WO1994023287A1 - Immunoassay and immunoassay cell used therefor - Google Patents

Immunoassay and immunoassay cell used therefor Download PDF

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Publication number
WO1994023287A1
WO1994023287A1 PCT/JP1994/000535 JP9400535W WO9423287A1 WO 1994023287 A1 WO1994023287 A1 WO 1994023287A1 JP 9400535 W JP9400535 W JP 9400535W WO 9423287 A1 WO9423287 A1 WO 9423287A1
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WO
WIPO (PCT)
Prior art keywords
antibody
electrodes
electrode
antigen
pair
Prior art date
Application number
PCT/JP1994/000535
Other languages
French (fr)
Japanese (ja)
Inventor
Isao Karube
Chien-Yuan Chen
Kaoru Yagiuda
Akihide Hemmi
Yasukazu Asano
Original Assignee
Dkk Corporation
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Publication date
Application filed by Dkk Corporation filed Critical Dkk Corporation
Priority to AU62924/94A priority Critical patent/AU6292494A/en
Publication of WO1994023287A1 publication Critical patent/WO1994023287A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the present invention is suitable for use in an immunoassay method for measuring the concentration of a test substance such as a drug in a body fluid such as urine or blood using an antigen-antibody reaction, for example, a drug, and the like. And a cell for immunoassay. More specifically, the present invention measures the concentration of a test substance in a test wave by measuring a change in electrical conductivity caused by an antigen-antibody reaction between the test substance and a reagent that specifically reacts with the test substance. And a cell for immunoassay suitable for use in such an immunoassay.
  • the present invention can be used to measure drugs such as ***e and methanephthalamine and stimulants in a short time, with high accuracy, and easily, and is useful not only at customs and police stations, but also on-site. It is. Background art
  • test substance in a test wave is carried out using an antigen-antibody reaction.
  • a measurement method is generally called an immunoassay or biosensing.
  • An immunoassay for measuring the concentration of a test substance in a test solution is performed by selectively reacting an indicator, which is an antibody against the test substance (protein, drug, etc.), with the test substance and reacting with the antigen.
  • An antibody reaction complex is formed.
  • the concentration of the test substance is detected by measuring a physicochemical or electrochemical change caused by the formation of the antigen-antibody reaction complex.
  • the test substance is an antibody
  • an antigen that specifically reacts with the antibody can be used as a reagent.
  • the description will be focused on the case where an antibody against the test substance (antigen) is used.
  • the non-labeling method is a method for directly detecting an antigen-antibody reaction complex without using a labeling agent.
  • an antibody is immobilized on a solid matrix
  • an antigen-antibody reaction complex is formed on the surface of the immobilized matrix, and changes in the physical properties of the solid matrix before and after the formation of the antigen-antibody complex, such as membrane potential, electrode potential, It determines the concentration of the test substance from changes in the piezoelectric characteristics, optical characteristics, and the like.
  • the labeling method is a method for detecting an antigen-antibody complex using a radioisotope (RI), a fluorescent molecule, an enzyme, or the like as a labeling agent.
  • RI radioisotope
  • RI fluorescent molecule
  • enzyme enzyme
  • PIA method particle immunoassay method
  • the final measurement of the concentration of the test substance is to initiate the antigen-antibody reaction and then measure this antigen-antibody reaction Therefore, additional steps such as a labeling process and a discoloration process are required. If you want to measure by weight change, you must take out the electrode or device and measure the weight. On the other hand, since the electrode potential or the membrane potential is easily changed by various factors such as temperature and pH, it is powerful to obtain reliable measurement results. Furthermore, when measuring a color change or a turbidity change, the force that requires an optical device for measuring the change is used. ⁇ In general, since these devices are large in size, the devices used for immunoassay must be used. There was a problem such as an increase in size.
  • the concentration of a test substance in a test liquid can be determined by a simple operation in a short time. It is an object of the present invention to provide an immunoassay method capable of performing accurate and accurate measurement. Another object of the present invention is to provide an immunoassay cell suitable for use in such an immunoassay.
  • the present inventors have conducted intensive studies to achieve the above object, and as a result, have used a pair of electrically conductive electrodes, immobilized a reagent that reacts with a test substance on the surface of the pair of electrodes, By arranging this electrode at a distance from the test wave and measuring the electrical conductivity between the electrodes during the formation of the antigen-antibody complex, we found that the concentration of the test substance could be measured easily and accurately. Was.
  • An immunoassay method for measuring the concentration of a test substance in a test wave, and a cell body having a test liquid injection section, and an antigen or antibody reacting with the test substance immobilized on the surface comprises a pair of electrically conductive electrodes which are spaced apart from the cell to be detected of the cell body and a connection means for connecting the pair of electrodes to a measuring instrument for measuring the electrical conductivity between these electrodes.
  • the present invention simply and simply connects to an existing simple and small measuring instrument that can measure electrical conductivity, without the use of special measurement techniques or dangerous reagents, etc.
  • the concentration can be measured. That is, in the present invention, an antibody or antigen on the surfaces of both electrodes is reacted with a test substance in a test solution to form an antigen-antibody reaction complex.
  • the change in electrical conductivity between the two electrodes before and after the formation of the antigen-antibody reaction complex has a correlation with the concentration of the test substance in the test liquid. Therefore, by measuring the change in electrical conductivity between the two electrodes before and after the formation of the antigen-antibody reaction complex, the concentration of the test substance in the test solution can be determined from the change. Also, this electric conductivity Can be easily measured by electrically connecting the immunoassay cell to an existing measuring instrument. Also, the measurement of electrical conductivity is a highly reliable measurement that can be performed extremely stably.
  • FIG. 1 is a cross-sectional view showing one embodiment of the immunoassay cell of the present invention.
  • FIG. 2 is a cross-sectional view showing one embodiment of an electrode structure used for the immunoassay cell of the present invention.
  • FIG. 3 is a plan view showing another embodiment of the electrode structure used for the immunoassay cell of the present invention.
  • FIG. 4 is a plan view i> showing another embodiment of the electrode structure used in the immunoassay cell of the present invention.
  • FIG. 5 is a plan view showing another embodiment of the electrode structure used in the immunoassay cell of the present invention.
  • FIG. 6 is a plan view showing another embodiment of the electrode structure used for the immunoassay cell of the present invention.
  • FIG. 7 is a process chart schematically showing a reaction mechanism in the immunoassay of the present invention.
  • FIG. 8 is a cross-sectional view showing one embodiment of the immunoassay cell of the present invention.
  • FIG. 9 is a diagram showing a calibration curve between ***e concentration and the rate of change in electrical conductivity (%).
  • FIG. 10 is a diagram showing a calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%).
  • FIG. 11 is a diagram showing another calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%).
  • FIG. 12 is a diagram showing a calibration curve between the mouse IgG concentration and the rate of change in electrical conductivity (%).
  • FIG. 13 is a diagram showing yet another calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%). Detailed description of the invention
  • a pair of electrodes that are electrically conductive and have a surface on which an antibody that reacts with a test substance (well) is immobilized is used as a test liquid.
  • the pair of electrodes is separated from each other for a time sufficient for the antibody fixed on the electrode surface to form an antigen-antibody reaction complex with a test substance (antigen) in the test solution. It is held during the test wave, and then the electric conductivity between the pair of electrodes is measured.
  • Test substances (antigens) include macromolecules such as proteins, which are always large in molecular weight, and relatively low molecular weight substances such as narcotics and stimulants.
  • Drugs and stimulants include various substances such as ***e, methamphetamine, amphetamine, marijuana, heroin, and morphine.
  • a monoclonal antibody or a polyclonal antibody can be used without particular limitation.
  • Monoclonal antibodies for example, Koehler
  • a hybridoma that fuses antibody-producing cells derived from spleen cells of a mammal such as a mouse immunized with an antigen with myeloma cells of a mammal such as a mouse and produces an antibody in a selective medium
  • the desired hybrid doroma is obtained by screening, and the ability to produce the monoclonal antibody by culturing it is obtained, or the hybridoma is administered intraperitoneally to a mammal such as a mouse, and the monoclonal antibody is purified from the ascites.
  • a monoclonal antibody can be obtained by purification.
  • the serum is conjugated with peroxy serum albumin (BSA) or human serum albumin as a carrier, and then immunized in the same manner as above. By doing so, an antibody against the antigen can be produced.
  • BSA peroxy serum albumin
  • human serum albumin as a carrier
  • an antibody against the antigen can be produced.
  • methamphetamine molecular weight: 149.22
  • ***e molecular weight: 30.335
  • the polyclonal antibody can be obtained, for example, by subcutaneously or intraperitoneally administering an antigen to a mammal such as a mouse, obtaining serum from the mammal, and purifying the serum to obtain a polyclonal antibody.
  • the pair of electrodes used in the present invention can be used without particular limitation as long as they can conduct.
  • an electrode material for example, metal, carbon or a semiconductor is used.
  • the metal include platinum, gold, silver, copper, Niggel, iron, aluminum, and stainless steel.
  • the semiconductor include a metal oxide semiconductor. Examples of such a semiconductor include tin oxide, titanium oxide, zinc oxide, tungsten oxide, iridium oxide, and rhodium oxide. Further, a combination of these may be used.
  • platinum, gold, copper and carbon are preferred from the viewpoint of stability of measurement accuracy and availability.
  • the electrode material used in the present invention can use the electrode material as it is, it can also be prepared by coating the electrode material on a glass substrate or a printed substrate such as a plastic substrate.
  • the coating method include a method of forming an electrode material coating on a printed board by vapor deposition or sputtering. Further, it can be formed by applying a granular metal or the like or an oxide thereof in the form of a paste to a print substrate and then sintering. According to the latter method, a porous electrode can be formed.
  • Immobilization of the antibody on the electrode can be performed by physically or chemically adsorbing or binding to the electrode surface, or by a combination thereof.
  • the binding strength of the antibody is very low, and the amount of the antibody that can be immobilized is limited.
  • a carboxylic acid is applied to the electrode surface via a sulfide by using a force coupling agent such as an organic thiol compound having a functional group such as a sulfoxyl group (for example, mercaptopropionic acid).
  • a functional group such as a thiol group is provided, and this is used to bind to an amino group or a carboxyl group of an antibody protein.
  • a metal that is difficult to oxidize is gold.
