized electrode. Then, the conductivity before the antibody-antigen reaction was determined. Next, an antibody-antigen reaction was performed using 10 μg / ml of N-methylphenethylamine as an antigen, and the conductivity of pure water between two adjacent electrodes of each of the 20 electrodes was measured again. Was determined. And the rate of change was calculated by the following equation

## EQU1 ## Rate of change (%) = {(c / a−b / a)} b /
a｝ × 100 a: Background value (mV) b: Conductivity before antigen-antibody reaction (mV) c: Conductivity after antigen-antibody reaction (mV)

FIG. 4 shows the measurement results at 10 V and 80 Hz. The change rate of the conductivity of pure water before and after the antibody-antigen reaction showed a higher value as measured between the electrodes at the end of the substrate, and showed a relatively low value between the electrodes located in the middle of the substrate. .

That is, in the present invention, No. 1 in FIG.
No. 1 to No. 1 As shown in FIG. 3 (d), the monoclonal antibody 8C
1 and bovine serum albumin, and an antibody-containing protein layer is formed. This layer does not show conductivity enough to short-circuit between electrodes. No. 1 electrode and No. 1 electrode. 2
When a voltage is applied between the electrodes, the maximum sensitivity (rate of change%) is exhibited as shown in the data of FIG. The reason for this is
No. No. 2 electrode, that is, the portion to the right of the electrode No. 2 Two electrodes,
No. 2 and No. An antibody-containing protein layer between the three electrodes,
No. No. 3 electrode, no. No. 3 electrode and no. No. 4 antibody-containing protein layer between the four electrodes,. 19 and no. No. 20, the antibody-containing protein layer between the electrodes; A phenomenon close to the shape in which the electrodes of No. 20 were integrated appeared. 2
No. ~ No. This is probably because the 20 electrode portions functioned as one electrode. N in this case
o. Assuming that the area of one electrode is 1, no. It can be said that the area on the two electrode side is similar to an electrode having an area 37 times larger.

On the other hand, no. 9 and no. When a voltage was applied to the electrode of No. 10, The electrode side of No. 9 is N
o. No. 1 to No. 1 The portion up to the electrode of No. 9 shows the function of one electrode. The electrode side of No. 10 is No. 10
From the electrode No. The portion up to the 20 electrodes shows the function of one electrode, and the area ratio between the two is quite close to 1. No. 1 and No. As shown in the data of FIG. 4, the detection sensitivity is lower than that when the voltage is applied to the second electrode.

FIG. 5 shows No. 3 in FIG. 1 to No.
The measurement results when the same apparatus as in FIG. 2 was used except that the electrode 20 was replaced with an electrode on which no antibody was immobilized were shown. Comparison between the values measured on the electrode on which the antibody was not immobilized and the electrode on which the antibody was immobilized, that is, from the comparison between FIGS. 4 and 5, the change in the conductivity of pure water before and after the antibody-antigen reaction showed that It proves that it is due to the reaction.

Example 1-7: Preparation of Antibody-Immobilized Electrode Section Having Reference Electrode and Guard Electrode (1) Amino group on the surface of antibody-immobilized electrode section having antibody-immobilized electrode, reference electrode and guard electrode As shown in FIG. 6, gold-plated copper foil electrodes 1 and 2 having a pole width of 1 mm were etched in the center on a glass epoxy substrate 10 having a length of 66 mm and a width of 21 mm in the same manner as in Example 1-5 (1). 3 are formed vertically, and a gold-plated copper foil electrode having a pole width of 1 mm is arranged around the guard electrode 4, and a resist layer 5 made of an epoxy resin is printed on a portion indicated by oblique lines on the lower left in FIG. 6. Thus, an electrode substrate was prepared. The electrode substrate was cleaned of fat-soluble stains on the surface with acetone, chloroform and tetrahydrofuran, dried and immersed in 0.1N HCl for 5 minutes to oxidize the surface.
Then, after applying a Teflon (trade name) tape to a portion indicated by oblique lines on the lower right side in FIG. 6 and masking, the electrode whose surface was oxidized was immersed in a 2% acetone solution of δ-aminopropyltriethoxysilane. , 120 ° C, 1
By heating for 8 hours, amino groups were introduced on the electrode surface.

(2) Immobilization of Antibody on Electrode Surface The monoclonal antibody 8Cl purified in Example 1-3 was
It was adjusted to be 00 mg / ml. Glutaraldehyde is added to this solution to a concentration of 0.1 mg / ml, and the electrode (amino group-introduced) substrate prepared in (1) of Example 1-7 is immersed in the solution. After reacting for 4 hours, the antibody was immobilized on the electrode surface and on the FRP surface which had not been resisted, that is, on the region 6.

(3) Blocking treatment In order to prevent non-specific adsorption of proteins and the like to the electrode surface,
The FRP substrate treated in the above paragraph (1) was immersed in a 1 wt% bovine serum albumin solution for 18 hours, and bovine serum albumin was fixed between the monoclonal antibodies 8Cl fixed on the FRP surface between the electrodes. As a result, an antibody-containing protein layer was formed at the electrode 1 and the electrode 2 and at a predetermined portion between them, that is, in the region 6. Thus, electrodes 1 and 2 became antibody-immobilized electrodes. The portion indicated by the diagonally right slanted line functions as the electrode portion in the present embodiment. The electrode 1 and the electrode 2 in this portion become the antibody-immobilized electrodes 1 and 2, of which the antibody-immobilized electrode 2 is used as the reference electrode 2. An antibody-containing protein layer is formed between the reference electrode 2 (that is, the antibody-immobilized electrode 2) and the antibody-immobilized electrode 1.