  • the electrode material is made of porous carbon, for example, glass carbon. The material can also be impregnated inside by suction.
  • a silane coupling agent such as aminopropyltriethoxysilane ( ⁇ -APTES) is used to provide a functional group for binding antibodies to the electrode surface using the hydroxyl groups formed on the electrode surface. I do.
  • ⁇ -APTES aminopropyltriethoxysilane
  • an amino group is provided on the electrode surface through a siloxy group generated by a reaction between a hydroxyl group and a silyl group.
  • daltaraldehyde which reacts with an amino group, acts to give an aldehyde group via the amide group, and then the antibody protein is reacted.
  • 7-APTES is generally used as a 0.2-0.5% solution in a solvent such as acetone.
  • the antibody can be retained inside the electrode material by impregnating the antibody without directly reacting the antibody with the electrode surface.
  • it is effective when carbon is used as an electrode material or when a large amount of antibody is immobilized on an electrode.
  • a powder made of platinum, gold, carbon or the like having a particle size of 0.1 to 10 m is suspended in a medium such as water, and then a polyvinyl alcohol or Nafion solution is added to form a paste. Is applied to a porous fluororesin layer as an electrode substrate, and then sintered at about 220 ° C. to form a porous electrode material.
  • the antibody can be retained inside the electrode material.
  • the antibody may be immobilized after imparting affinity to the antibody with the silane coupling agent and then glutaraldehyde or the like to the sintered electrode material as described above.
  • the amount of the antibody immobilized on the electrode material is generally from 0.01 to 100 ng, preferably from 1 to 500 ng, per unit surface area (cm 2 ) of the electrode.
  • the concentration of the antibody suspension used when immobilized on the electrode material is generally from 0.01 to 10 g / liter, preferably from 0.1 to 1 g / liter.
  • the arrangement of the pair of electrodes is not particularly limited as long as these electrodes are kept in a state of being immersed in the test solution at a distance. These electrodes may be present on the same plane and separated from each other.
  • FIG. 1 longitudinal sectional view
  • the configuration of an electrode constituting a part of a cell for receiving a test solution is shown.
  • the facing two sides 2 and 3 constituting the rectangular column cell 1 constitute electrodes (lead wires are omitted).
  • the test solution is filled in the space surrounded by each side, and the electrodes 2 and 3 come into contact with the test solution.
  • FIG. 2 shows another type of electrode (lead wire is omitted).
  • This electrode is provided with electrodes 12 and 13 on both sides of the insulator 10.
  • the electrode of this embodiment is immersed in a cell containing a test solution, so that the electrodes 12 and 13 come into contact with the test wave.
  • FIG. 3 shows still another embodiment of the electrodes 22 and 23 (lead wires omitted).
  • the electrodes 22 and 23 are provided spirally around the cylindrical insulator 20.
  • the electrode of this embodiment is also immersed in the cell containing the test liquid, so that the electrodes 22 and 23 come into contact with the test liquid.
  • FIG. 4 shows still another embodiment of the electrodes 32 and 33 (lead wires omitted).
  • the electrodes 32 and 33 are alternately arranged at equal intervals.
  • the electrode of this embodiment is also immersed in the cell containing the test solution, and the electrodes 32 and 33 come into contact with the test solution.
  • FIG. 5 shows electrodes that are spaced apart on the same plane.
  • electrodes 42 and 43 are arranged concentrically via an insulator 40.
  • the electrode of this embodiment is used for a flat shallow cell, and comes into contact with the test solution contained in the cell by virtue of the electrodes 42, 43.
  • FIG. 6 shows another embodiment of the planar electrode.
  • the rectangular electrodes 52, 53 are arranged on the insulator 50 so as to be separated from each other.
  • the electrode of this embodiment is also used for a flat shallow cell, and is brought into contact with the test liquid contained in the cell by the electrodes 52 and 53.
  • a pair of antibody-immobilized electrodes arranged at regular intervals inside cylindrical glass or plastic can also be used.
  • an electrode flow type
  • the concentration of the test substance can be measured.
  • an outline of a typical immunoassay method of the present invention will be described with reference to FIG. First, an antibody against a test substance (standard sample) is immobilized on the electrodes 61 and 62 (A).
  • the immobilized electrodes 61 and 62 are sufficiently washed, placed in a cell containing pure water, and the electrical conductivity in that state is measured.
  • the electrode is removed from the cell, and a test liquid containing the test substance is introduced into the cell.
  • the electrodes 61 and 62 are immersed in the test wave and kept at 25 ° C. for 15 minutes to cause an antigen-antibody reaction and to form an antigen-antibody reaction complex (B :).
  • the electrodes 61 and 62 are taken out of the cell, washed sufficiently with pure water, and then the electrodes 61 and 62 are inserted into pure water, and the electric conductivity is specified again (C).
  • This operation is repeated by changing the concentration of the test substance to create a calibration curve for the test substance.
  • a calibration curve is created by plotting the rate of change (%) in electrical conductivity with respect to the concentration of the test substance.
  • the electric conductivity of the test liquid containing the test substance is measured in the same manner as above, and the concentration of the test substance in the test wave is calculated based on the calibration curve. Is calculated.
  • the slope of the calibration curve may be positive or negative depending on the type of the test substance.
  • antibodies are proteins, so immobilizing the antibody on the electrode increases the electrical conductivity compared to before immobilization.
  • the test substance is a protein
  • the electric conductivity increases as the antigen-antibody reaction progresses. This is probably because proteins are electrolytes (ampholytes).
  • the antigen is a non-electrolyte, for example, when it is a drug such as methanephenamine, the electric conductivity decreases as the antigen-antibody reaction force progresses. This is probably because the binding of the drug to the immobilized antibody protein prevents the passage of current.
  • Example 1
  • the immunoassay cell 71 shown in FIG. 8 was produced.
  • ⁇ 2 indicates a square cell body.
  • the cell body 72 includes a main part 73 and an insertion part 74 detachably attached to the main part 73.
  • Rectangular electrodes 75, 76 on which antibodies are immobilized are fixed to the opposite sides of the main part 73 and the insertion part 74, and when the insertion part 74 is attached to the main part 73, both electrodes are fixed.
  • the electrodes 75 and 76 are arranged so as to face each other, and a deep groove-shaped test solution injection portion 77 having a width w is formed between the electrodes 75 and 76.
  • the immunoassay cell 71 is provided with connection means for connecting the electrodes 75 and 76 to an electric conductivity meter, and the electric conductivity meter is connected to this connection means. Have been.
  • the electrode materials shown in Table 1 below were used.
  • the electrode area is 0.785 cm 2
  • the distance between the electrodes is 0.5 mm
  • the cell volume is 0.1. 9 3 4 3 cm 3, cell constant 0.0 6 der ivy.
  • BSA was conjugated to methanephthalamine as a carrier, and this was immunized as an antigen (concentration: 1 mg / ml), administered to nails of five puppies, 3 ml each, between the nail and skin.
  • injections were made twice a week, once every other month from the third month, and repeated three times to produce antibodies.
  • the carotid artery of the egret was then cut, whole blood was collected, and antibodies were collected using a dialysis membrane.
  • gel filtration was further performed to purify the anti-methamphetamine antibody.
  • the obtained antibody was stored frozen until use.
  • the antibody yield was 30 to 40% based on 0.5 g of methamphetamine. Immobilization of antibodies on electrodes
  • the surface of the platinum electrode was oxidized by leaving the platinum electrode unattended (introduction of hydroxyl groups).
  • a solution of a silane coupling agent (r-APTES) in acetone (0.4% acetone solution) was applied to the platinum surface, and dried at 120 ° C for 24 hours to introduce amino groups.
  • the obtained electrode was immersed in a 5% glutaraldehyde solution and left at room temperature for 2 hours. Then washed electrode with pure water, and c to remove unreacted glutaraldehyde, and dipped 12 hours at 4 ° C in phosphate buffered saline suspension of the electrode antibody, immobilized antibody Was done. Unreacted antibodies were removed by washing the electrodes with pure water.
  • the electrodes were immersed in a glycine solution (0.1 M solution) at 25 ° C for 30 minutes to inactivate the functional groups that had not reacted with the antibody.
  • the obtained electrode was stored at 4 ° C. in phosphate buffered saline at pH 7.4 until the cell for immunoassay was assembled as described above.
  • the gold electrode was washed with ethanol and dried at room temperature. Next, the electrode was immersed in a 0.5 m mercaptopropionic acid 80% ethanol solution for 30 minutes, washed with ethanol to remove mercaptopropionic acid, and dried at room temperature. Then, The antibody was immobilized on the surface of the gold electrode by performing the operations after the glutaraldehyde treatment in the same manner as in Method 1.
  • the gold electrode was washed with ethanol and dried at room temperature in the same manner as in Method 3 above. Then, a dodecylamine solution (30%) of dodecylamine was added to a 1 O mM polyion complex solution, and dodecylamine was added to the solution. % To introduce amino groups into the polyion complex. Next, a cholesterol solution in form of cholesterol (10%) was added to the mixture, and the mixture was applied to the surface of a gold electrode (a coating amount after drying was 2.5 ⁇ m Zcm 2 ). Then, by treating with tetrahydrofuran, cholesterol was removed and the electrode surface was made porous. Thereafter, the steps subsequent to the glutaraldehyde treatment were repeated in the same manner as in Method 1 described above to immobilize the antibody on the electrode surface.
  • the porous silica After 0.2 g of the porous silica was washed with pure water, it was treated with a 5% glutaraldehyde solution for 2 hours at room temperature. Then, after washing with pure water to remove unreacted glutaraldehyde, the obtained silica is immersed in a suspension of the antibody's phosphate buffer at 4 ° C for 12 hours to fix the antibody on the silica. It has become. Next, the silica was washed with a phosphate buffer to remove unreacted antibodies, and then treated with a glycine solution. The obtained suspension of silica was suction-filtered through porous carbon (glass carbon) as an electrode to impregnate the inside of the electrode with silica force, thereby forming an integral immobilized electrode. Creating a calibration curve
  • the immunoassay cell may be disassembled to wash the electrode surface.
  • the antigen-antibody complex was dissociated with a glycine buffer at pH 2.8 to remove the antigen bound to the immobilized antibody.
  • Electrode material Electrode shape Fixation method Test substance Quantitable range Drawing
  • Helix type refers to the shape of the electrode shown in Fig. 3,
  • the “comb shape” refers to the shape of the electrode shown in FIG.
  • “Face-to-face” refers to the shape of the electrode shown in Figure 2, Response time From the relationship between the antigen-antibody reaction time and the electrical conductivity in mouse IgG, it was found that the electrical conductivity was saturated in 15 to 20 minutes. Therefore, an immune reaction time of 15 minutes is considered to be sufficient. One minute was sufficient as the measurement time of the electric conductivity as a 90% response time. Therefore, it is considered that 20 minutes is sufficient for the time required to measure the concentration of the test substance, including the pretreatment time. Conventionally, such a measurement of a test substance required 2 to 3 hours, which indicates that the immunoassay of the present invention can be performed in a very short time. Lifetime of immunoassay cell
  • a predetermined amount of methanephthalamine was added to urine to prepare five kinds of test liquids.
  • an immunoassay cell (electrode shape is spiral) manufactured using platinum as the electrode material in accordance with Method 1 described above was used to measure the amount of methanephthalamine contained in the above five types of test solutions. The concentration was measured. On the other hand, the concentration of methamphetamine was also measured by GC-MS (Gas Chromatography-Mass Spectrometry), which is used as the official method for drugs, and the two were compared. The results are shown in Table 2 below. Table 2
  • the immunoassay cell of the example achieved simplification of the immunoassay method by using electric conductivity for detecting the antigen concentration. That is, the antibody against the test substance is immobilized on the electrode, the test solution is injected into the test wave injection part of the immunoassay cell, and the electric conductivity before and after the antigen-antibody reaction is measured. The antigen concentration in the test wave can be easily and quickly obtained based on the calibration curve.
  • the immunoassay cell of the present invention can be simply connected to an existing conductivity meter, so that the measurement operation does not require any special technique, and is easy to carry. Is also possible. Furthermore, if the immobilization method is constant, the immunoassay cell of the present invention has a small measurement variation, and can be used as a disposable cell.
  • the antigen concentration was measured by immobilizing the antibody on the electrode surface.
  • the antibody concentration may be measured by immobilizing the antigen on the electrode surface.

Abstract

This invention relates to an immunoassay for determining the concentration of a substance to be examined which is contained in a liquid to be examined, such as urine and blood, by utilizing an antigen-antibody reaction, and an immunoassay cell used for this method. According to the present invention, a pair of electrodes on the surfaces of which an antibody or an antigen which reacts with a substance to be examined is immobilized are disposed in a mutually spaced condition in a liquid to be examined, and these electrodes are retained in this liquid for a period of time long enough for the antibody or antigen immobilized to the surfaces of the electrode to form an antigen-antibody complex with the substance to be examined in the object liquid, the electric conductivity between these two electrodes being then measured. This invention also relates to a measuring cell provided with a cell body having a sample liquid injection portion, a pair of electrically conductive electrodes which have an antibody or an antigen immobilized to the surfaces thereof, and which are disposed in a mutually spaced condition in the sample injection portion of the cell body, and a connecting means for joining the two electrodes to an instrument for measuring the electric conductivity therebetween. According to the present invention, since the concentration of a substance to be examined is measured by electric conductivity, stable measurement results are obtained. Moreover, the concentration of a substance to be examined can be measured easily in a short period of time.

Description

明 細 書 免疫測定方法並.びにそれに使用する免疫測定用セル 産業上の利用分野  Description Immunoassay methods and immunoassay cells used for them Industrial applications
本発明は、 抗原抗体反応を利用して尿や血液等の体液の被検液中の成分、 例え ば薬物等の被検物質の濃度を測定する免疫測定方法及びその方法に使用するのに 適した免疫測定用セルに関する。 更に詳述すれば、 本発明は、 被検物質とそれと 特異的に反応する試薬との抗原抗体反応によって生じる電気伝導度の変化を測定 することによって、 被検波中における被検物質の濃度を測定する方法並びにその ような免疫測定方法に使用するのに適した免疫測定用セルに関する。 特に、 本発 明は、 コカインやメタンフヱタミン等の麻薬や覚醒剤等の測定を短時間でかつ高 精度でしかも簡易に実施できるので、 税関や警察署等におけるだけでなく、 現場 での測定にとっても有用である。 背景技術  INDUSTRIAL APPLICABILITY The present invention is suitable for use in an immunoassay method for measuring the concentration of a test substance such as a drug in a body fluid such as urine or blood using an antigen-antibody reaction, for example, a drug, and the like. And a cell for immunoassay. More specifically, the present invention measures the concentration of a test substance in a test wave by measuring a change in electrical conductivity caused by an antigen-antibody reaction between the test substance and a reagent that specifically reacts with the test substance. And a cell for immunoassay suitable for use in such an immunoassay. In particular, the present invention can be used to measure drugs such as ***e and methanephthalamine and stimulants in a short time, with high accuracy, and easily, and is useful not only at customs and police stations, but also on-site. It is. Background art
従来より、 被検波中における被検物質の測定を、 抗原抗体反応を利用して行う ことは既に公知である。 このような測定方法は、 一般には、 免疫測定又はバイオ センシングと呼ばれる。 被検液中の被検物質の濃度を測定する免疫測定は、 被検 物質 （タンパク質や薬物等） に対して抗体となる関係にある指示薬をその被検物 質と選択的に反応させ、 抗原抗体反応複合体を形成させる。 この抗原抗体反応複 合体の形成によつて生じる物理化学的又は電気化学的な変化を測定することによ り、 被検物質の濃度を検知する。 なお、 被検物質が抗体である場合には、 それと 特異的に反応する抗原を試薬として使用することもできる。 ここでは、 被検物質 (抗原） に対する抗体を使用する場合を中心にして説明する。  It has been known that measurement of a test substance in a test wave is carried out using an antigen-antibody reaction. Such a measurement method is generally called an immunoassay or biosensing. An immunoassay for measuring the concentration of a test substance in a test solution is performed by selectively reacting an indicator, which is an antibody against the test substance (protein, drug, etc.), with the test substance and reacting with the antigen. An antibody reaction complex is formed. The concentration of the test substance is detected by measuring a physicochemical or electrochemical change caused by the formation of the antigen-antibody reaction complex. When the test substance is an antibody, an antigen that specifically reacts with the antibody can be used as a reagent. Here, the description will be focused on the case where an antibody against the test substance (antigen) is used.
従来より、 免疫測定は、 抗原抗体反応の特異性を生かして被検物質の濃度を測 定するものとして種々の態様のものが開発されている。 これらの免疫測定方法は これまで主に医療分野、 特に、 診断分野で広く利用されてきている。  Hitherto, various forms of immunoassay have been developed for measuring the concentration of a test substance by making use of the specificity of an antigen-antibody reaction. Until now, these immunoassay methods have been widely used mainly in the medical field, particularly in the diagnostic field.
抗原抗体反応複合体の形成を利用する公知の免疫測定方法には、 非標識法と標 識法とに大別される。 非標識法は、 標識剤を用いずに抗原抗体反応複合体を直接 検出する方法である。 通常、 固体マトリクスに抗体を固定し、 この固定マトリク ス表面で抗原抗体反応複合体を形成させ、 抗原抗体複合体の形成前後の固体マト リクスの物理的性質の変化、 例えば膜電位、 電極電位、 圧電特性、 光学特性等の 変化から、 被検物質の濃度を求めるものである。 Known immunoassays utilizing the formation of an antigen-antibody reaction complex include unlabeled and standard It is roughly divided into intellectual methods. The non-labeling method is a method for directly detecting an antigen-antibody reaction complex without using a labeling agent. Usually, an antibody is immobilized on a solid matrix, an antigen-antibody reaction complex is formed on the surface of the immobilized matrix, and changes in the physical properties of the solid matrix before and after the formation of the antigen-antibody complex, such as membrane potential, electrode potential, It determines the concentration of the test substance from changes in the piezoelectric characteristics, optical characteristics, and the like.
一方、 標識法は、 放射性同位元素 （R I ) 、 蛍光分子、 酵素などを標識剤とし て抗原抗体複合体を検出する方法であり、 ラジオィムノアツセィ法 （R I A法） 、 ェンザィムィムノアツセィ法 （E I A法） 、 パーティクルイムノアッセィ法 （P I A法） などが知られている。  On the other hand, the labeling method is a method for detecting an antigen-antibody complex using a radioisotope (RI), a fluorescent molecule, an enzyme, or the like as a labeling agent. The radioimmunoassay method (RIA method), the enzymimno method Atssay method (EIA method) and particle immunoassay method (PIA method) are known.
しかしながら、 従来の免疫測定方法は、 被検波中に、 抗体を固定した単一の電 極又は抗原抗体反応を起こさせる装置、 及び差動型の場合には、 更に参照電極を 挿入し、 そこで一定時間保持して、 抗原抗体反応を行わせた後、 固定化電極又は 装置における電位や、 膜電位、 重量変化、 被検液の濁度変化、 色変化、 放射性濃 度変化等を測定することによって被検物質の濃度を測定するものである。 この場 合、 電極電位又は膜電位の測定の場合を除いては、 最終的に被検物質の濃度を測 定するには、 抗原抗体反応を起こさせた後、 この抗原抗体反応の測定をするため に、 標識化処理や、 変色処理等の付加的な工程が必要となる。 また、 重量変化に よって測定しょうとする場合は、 電極又は装置を一旦取り出して、 重量測定をし なければならない。 一方、 電極電位又は膜電位は、 温度や、 pH等の種々の因子に よって変化し易いために、 信頼性の置ける測定結果を得ること力，しい。 更に、 色変化又は濁度変化等を測定する場合には、 その変化を測定するための光学装置 が必要となる力《、 一般にこれらの装置は大型であるため、 免疫測定に使用する装 置が大型化するなど問題となっていた。 更に、 放射線標識剤は、 放射線を放出す るので、 人体に有害であり、 そのような試薬を使用することはできるだけさける ことが望ましい。 そのため、 従来より、 被検液中の被検物質の濃度を短時間でか つ高精度にしかも簡易に測定できる方法及び装置が強く要望されていた。 発明の要約  However, in the conventional immunoassay method, a single electrode on which an antibody is immobilized or a device for causing an antigen-antibody reaction during the test wave, and in the case of the differential type, a reference electrode is further inserted, After performing the antigen-antibody reaction for a period of time, the potential at the immobilized electrode or device, membrane potential, weight change, turbidity change, color change, radioactive concentration change, etc. of the test solution are measured. It measures the concentration of the test substance. In this case, except for the case of measuring the electrode potential or membrane potential, the final measurement of the concentration of the test substance is to initiate the antigen-antibody reaction and then measure this antigen-antibody reaction Therefore, additional steps such as a labeling process and a discoloration process are required. If you want to measure by weight change, you must take out the electrode or device and measure the weight. On the other hand, since the electrode potential or the membrane potential is easily changed by various factors such as temperature and pH, it is powerful to obtain reliable measurement results. Furthermore, when measuring a color change or a turbidity change, the force that requires an optical device for measuring the change is used. <In general, since these devices are large in size, the devices used for immunoassay must be used. There was a problem such as an increase in size. Furthermore, radiolabels are harmful to the human body because they emit radiation, and it is desirable to avoid using such reagents as much as possible. Therefore, conventionally, there has been a strong demand for a method and apparatus capable of measuring the concentration of a test substance in a test liquid in a short time, with high accuracy, and easily. Summary of the Invention
従って、 本発明は、 被検液中の被検物質の濃度を簡単な操作で、 しかも短時間 でかつ精度よく測定できる免疫測定法を提供することを目的とする。 また、 本発 明は、 このような免疫測定法に使用するのに適した免疫測定用セルを提供するこ とを目的とする。 Therefore, according to the present invention, the concentration of a test substance in a test liquid can be determined by a simple operation in a short time. It is an object of the present invention to provide an immunoassay method capable of performing accurate and accurate measurement. Another object of the present invention is to provide an immunoassay cell suitable for use in such an immunoassay.