Example 1-8: Examination of the influence of the antibody-containing layer existing between the two antibody-immobilized electrodes on the sensitivity The antibody-immobilized electrodes 1 and 2 produced in Example 1-7 and the reference electrode 3 Using the antibody-immobilized electrode portion having the guard electrode 4, the structure of the antibody-immobilized electrode required for obtaining sensitivity was examined. The immobilized antibody on each electrode of the antibody-immobilized electrode or the antibody-containing layer between the electrodes was physically removed using a spatula, and the reference electrode (antibody-immobilized electrode 2 was removed in pure water at 10 V and 80 Hz). The electric conductivity between the reference electrode) and the antibody-immobilized electrode 1 was measured as a potential when the cell constant was assumed to be 1.

The results are shown in FIG. When the antibody-containing protein layer between the reference electrode 2 and the antibody-immobilized electrode 1 is deleted, the potential becomes extremely small, and in order to obtain sensitivity, the antibody-containing protein layer between the reference electrode 2 and the antibody-immobilized electrode 1 is required. Proved essential.

Example 2 Detection of N-Methylphenethylamine Using an Immunosensor Having a Reference Electrode and a Guard Electrode in the Electrode Section The antibody-immobilized electrodes 1 and 2 produced in Example 1-7, the reference electrode 3, Using the antibody-immobilized electrode section having the guard electrode 4, N-methylphenethylamine, in which the monoclonal antibody 8Cl reacts similarly to methamphetamine, was detected. First, the conductivity of pure water between the reference electrode 2 and the antibody-immobilized electrode 1 was measured. Next, an antibody-antigen reaction was performed for 5 minutes using 1 μg / ml and 10 μg / ml of N-methylphenethylamine, and the conductivity of pure water between the reference electrode 2 and the antibody-immobilized electrode 1 was measured. Each value is calculated as the ratio to the conductivity (background value) of pure water measured using an electrode of the same design without surface treatment, and the rate of change in conductivity of pure water before and after antibody-antigen reaction. did.

FIG. 8 shows the measurement results at 10 V and 80 Hz. The reason why there are two black spots at the same concentration of N-methylphenethylamine is that the same experiment was performed twice.
The immunosensor of the present invention can accurately detect N-methylphenethylamine even at low concentrations of 1 μg / ml and 10 μg / ml, and the rate of change shows a high value depending on the concentration of N-methylphenethylamine. Was.

[0057]

[Effect] An inexpensive and simple method for detecting an antibody-antigen reaction by measuring the electrical change between the antibody-immobilized electrodes or between the antigen-immobilized electrodes due to the antibody-antigen reaction using a general-purpose measuring instrument. Can be provided. In addition, the present invention can not only shorten the measurement time because it does not use a means for measuring radioactivity or enzyme activity, but also can measure an antigen (hapten) having a small molecular weight without using a competitive method, and has high sensitivity. Measurement can be realized.

[Brief description of the drawings]

FIG. 1 is a graph showing the reaction specificity (reaction selectivity) of various display reagents when a monoclonal antibody produced by hybridoma cell 8Cl is used, as compared with the relative absorbance at 415 nm of each reaction solution.

FIG. 2 is a plan view of an FRP substrate provided with a comb-shaped antibody-immobilized electrode used in Example 1.

FIG. 3 is a partial cross-sectional view for explaining an electrode formed by forming a copper-gold layer at a predetermined position on an FRP substrate and a step of immobilizing an antibody on the FRP substrate. Shows a state in which an electrode made of copper-gold is formed on top,
(B) shows a state in which an amino group is introduced on the electrode surface,
(C) shows a state in which a monoclonal antibody is bound to the amino group via glutaraldehyde, and a monoclonal antibody is bound to the surface of the FRP substrate.
The state in which bovine serum albumin (BSA) is fixed between the monoclonal antibodies on the surface of the RP substrate is modeled.

FIG. 4 is a graph showing how the difference in area (area ratio) of a pair of antibody-immobilized electrodes performed in Example 1-6 affected the measurement sensitivity.

FIG. 5 is a graph showing the measurement sensitivity when a simple electrode on which no antibody is immobilized is used instead of the antibody-immobilized electrode in FIG.

FIG. 6 is a plan view showing a structure of an electrode unit according to Example 1-7.

FIG. 7 is a graph showing the influence on the measurement sensitivity depending on the presence or absence of a conductor layer containing an antibody.

FIG. 8 is a graph showing the ease of detection at each concentration of N-methylphenethylamine in Example 2.

[Explanation of symbols]

1 electrode (only the lower-right hatched portion is an antibody-immobilized electrode) 2 electrode (only the lower-right hatched portion is an antibody-immobilized electrode) (reference electrode) 3 reference electrode 4 guard electrode 5 resist layer 6 antibody-immobilized treatment Area 10 FRP substrate

Claims (5)

[Claims] (A) having a pair of antibody-immobilized electrodes or antigen-immobilized electrodes on the surface of which an antibody or antigen is immobilized,
Between the antibody-immobilized electrode or the antigen-immobilized electrode, an electrode portion electrically connected by a protein layer containing an antibody or an antigen, and (B) a measuring instrument for measuring electric properties between these electrodes An immunosensor characterized by being constituted. 2. The immunosensor according to claim 1, wherein the protein layer containing the antibody or the antigen has a conductivity of 10 ⁻⁴ S / cm or less. 3. The immunosensor according to claim 1, wherein the pair of antibody-immobilized electrodes or antigen-immobilized electrodes has different areas. 4. The immunosensor according to claim 1, further comprising (C) a reference electrode. 5. The immunosensor according to claim 1, further comprising (D) a guard electrode.