本発明者は、 上記目的を達成するため、 鋭意検討した結果、 電気的に導通可能 な一対の電極を使用し、 この一対の電極の表面に被検物質に対して反応する試薬 を固定化し、 この電極を被検波に離隔して配置して、 抗原抗体複合体の形成に伴 う電極間の電気伝導度を測定することにより、 被検物質の濃度を容易にしかも精 度よく測定できることを見出した。  The present inventors have conducted intensive studies to achieve the above object, and as a result, have used a pair of electrically conductive electrodes, immobilized a reagent that reacts with a test substance on the surface of the pair of electrodes, By arranging this electrode at a distance from the test wave and measuring the electrical conductivity between the electrodes during the formation of the antigen-antibody complex, we found that the concentration of the test substance could be measured easily and accurately. Was.
即ち、 本発明は、  That is, the present invention
電気的に導通可能な一対の電極であつて、 前記電極表面に被検物質と反応する 試薬 （抗原又は抗体） を固定化した一対の電極を被検液に離隔して配置し、 前記電極表面に固定された前記抗原又は抗体が、 前記被検液中の抗体又は抗原 と抗原抗体複合体を形成するに十分な時間、 前記一対の電極を前記被検液中に保 持し、 次いで  A pair of electrodes that are electrically conductive, and a pair of electrodes on which a reagent (antigen or antibody) that reacts with a test substance is immobilized on a surface of the electrode; Holding the pair of electrodes in the test solution for a time sufficient for the antigen or antibody immobilized to form an antigen-antibody complex with the antibody or antigen in the test solution;
前記一対の電極間の電気伝導度を測定する、  Measuring the electrical conductivity between the pair of electrodes,
ことを特徴とする被検波中の被検物質の濃度を測定する免疫測定方法、 並びに 被検液注入部を有するセル本体と、 表面に被検物質と反応する抗原又は抗体が 固定化されかつ前記セル本体の被検波注人部に離間配置された電気的に導通可能 な一対の電極と、 前記一対の電極をこれら電極間の電気伝導度を計測する計測器 に接続する接続手段とを具備する免疫測定用セル、 An immunoassay method for measuring the concentration of a test substance in a test wave, and a cell body having a test liquid injection section, and an antigen or antibody reacting with the test substance immobilized on the surface, and It comprises a pair of electrically conductive electrodes which are spaced apart from the cell to be detected of the cell body and a connection means for connecting the pair of electrodes to a measuring instrument for measuring the electrical conductivity between these electrodes. Immunoassay cell,
に関する。 About.
本発明は、 電気伝導度を測定することのできる簡易かつ小型の既存の計測器に 接続するだけで、 特殊な測定技術や、 危険な試薬等を使用するとなく、 簡便かつ 迅速に被検物質の濃度を測定することがでる。 即ち、 本発明においては、 両電極 表面の抗体又は抗原と被検液中の被検物質とを反応させて、 抗原抗体反応複合体 を形成させる。 この場合、 抗原抗体反応複合体形成の前後の両電極間の電気伝導 度の変化は、 被検液中の被検物質の濃度と相関を有する。 従って、 抗原抗体反応 複合体形成前後の両電極間の電気伝導度の変化を計測することにより、 その変化 量から被検液中の被検物質の濃度を求めることができる。 また、 この電気伝導度 の測定は、 既存の計測器に免疫測定セルを電気的に接続することによって容易に 測定することができる。 また、 電気伝導度の測定は、 極めて安定に行うことがで きる信頼性の高い測定である。 図面の簡単な説明 The present invention simply and simply connects to an existing simple and small measuring instrument that can measure electrical conductivity, without the use of special measurement techniques or dangerous reagents, etc. The concentration can be measured. That is, in the present invention, an antibody or antigen on the surfaces of both electrodes is reacted with a test substance in a test solution to form an antigen-antibody reaction complex. In this case, the change in electrical conductivity between the two electrodes before and after the formation of the antigen-antibody reaction complex has a correlation with the concentration of the test substance in the test liquid. Therefore, by measuring the change in electrical conductivity between the two electrodes before and after the formation of the antigen-antibody reaction complex, the concentration of the test substance in the test solution can be determined from the change. Also, this electric conductivity Can be easily measured by electrically connecting the immunoassay cell to an existing measuring instrument. Also, the measurement of electrical conductivity is a highly reliable measurement that can be performed extremely stably. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 本発明の免疫測定用セルの 1態様を示す断面図である。  FIG. 1 is a cross-sectional view showing one embodiment of the immunoassay cell of the present invention.
図 2は、 本発明の免疫測定用セルに使用する電極構造の 1態様を示す断面図で ある。  FIG. 2 is a cross-sectional view showing one embodiment of an electrode structure used for the immunoassay cell of the present invention.
図 3は、 本発明の免疫測定用セルに使用する電極構造の別の態様を示す平面図 ?¾>る。  FIG. 3 is a plan view showing another embodiment of the electrode structure used for the immunoassay cell of the present invention.
図 4は、 本発明の免疫測定用セルに使用する電極構造の別の態様を示す平面図 i> 。  FIG. 4 is a plan view i> showing another embodiment of the electrode structure used in the immunoassay cell of the present invention.
図 5は、 本発明の免疫測定用セルに使用する電極構造の別の態様を示す平面図 あ <©。  FIG. 5 is a plan view showing another embodiment of the electrode structure used in the immunoassay cell of the present invention.
図 6は、 本発明の免疫測定用セルに使用する電極構造の別の態様を示す平面図 である。  FIG. 6 is a plan view showing another embodiment of the electrode structure used for the immunoassay cell of the present invention.
図 7は、 本発明の免疫測定における反応機構を模式的に示す工程図である。 図 8は、 本発明の免疫測定用セルの 1態様を示す断面図である。  FIG. 7 is a process chart schematically showing a reaction mechanism in the immunoassay of the present invention. FIG. 8 is a cross-sectional view showing one embodiment of the immunoassay cell of the present invention.
図 9は、 コカイン濃度と、 電気伝導度の変化率 (%) との間の検量線を示す図 である。  FIG. 9 is a diagram showing a calibration curve between ***e concentration and the rate of change in electrical conductivity (%).
図 1 0は、 メタンフヱタミン濃度と、 電気伝導度の変化率 （％) との間の検量 線を示す図である。  FIG. 10 is a diagram showing a calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%).
図 1 1は、 メタンフヱタミン濃度と、 電気伝導度の変化率 （％) との間の別の 検量線を示す図である。  FIG. 11 is a diagram showing another calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%).
図 1 2は、 マウス IgG濃度と、 電気伝導度の変化率 （％) との間の検量線を示 す図である。  FIG. 12 is a diagram showing a calibration curve between the mouse IgG concentration and the rate of change in electrical conductivity (%).
図 1 3は、 メタンフヱタミン濃度と、 電気伝導度の変化率 （％) との間の更に 別の検量線を示す図である。 発明の詳細な説明 FIG. 13 is a diagram showing yet another calibration curve between the methanephthalamine concentration and the rate of change in electrical conductivity (%). Detailed description of the invention
本発明の免疫測定方法においては、 電気的に導通可能な一対の電極であって、 前記電極表面に被検物質 （坑原） と反応する抗体が固定化された一対の電極を被 検液に離隔して配置し、 前記電極表面に固定された前記抗体が、 前記被検液中の 被検物質 （抗原） と抗原抗体反応複合体を形成するに十分な時間、 前記一対の電 極を前記被検波中に保持し、 次いで、 前記一対の電極間の電気伝導度を測定する。 被検物質 （抗原） としては、 分子量カ琲常に大きいタンパク質等の高分子や、 麻薬、 覚醒剤等の比較的低分子量の物質が含まれる。 麻薬や覚醒剤としては、 例 えば、 コカイン、 メタンフェタミン、 アンフェタミン、 マリファナ、 ヘロイン、 モルヒネ等種々の物質が含まれる。  In the immunoassay method of the present invention, a pair of electrodes that are electrically conductive and have a surface on which an antibody that reacts with a test substance (well) is immobilized is used as a test liquid. The pair of electrodes is separated from each other for a time sufficient for the antibody fixed on the electrode surface to form an antigen-antibody reaction complex with a test substance (antigen) in the test solution. It is held during the test wave, and then the electric conductivity between the pair of electrodes is measured. Test substances (antigens) include macromolecules such as proteins, which are always large in molecular weight, and relatively low molecular weight substances such as narcotics and stimulants. Drugs and stimulants include various substances such as ***e, methamphetamine, amphetamine, marijuana, heroin, and morphine.
被検物質に対する抗体は、 モノクローナル抗体でも、 ポリクローナル抗体でも 特に制限なく使用することができる。 モノクローナル抗体は、 例えば、 ケーラー As the antibody against the test substance, a monoclonal antibody or a polyclonal antibody can be used without particular limitation. Monoclonal antibodies, for example, Koehler
• ミルシュタイ '(Kohler、 i lstein) の方法に従って当業者には容易に製造する ことができる。 詳述すれば、 一般に、 抗原で免疫したマウス等の哺乳類の脾臓細 胞等に由来する抗体産生細胞と、 マウス等の哺乳類のミエローマ細胞とを融合し、 選択培地によって、 抗体を産生するハイプリ ドーマをスクリーニングすることに よって、 所望のハイプリ ドーマを入手し、 これを培養してモノクローナル抗体を 産生させる力、、 又はハイプリ ドーマをマウス等の哺乳類の腹腔内に投与し、 腹水 からモノクローナノレ抗体を精製することによってモノクローナル抗体を入手する ことができる。 • It can be easily manufactured by those skilled in the art according to the method of Milstein (Kohler, Ilstein). More specifically, in general, a hybridoma that fuses antibody-producing cells derived from spleen cells of a mammal such as a mouse immunized with an antigen with myeloma cells of a mammal such as a mouse and produces an antibody in a selective medium The desired hybrid doroma is obtained by screening, and the ability to produce the monoclonal antibody by culturing it is obtained, or the hybridoma is administered intraperitoneally to a mammal such as a mouse, and the monoclonal antibody is purified from the ascites. A monoclonal antibody can be obtained by purification.
抗原が、 低分子量のもの、 例えば薬物や覚醒剤である場合には、 その抗原に、 キヤリャ一としてゥシ血清アルブミン （BSA)や、 ヒト血清アルブミン等を結合し た後、 上記と同様にして免疫することによって、 抗原に対する抗体を産生させる ことができる。 薬物又は覚醒剤として、 例えば、 メタンフェタミン （分子量： 1 4 9 . 2 2 ) や、 コカイン （分子量： 3 0 3 . 3 5 ) 等の場合には、 このよう な方法が好適である。  When the antigen is of low molecular weight, for example, a drug or stimulant, the serum is conjugated with peroxy serum albumin (BSA) or human serum albumin as a carrier, and then immunized in the same manner as above. By doing so, an antibody against the antigen can be produced. In the case of, for example, methamphetamine (molecular weight: 149.22) or ***e (molecular weight: 30.335) as a drug or stimulant, such a method is suitable.
ポリクローナル抗体は、 例えば、 抗原をマウス等の哺乳類に皮下又は腹腔内投 与して、 その哺乳類から血清を入手し、 精製することによってポリクロ一ナル抗 体を取得することができる。 本発明において使用される一対の電極としては、 導通可能なものであれば、 特 に制限なく使用することができる。 このような電極材料としては、 例えば、 金属、 炭素又は半導体が使用される。 金属としては、 例えば、 白金や、 金、 銀、 銅、 二 ッゲル、 鉄、 アルミニウム、. ステンレス等が含まれる。 また、 半導体としては、 金属酸化物型の半導体が挙げられる。 このような半導体としては、 例えば、 酸化 錫や、 酸化チタン、 酸化亜鉛、 酸化タングステン、 酸化イリジウム、 酸化ロジゥ ム等が挙げられる。 また、 これらを複合して使用したものでもよい。 これらの電 極の内、 測定精度の安定性や入手容易性等の観点から、 白金、 金、 銅及び炭素が 好ましい。 The polyclonal antibody can be obtained, for example, by subcutaneously or intraperitoneally administering an antigen to a mammal such as a mouse, obtaining serum from the mammal, and purifying the serum to obtain a polyclonal antibody. The pair of electrodes used in the present invention can be used without particular limitation as long as they can conduct. As such an electrode material, for example, metal, carbon or a semiconductor is used. Examples of the metal include platinum, gold, silver, copper, Niggel, iron, aluminum, and stainless steel. Examples of the semiconductor include a metal oxide semiconductor. Examples of such a semiconductor include tin oxide, titanium oxide, zinc oxide, tungsten oxide, iridium oxide, and rhodium oxide. Further, a combination of these may be used. Of these electrodes, platinum, gold, copper and carbon are preferred from the viewpoint of stability of measurement accuracy and availability.
本発明で使用する電極は、 電極材料をそのまま使用することもできるが、 ガラ ス基板や、 プラスチック基板等のプリント基板に電極材料を塗装することによつ ても調製することができる。 塗装方法としては、 例えば、 プリント基板に蒸着や、 スパッタリングによって電極材料被膜を形成する方法が挙げられる。 また、 プリ ント基板に、 粒状の金属等又はその酸化物をべ一ストの形態で塗布した後、 焼成 することによって形成させることができる。 後者の方法によれば、 多孔質の電極 を形成することができる。  Although the electrode material used in the present invention can use the electrode material as it is, it can also be prepared by coating the electrode material on a glass substrate or a printed substrate such as a plastic substrate. Examples of the coating method include a method of forming an electrode material coating on a printed board by vapor deposition or sputtering. Further, it can be formed by applying a granular metal or the like or an oxide thereof in the form of a paste to a print substrate and then sintering. According to the latter method, a porous electrode can be formed.
電極への抗体の固定化は、 電極表面に対して物理的又は化学的に吸着又は結合 させること、 又はこれらの組合せによって行うことができる。 金属表面に直接抗 体を結合する場合には、 抗体の結合力が大変に小さく、 抗体の固定できる量に限 界があるので、 電極表面を抗体に対して親和性を持たせた後、 抗体を電極表面に 固定すること力好ましい。 例えば、 白金や、 銅のような酸化し易い金属の場合に は、 空気中に放置するだけで、 酸化して、 電極表面に水酸基を生ずるので、 この 官能基を使用して、 抗体を固定化する。 一方、 酸化し難い金属等の場合には、 力 ルポキシル基等の官能基を有する有機チオール化合物 （例えば、 メルカプトプロ ピオン酸） 等の力ップリング剤を使用して電極表面に硫化物を介してカルボキシ ル基等の官能基を付与し、 これを利用して、 抗体であるタンパク質のアミノ基又 はカルボキシル基と結合させること力通常行われる。 このような酸化し難い金属 としては、 例えば、 金が挙げられる。 更に、 シリカや、 金属又は炭素粒子等の粒 状担体に抗体を担持した後、 多孔質炭素、 例えばグラシカ一ボンからなる電極材 料に吸引によつてその内部に含浸させることもできる。 Immobilization of the antibody on the electrode can be performed by physically or chemically adsorbing or binding to the electrode surface, or by a combination thereof. When binding an antibody directly to a metal surface, the binding strength of the antibody is very low, and the amount of the antibody that can be immobilized is limited. It is preferable to fix the electrode to the electrode surface. For example, in the case of easily oxidizable metals such as platinum and copper, simply leaving them in the air will cause them to oxidize and produce hydroxyl groups on the electrode surface, and this functional group is used to immobilize the antibody. I do. On the other hand, in the case of a metal or the like which is hardly oxidized, a carboxylic acid is applied to the electrode surface via a sulfide by using a force coupling agent such as an organic thiol compound having a functional group such as a sulfoxyl group (for example, mercaptopropionic acid). A functional group such as a thiol group is provided, and this is used to bind to an amino group or a carboxyl group of an antibody protein. An example of such a metal that is difficult to oxidize is gold. Further, after the antibody is supported on a particulate carrier such as silica, metal, or carbon particles, the electrode material is made of porous carbon, for example, glass carbon. The material can also be impregnated inside by suction.
電極表面に形成した水酸基を利用して、 電極表面に抗体を結合するための官能 基を付与するためには、 ァ一ァミノプロピルトリエトキシシラン （ァ一 APTES)等 のシランカップリング剤を使用する。 この処理によって、 電極表面には、 水酸基 とシリル基との反応によって生じたシロキシ基を介してァミノ基を付与する。 次 いで、 ァミノ基と反応するダルタルアルデヒドを作用させて、 アミ ド基を介して アルデヒド基を付与した後、 抗体であるタンパク質を反応させる。  A silane coupling agent, such as aminopropyltriethoxysilane (α-APTES), is used to provide a functional group for binding antibodies to the electrode surface using the hydroxyl groups formed on the electrode surface. I do. By this treatment, an amino group is provided on the electrode surface through a siloxy group generated by a reaction between a hydroxyl group and a silyl group. Next, daltaraldehyde, which reacts with an amino group, acts to give an aldehyde group via the amide group, and then the antibody protein is reacted.
7 -APTES は、 一般にアセトンのような溶剤に 0. 2〜 0. 5 %の溶液として使用 する。  7-APTES is generally used as a 0.2-0.5% solution in a solvent such as acetone.
また、 電極への固定化は、 電極表面と直接抗体を反応させることなく、 電極材 料中に抗体を含浸等によって、 内部に保持させることもできる。 例えば、 炭素を 電極材料として使用する場合や、 抗体を多量に電極に固定化する場合に有効であ る。 例えば、 粒径が 0. 1〜1 0 mの白金、 金又は炭素等からなる粉末を水等の 媒体に懸濁した後、 ポリビニルアルコールやナフィヨン溶液を添加して、 ペース ト状にし、 このペーストを電極基板としての多孔質フッ素樹脂層に塗布した後、 2 2 0 °C前後で焼結させることによって、 多孔質の電極材料を形成させ、 次いで、 抗体を含有する懸濁液を、 必要に応じて吸引等によってこの多孔質電極材料に含 浸させることによって、 電極材料内部に抗体を保持させることができる。 また、 このような焼結した電極材料に、 上記と同様に、 シランカップリング剤、 次いで グルタルアルデヒド等によって抗体に対する親和性を付与した後、 抗体を固定し てもよい。  Further, for immobilization to the electrode, the antibody can be retained inside the electrode material by impregnating the antibody without directly reacting the antibody with the electrode surface. For example, it is effective when carbon is used as an electrode material or when a large amount of antibody is immobilized on an electrode. For example, a powder made of platinum, gold, carbon or the like having a particle size of 0.1 to 10 m is suspended in a medium such as water, and then a polyvinyl alcohol or Nafion solution is added to form a paste. Is applied to a porous fluororesin layer as an electrode substrate, and then sintered at about 220 ° C. to form a porous electrode material. Accordingly, by impregnating the porous electrode material by suction or the like, the antibody can be retained inside the electrode material. In addition, the antibody may be immobilized after imparting affinity to the antibody with the silane coupling agent and then glutaraldehyde or the like to the sintered electrode material as described above.
電極材料に固定化する抗体の量は、 一般に、 電極の単位表面積 （cm2 ) 当たり 0. 0 1〜 1 0 0 0 ng、 好ましくは 1〜 5 0 0 ngである。 The amount of the antibody immobilized on the electrode material is generally from 0.01 to 100 ng, preferably from 1 to 500 ng, per unit surface area (cm 2 ) of the electrode.
電極材料に固定する際に使用する抗体懸濁液の濃度は、 一般に 0. 0 1〜1 0 g/ リットル、 好ましくは 0. l〜l g/リットルである。  The concentration of the antibody suspension used when immobilized on the electrode material is generally from 0.01 to 10 g / liter, preferably from 0.1 to 1 g / liter.
一対の電極の配置は、 これらの電極が被検液中に離隔して浸漬した状態に保持 される限り、 特に限定されるものではない。 これらの電極は、 同一平面上に離隔 した状態で存在して 、ていもよい。  The arrangement of the pair of electrodes is not particularly limited as long as these electrodes are kept in a state of being immersed in the test solution at a distance. These electrodes may be present on the same plane and separated from each other.
このような電極又はそれを含有するセルの例を以下に図面により説明する。 図 1 (縦断面図） の態様においては、 被検液を受容するセルの一部を構成する 電極の構成が示されている。 この態様においては、 四角柱のセル 1を構成する対 面する 2辺 2、 3が電極 （リード線を省略） を構成している。 この態様において は、 被検液が各辺で囲まれる空間に充塡され、 電極 2、 3がその被検液と接触す るようになっている。 Examples of such an electrode or a cell containing the same will be described below with reference to the drawings. In the embodiment shown in FIG. 1 (longitudinal sectional view), the configuration of an electrode constituting a part of a cell for receiving a test solution is shown. In this embodiment, the facing two sides 2 and 3 constituting the rectangular column cell 1 constitute electrodes (lead wires are omitted). In this embodiment, the test solution is filled in the space surrounded by each side, and the electrodes 2 and 3 come into contact with the test solution.
図 2には、 別の態様の電極 （リード線を省略） が示されている。 この電極は、 絶縁体 1 0を挟んで両側に電極 1 2、 1 3が設けられている。 この態様の電極は、 被検液の入ったセルに浸漬され、 電極 1 2、 1 3が被検波と接触するようになつ ている。  FIG. 2 shows another type of electrode (lead wire is omitted). This electrode is provided with electrodes 12 and 13 on both sides of the insulator 10. The electrode of this embodiment is immersed in a cell containing a test solution, so that the electrodes 12 and 13 come into contact with the test wave.
図 3には、 更に別の態様の電極 2 2、 2 3 (リード線省略） が示されている。 この態様においては、 電極 2 2、 2 3は、 円筒状の絶縁体 2 0の回りを螺旋状に 設けられている。 この態様の電極も、 被検液を収めたセル中に浸漬され、 電極 2 2、 2 3が被検液と接触するようになっている。  FIG. 3 shows still another embodiment of the electrodes 22 and 23 (lead wires omitted). In this embodiment, the electrodes 22 and 23 are provided spirally around the cylindrical insulator 20. The electrode of this embodiment is also immersed in the cell containing the test liquid, so that the electrodes 22 and 23 come into contact with the test liquid.
図 4には、 更に別の態様の電極 3 2、 3 3 (リード線省略） が示されている。 この態様においては、 電極 3 2、 3 3は、 交互に等間隔に配列されている。 この 態様の電極も、 被検液を収容するセルに浸漬され、 電極 3 2、 3 3が被検液と接 触するようになっている。  FIG. 4 shows still another embodiment of the electrodes 32 and 33 (lead wires omitted). In this embodiment, the electrodes 32 and 33 are alternately arranged at equal intervals. The electrode of this embodiment is also immersed in the cell containing the test solution, and the electrodes 32 and 33 come into contact with the test solution.
図 5には、 同一平面に離隔して配置された電極が示されている。 この態様にお いては、 同心円状に、 絶縁体 4 0を介して、 電極 4 2、 4 3が配置されている。 この態様の電極においては、 平面状の浅いセルに使用され、 セルに収容された被 検液と電極 4 2、 4 3力く接触するようになっている。  FIG. 5 shows electrodes that are spaced apart on the same plane. In this embodiment, electrodes 42 and 43 are arranged concentrically via an insulator 40. The electrode of this embodiment is used for a flat shallow cell, and comes into contact with the test solution contained in the cell by virtue of the electrodes 42, 43.
図 6には、 別の態様の平面型電極が示されている。 この態様においては、 長方 形の電極 5 2、 5 3力《絶縁体 5 0上に相互に離隔するように配置されている。 こ の態様の電極も、 平面状の浅いセルに使用され、 セルに収容された被検液と電極 5 2、 5 3力《接触するようになっている。  FIG. 6 shows another embodiment of the planar electrode. In this embodiment, the rectangular electrodes 52, 53 are arranged on the insulator 50 so as to be separated from each other. The electrode of this embodiment is also used for a flat shallow cell, and is brought into contact with the test liquid contained in the cell by the electrodes 52 and 53.
なお、 円筒状のガラス又はプラスチック （例えば、 テフロン） の内側に一定の 間隔で一対の抗体固定化電極を配置したものも使用することができる。 このよう な電極 （フロー型） を被検液中に浮遊させることによって、 この一対の電極間で 電気伝導度を測定することによって、 被検物質の濃度を測定することもできる。 更に、 図 7を参照しながら、 典型的な本発明の免疫測定方法の概略を説明する。 まず、 電極 6 1及び 6 2に、 被検物質 （標準試料） に対する抗体を固定化する (A)。 It should be noted that a pair of antibody-immobilized electrodes arranged at regular intervals inside cylindrical glass or plastic (for example, Teflon) can also be used. By suspending such an electrode (flow type) in a test solution, and measuring the electrical conductivity between the pair of electrodes, the concentration of the test substance can be measured. Further, an outline of a typical immunoassay method of the present invention will be described with reference to FIG. First, an antibody against a test substance (standard sample) is immobilized on the electrodes 61 and 62 (A).
次に、 この固定化した電極 6 1、 6 2を十分に洗浄し、 純水を収容するセルに 入れ、 その状態での電気伝導度を測定する。  Next, the immobilized electrodes 61 and 62 are sufficiently washed, placed in a cell containing pure water, and the electrical conductivity in that state is measured.
次いで、 セルから電極を取り出し、 セルに被検物質を含む被検液を導入する。 この被検波に電極 6 1、 6 2を浸漬し、 2 5 °Cで 1 5分間保持し、 抗原抗体反応 を起こさせ、 抗原抗体反応複合体の形成を起こさせる （B：) 。  Next, the electrode is removed from the cell, and a test liquid containing the test substance is introduced into the cell. The electrodes 61 and 62 are immersed in the test wave and kept at 25 ° C. for 15 minutes to cause an antigen-antibody reaction and to form an antigen-antibody reaction complex (B :).
次いで、 電極 6 1、 6 2をセルから取り出し、 十分純水で洗浄した後、 電極 6 1、 6 2を純水に挿入し、 再び電気伝導度を特定する （C) 。  Next, the electrodes 61 and 62 are taken out of the cell, washed sufficiently with pure water, and then the electrodes 61 and 62 are inserted into pure water, and the electric conductivity is specified again (C).
この操作を被検物質の濃度を変えて、 繰り返し行い、 被検物質に対する検量線 を作成する。 通常、 検量線は、 被検物質の濃度に対する電気伝導度の変化率 （％) をプロットして作成される。  This operation is repeated by changing the concentration of the test substance to create a calibration curve for the test substance. Normally, a calibration curve is created by plotting the rate of change (%) in electrical conductivity with respect to the concentration of the test substance.
実際の被検物質の濃度測定に当たっては、 被検物質を含む被検液に対して上記 と同様にして電気伝導度を測定した後、 この検量線に基づいて、 被検波における 被検物質の濃度を算出する。  In the actual measurement of the concentration of the test substance, the electric conductivity of the test liquid containing the test substance is measured in the same manner as above, and the concentration of the test substance in the test wave is calculated based on the calibration curve. Is calculated.
なお、 温度や湿度等の周囲環境の変化による影響を最小化するために、 差動型 とし、 一対の参照電極 （抗体を固定化していないもの） を同一の被検液中に揷入 してもよい。  To minimize the effects of changes in the surrounding environment such as temperature and humidity, use a differential type and insert a pair of reference electrodes (without immobilized antibodies) into the same test solution. Is also good.
本発明の免疫測定方法においては、 被検物質の種類によって、 検量線の勾配が 正となる場合や、 負となる場合がある。 一般に、 抗体はタンパク質なので、 抗体 を電極への固定化すると、 固定化前に比べて、 電気伝導度が上がる。 また、 被検 物質 （抗原） がタンパク質である場合には、 抗原抗体反応が進行するに従って、 電気伝導度が上昇する。 これは、 タンパク質が電解質 （両性電解質） であるため と考えられる。 これに対して、 抗原が非電解質である場合、 例えば、 メタンフエ タミンのような薬物である場合には、 抗原抗体反応力進行するに従って、 電気伝 導度が低下する。 これは、 固定化された抗体タンパク質に薬物が結合することに よって、 電流の通過が妨げられるからと考えられる。 好ましい態様 In the immunoassay method of the present invention, the slope of the calibration curve may be positive or negative depending on the type of the test substance. In general, antibodies are proteins, so immobilizing the antibody on the electrode increases the electrical conductivity compared to before immobilization. When the test substance (antigen) is a protein, the electric conductivity increases as the antigen-antibody reaction progresses. This is probably because proteins are electrolytes (ampholytes). On the other hand, when the antigen is a non-electrolyte, for example, when it is a drug such as methanephenamine, the electric conductivity decreases as the antigen-antibody reaction force progresses. This is probably because the binding of the drug to the immobilized antibody protein prevents the passage of current. Preferred embodiment
次に、 実施例により本発明を更に詳細に説明する。 但し、 本発明の範囲は、 れらの実施例によって限定されるものではない。 実施例 1 ：  Next, the present invention will be described in more detail by way of examples. However, the scope of the present invention is not limited by these examples. Example 1:
免疫測定用セルの製造 図 8に示す免疫測定用セル 7 1を製造した。 この免疫測定用セル 7 1において は、 Ί 2は角型のセル本体を示す。 このセル本体 7 2は、 主部 7 3と主部 7 3に 着脱可能に取り付けられた挿入部 7 4とから構成されている。 主部 7 3及び挿入 部 7 4の対面する側には、 抗体を固定化した長方形の電極 7 5、 7 6が固定され ており、 主部 7 3に揷入部 7 4を取り付けたときに両電極 7 5、 7 6力互いに対 向配置されるとともに、 両電極 7 5、 7 6間に幅が wの深溝状被検液注入部 7 7 力《形成されるようになっている。 また、 図示していないが、 免疫測定用セル 7 1 には、 両電極 7 5、 7 6を電気伝導度計に接続するための接続手段が設けられ、 この接続手段に電気伝導度計が接続されている。 Production of Immunoassay Cell The immunoassay cell 71 shown in FIG. 8 was produced. In the immunoassay cell 71, Ί2 indicates a square cell body. The cell body 72 includes a main part 73 and an insertion part 74 detachably attached to the main part 73. Rectangular electrodes 75, 76 on which antibodies are immobilized are fixed to the opposite sides of the main part 73 and the insertion part 74, and when the insertion part 74 is attached to the main part 73, both electrodes are fixed. The electrodes 75 and 76 are arranged so as to face each other, and a deep groove-shaped test solution injection portion 77 having a width w is formed between the electrodes 75 and 76. Although not shown, the immunoassay cell 71 is provided with connection means for connecting the electrodes 75 and 76 to an electric conductivity meter, and the electric conductivity meter is connected to this connection means. Have been.
この免疫測定用セルにおいては、 電極材料は以下の表 1に示す材料を使用した。 電極面積は 0. 7 8 5 cm2 、 電極間距離 （被検液注入部 7 7の幅 w) は 0. 5■、 セ ル容量 （被検液注入部 7 7の容量） は 0. 1 9 3 4 3 cm3 、 セル定数は 0. 0 6であ つた。 抗体の入手 In this immunoassay cell, the electrode materials shown in Table 1 below were used. The electrode area is 0.785 cm 2 , the distance between the electrodes (width w of the sample injection part 77) is 0.5 mm, and the cell volume (the capacity of the sample injection part 77) is 0.1. 9 3 4 3 cm 3, cell constant 0.0 6 der ivy. Obtaining antibodies
(1)マウスに対する IgGは、 市販品 （シグマ社製） を使用した。  (1) A commercially available product (manufactured by Sigma) was used as IgG for mice.
(2)コカインに対する抗体は、 市販品 （ケンブリッジメジイカル社製） を使用した。  (2) A commercially available antibody (manufactured by Cambridge Medical) was used as an antibody against ***e.
(3)メタンフヱタミンに対する抗体は、 以下のようにして製造した。  (3) An antibody against methanephthalamine was produced as follows.
メタンフヱタミンにキヤリャ一として BSAを結合し、 これを抗原 （濃度 1 mg/ ml) として、 ゥサギ 5羽に対してそれぞれ 3 ml、 爪と皮膚の間に投与することに よって、 免疫した。 最初の 2か月は、 1週間に 2回注射し、 3か月目から 1か月 置きに 1回注射して、 これを 3回繰り返して抗体を産生させた。 次いで、 ゥサギの頸動脈を切断し、 全血液を採取し、 透析膜を使用して、 抗体 を採集した。 ここで、 得られた抗体には、 BSAに対する抗体が含まれるので、 更 にゲルろ過を行い、 抗メタンフヱタミン抗体を精製した。 得られた抗体は、 使用 時まで凍結保存した。 抗体の収率は、 メタンフェタミン 0. 5 gに対して、 3 0〜 4 0 %であった。 抗体の電極への固定化 BSA was conjugated to methanephthalamine as a carrier, and this was immunized as an antigen (concentration: 1 mg / ml), administered to nails of five puppies, 3 ml each, between the nail and skin. In the first two months, injections were made twice a week, once every other month from the third month, and repeated three times to produce antibodies. The carotid artery of the egret was then cut, whole blood was collected, and antibodies were collected using a dialysis membrane. Here, since the obtained antibody includes an antibody against BSA, gel filtration was further performed to purify the anti-methamphetamine antibody. The obtained antibody was stored frozen until use. The antibody yield was 30 to 40% based on 0.5 g of methamphetamine. Immobilization of antibodies on electrodes
(1)方法 1  (1) Method 1
白金電極を自然放置することによって、 その白金表面を酸化させた （水酸基の 導入） 。 この白金表面にシランカップリング剤 ( r - A P T E S ) のアセトン溶 液 （0. 4 %ァセトン溶液） を塗布し、 1 2 0 °Cで一昼夜乾燥し、 ァミノ基を導入 した。 得られた電極を、 5 %グルタルアルデヒド溶液に浸潰し、 室温で 2時間放 置した。 次いで、 電極を純水で洗浄し、 未反応のグルタルアルデヒドを除去した c 次いで、 この電極を抗体のリン酸緩衝生理食塩水懸濁液に 4 °Cで 1 2時間浸漬し、 抗体の固定化を行った。 未反応の抗体は、 電極を純水で洗浄することによって除 去した。 更に、 抗体と未反応の官能基を不活性化するために、 電極をグリシン溶 液 （0. 1 M溶液） に 2 5 °Cで 3 0分間浸漬した。 得られた電極は、 上記のように 免疫測定用セルを組み立てるまで、 4 °Cにおいて、 pH 7. 4のリン酸緩衝生理食塩 水中に保存した。 The surface of the platinum electrode was oxidized by leaving the platinum electrode unattended (introduction of hydroxyl groups). A solution of a silane coupling agent (r-APTES) in acetone (0.4% acetone solution) was applied to the platinum surface, and dried at 120 ° C for 24 hours to introduce amino groups. The obtained electrode was immersed in a 5% glutaraldehyde solution and left at room temperature for 2 hours. Then washed electrode with pure water, and c to remove unreacted glutaraldehyde, and dipped 12 hours at 4 ° C in phosphate buffered saline suspension of the electrode antibody, immobilized antibody Was done. Unreacted antibodies were removed by washing the electrodes with pure water. In addition, the electrodes were immersed in a glycine solution (0.1 M solution) at 25 ° C for 30 minutes to inactivate the functional groups that had not reacted with the antibody. The obtained electrode was stored at 4 ° C. in phosphate buffered saline at pH 7.4 until the cell for immunoassay was assembled as described above.
(2)方法 2  (2) Method 2
粒径 0. 1〜 1 0 〃mの白金、 金又は炭素、 0. 1 gを 0. 2 mlの純水に懸濁した後、 ポリビニルアルコーノレ又はナフィヨン溶液 0. 2 mlを混合して、 ペーストを形成し た。 直径 3 7 mmの多孔質フッ素樹脂の基板に、 上記ペーストを乾燥後の重量で 1 8. 6 mg/cm2塗布し、 2 2 0 °Cで 1時間焼結した。 次いで、 方法 1と同様にして シランカツプリング剤処理以降の操作を行って、 電極表面に抗体を固定化した。 After suspending 0.1 g of platinum, gold, or carbon having a particle size of 0.1 to 10 μm in 0.2 ml of pure water, mixing 0.2 ml of a polyvinyl alcohol solution or Nafyon solution, A paste was formed. The above paste was applied at 18.6 mg / cm 2 on a porous fluororesin substrate having a diameter of 37 mm by weight after drying, and sintered at 220 ° C. for 1 hour. Next, the same procedure as in Method 1 was performed to perform the operations after the silane coupling agent treatment, thereby immobilizing the antibody on the electrode surface.
(3)方法 3  (3) Method 3
金電極をエタノールで洗浄した後、 室温で乾燥した。 次に、 0. 5 m メルカプト プロピオン酸 8 0 %エタノール溶液に 3 0分電極を浸漬した後、 メルカプトプ 口ピオン酸を除去するためエタノールで洗浄し、 室温で乾燥した。 次いで、 上記 方法 1と同様にして、 グルタルアルデヒド処理以降の操作を行って、 金電極表面 に抗体を固定化した。 The gold electrode was washed with ethanol and dried at room temperature. Next, the electrode was immersed in a 0.5 m mercaptopropionic acid 80% ethanol solution for 30 minutes, washed with ethanol to remove mercaptopropionic acid, and dried at room temperature. Then, The antibody was immobilized on the surface of the gold electrode by performing the operations after the glutaraldehyde treatment in the same manner as in Method 1.
(4)方法 4  (4) Method 4
上記方法 3と同様にして、 金電極をエタノールで洗浄した後、 室温で乾燥した c 次いで、 ドデシルァミンのクロ口ホルム溶液 （3 0 %) を 1 O mMのポリイオンコ ンプレックス溶液に、 ドデシルァミンが 1 0 %の量となるように添加し、 ポリイ オンコンプレックスにアミノ基を導入した。 次いで、 この混合物に、 コレステロ ールのクロ口ホルム溶液 ( 1 0 %) を加えた後、 金電極表面に塗布した （乾燥後 の塗布量として、 2. 5〃m Zcm2 ) 。 次いで、 テトラヒドロフランで処理するこ とにより、 コレステロールを除去し、 電極表面を多孔質化した。 この後、 上記方 法 1と同様にして、 グルタルアルデヒド処理以降の工程を繰り返して、 電極表面 に抗体を固定化した。 The gold electrode was washed with ethanol and dried at room temperature in the same manner as in Method 3 above. Then, a dodecylamine solution (30%) of dodecylamine was added to a 1 O mM polyion complex solution, and dodecylamine was added to the solution. % To introduce amino groups into the polyion complex. Next, a cholesterol solution in form of cholesterol (10%) was added to the mixture, and the mixture was applied to the surface of a gold electrode (a coating amount after drying was 2.5 μm Zcm 2 ). Then, by treating with tetrahydrofuran, cholesterol was removed and the electrode surface was made porous. Thereafter, the steps subsequent to the glutaraldehyde treatment were repeated in the same manner as in Method 1 described above to immobilize the antibody on the electrode surface.
(5)方法 5  (5) Method 5
多孔質シリカ 0. 2 gを純水で洗浄した後、 5 %グルタルアルデヒド溶液で 2時 間室温で処理した。 次いで、 純水で洗浄し、 未反応グルタルアルデヒドを除去し た後、 抗体のリン酸緩衝液の懸濁液に、 得られたシリカを 4 °Cで 1 2時間浸漬し、 シリカに抗体を固定化した。 次いで、 シリカをリン酸緩衝液で洗浄して、 未反応 の抗体を除去した後、 グリシン溶液で処理した。 得られたシリカの懸濁液を、 電 極としての多孔質カーボン （グラシカーボン） に吸引ろ過して、 電極内部にシリ 力を含浸させ、 一体型の固定化電極を形成した。 検量線の作成  After 0.2 g of the porous silica was washed with pure water, it was treated with a 5% glutaraldehyde solution for 2 hours at room temperature. Then, after washing with pure water to remove unreacted glutaraldehyde, the obtained silica is immersed in a suspension of the antibody's phosphate buffer at 4 ° C for 12 hours to fix the antibody on the silica. It has become. Next, the silica was washed with a phosphate buffer to remove unreacted antibodies, and then treated with a glycine solution. The obtained suspension of silica was suction-filtered through porous carbon (glass carbon) as an electrode to impregnate the inside of the electrode with silica force, thereby forming an integral immobilized electrode. Creating a calibration curve
1. 図 8に示す免疫測定用セルの被検波注入部に純水を 1 8 0 1注入し、 両電 極間のベースの電気伝導度 （抗原抗体反応前の電気伝導度） を測定した （図 7 (A) の状態） 。  1. 1801 of pure water was injected into the test cell injection part of the immunoassay cell shown in Fig. 8, and the base electrical conductivity between the two electrodes (the electrical conductivity before the antigen-antibody reaction) was measured ( Fig. 7 (A).
2. 被検液注入部から純水を除去し、 その中に検量線作成用の所定濃度で所定抗 原を含有するサンプルを 1 8 0 1注入した。  2. Pure water was removed from the injection portion of the test solution, and a sample containing a predetermined antigen at a predetermined concentration for preparing a calibration curve was injected into the purified water.
3. 室温で 1 5分間静置し、 抗原抗体反応を行わせた （図 7 ( B ) の状態） 。  3. The mixture was allowed to stand at room temperature for 15 minutes to allow an antigen-antibody reaction (state in FIG. 7 (B)).
4. サンプルを排出した後、 被検液注入部を純水でよく洗浄し、 未反応の抗原を 除去した （BZF (結合 Z遊離） 分離） 。 この場合、 免疫測定用セルを分解し て電極表面を洗浄してもよい。 4. After draining the sample, thoroughly wash the test solution injection part with pure water to remove unreacted antigen. Removed (BZF (bound Z release) separation). In this case, the immunoassay cell may be disassembled to wash the electrode surface.
5. 被検液注入部に純水を 1. 8 0〃 1注入し、 両電極間の抗原抗体反応後の電気 伝導度を測定した （図 7 (C) の状態） 。  5. 1.81 μl of pure water was injected into the injection part of the test solution, and the electrical conductivity after the antigen-antibody reaction between both electrodes was measured (the state of Fig. 7 (C)).
6. pH2. 8のグリシン緩衝液で抗原抗体複合体を解離し、 固定化抗体に結合し た抗原を除去した。  6. The antigen-antibody complex was dissociated with a glycine buffer at pH 2.8 to remove the antigen bound to the immobilized antibody.
7. 上記操作 2〜 6をサンプル中の抗原の濃度を変化させながら、 繰り返した。 以上の操作を、 電極材料、 電極の形状、 固定化方法及び被検物質の種類を以下 の表 1のように変えて行い、 それぞれ対応する抗原に関する検量線を得た （図 9 〜1 3 ) 。 この場合、 検量線の作成においては、 被検物質の濃度に対して、 電気 伝導度の変化率 （％) をプロットした。 表 1  7. The above operations 2 to 6 were repeated while changing the concentration of the antigen in the sample. The above operation was performed by changing the electrode material, electrode shape, immobilization method and type of test substance as shown in Table 1 below, and calibration curves for the corresponding antigens were obtained (Figures 9 to 13). . In this case, in preparing the calibration curve, the change rate (%) of the electric conductivity was plotted against the concentration of the test substance. table 1
電極材料 電極の形状 固定化方法 被検物質 定量可能範囲 図面 Electrode material Electrode shape Fixation method Test substance Quantitable range Drawing
(方法 No. ) (mg/1)  (Method No.) (mg / 1)
白金 螺旋型 コカイン 0. 5 〜10 1 0 白金 櫛型 メタンフヱ 0. 5 〜10 1 1 タミン  Platinum spiral ***e 0.5 to 10 10 platinum comb methane 0.5 to 10 1 1
銅 螺旋型 メタンフエ 0. 5 〜10 1 2 タミン  Copper spiral methane fu 0.5 to 10 1 2 Tamine
白金 対面型 1 IgG 2x10―1〜 5xl03 1 4 金 櫛型 3 メタンフヱ 1〜20 1 5 注） 「螺旋型」 とは、 図 3で示す電極の形状をいう, Platinum Face-to-face type 1 IgG 2x10- 1 to 5xl0 3 1 4 Gold Comb type 3 Methane 1 to 20 15 Note) “Helix type” refers to the shape of the electrode shown in Fig. 3,
「櫛型」 とは、 図 4で示す電極の形状をいう。  The “comb shape” refers to the shape of the electrode shown in FIG.
「対面型」 とは、 図 2で示す電極の形状をいう, 応答時間 マウス IgGにおける抗原抗体反応時間と電気伝導度との関係から、 1 5〜2 0 分で電気伝導度が飽和することが分かった。 従って、 免疫反応時間は 1 5分で十 分であると考えられる。 電気伝導度の測定時間は、 9 0 %応答時間として 1分で 十分であった。 従って、 被検物質の濃度測定に至る所要時間は、 前処理時間を含 めると、 2 0分で十分であると考えられる。 従来、 このような被検物質の測定に は、 2〜3時間必要であったことから、 本発明の免疫測定方法は、 非常に短時間 で測定できることが分かる。 免疫測定用セルの寿命 “Face-to-face” refers to the shape of the electrode shown in Figure 2, Response time From the relationship between the antigen-antibody reaction time and the electrical conductivity in mouse IgG, it was found that the electrical conductivity was saturated in 15 to 20 minutes. Therefore, an immune reaction time of 15 minutes is considered to be sufficient. One minute was sufficient as the measurement time of the electric conductivity as a 90% response time. Therefore, it is considered that 20 minutes is sufficient for the time required to measure the concentration of the test substance, including the pretreatment time. Conventionally, such a measurement of a test substance required 2 to 3 hours, which indicates that the immunoassay of the present invention can be performed in a very short time. Lifetime of immunoassay cell
抗体を固定化した白金電極を冷蔵庫に入れて保存しながら、 定期的にメタンフ エタミン標準液で応答を調べた。 その結果、 1年経過後においても、 免疫測定用 セルとしての機能には変化はなかった。 尿中の薬物濃度の検査への応用  The response was periodically examined using a methane phthalamine standard solution while storing the platinum electrode with the antibody immobilized in a refrigerator. As a result, the function as an immunoassay cell did not change even after one year. Application of urine drug concentration to testing
尿に所定量のメタンフヱタミンを添加して、 5種類の被検液を作成した。 この 被検液について、 上記方法 1に従って、 電極材料として白金を使用して製造した 免疫測定用セル （電極の形状は螺旋型） を使用して、 上記 5種類の被検液に含ま れるメタンフヱタミンの濃度を測定した。 一方、 ドラッグ用公定法として使用さ れている、 GC- MS (ガスクロマトグラフィ一一質量分析） 法によってもメタンフ ェタミンの濃度を測定し、 両者を比較した。 結果を以下の表 2に示す。 表 2  A predetermined amount of methanephthalamine was added to urine to prepare five kinds of test liquids. Using this test solution, an immunoassay cell (electrode shape is spiral) manufactured using platinum as the electrode material in accordance with Method 1 described above was used to measure the amount of methanephthalamine contained in the above five types of test solutions. The concentration was measured. On the other hand, the concentration of methamphetamine was also measured by GC-MS (Gas Chromatography-Mass Spectrometry), which is used as the official method for drugs, and the two were compared. The results are shown in Table 2 below. Table 2
被検液 GC-MS 本発明のセル  Test liquid GC-MS Cell of the present invention
1 5 5 5 0  1 5 5 5 0
2 2. 5 2  2 2.5.2
3 4 2  3 4 2
4 6 8 5 5  4 6 8 5 5
5 6 6 5 6 上記表 2から、 本発明の免疫測定用セルを使用して測定した場合と GC-MS法を 使用した場合との相関性 、 回帰式： Y = 0. 7 5 X 1. 1 6であり、 相関係数はァ = 0. 9 9 6となり、 両 で得られた測定結果は良く相関することが分かる。 実施例の免疫測定用セルは、 抗原濃度の検出に電気伝導度を用いたことにより、 免疫測定方法の簡便化を達成した。 即ち、 電極に被検物質に対する抗体を固定化 し、 免疫測定用セルの被検波注入部に被検液を注入し、 抗原抗体反応の前後の電 気伝導度を測定するだけで、 予め調べておいた検量線に基づいて被検波中の抗原 濃度を簡単にしかも短時間で求めることができる。 これまでに電気伝導度の変化 を指標とした免疫測定の例はなかった。 また、 本発明の免疫測定用セルは、 既存 の電気伝導度測定計に接続すればよいので簡便であり、 また測定操作も特殊な技 術を必要とせず容易である上、 携帯して持ち歩くことも可能である。 更に、 本発 明の免疫測定用セルは、 固定化方法が一定であれば、 測定のばらつきが小さく、 使い捨てのセルとしても使用することができる。 5 6 6 5 6 From Table 2 above, the correlation between the case of using the immunoassay cell of the present invention and the case of using the GC-MS method, regression equation: Y = 0.75 X 1.16, The correlation coefficient is a = 0.996, indicating that the measurement results obtained in both cases correlate well. The immunoassay cell of the example achieved simplification of the immunoassay method by using electric conductivity for detecting the antigen concentration. That is, the antibody against the test substance is immobilized on the electrode, the test solution is injected into the test wave injection part of the immunoassay cell, and the electric conductivity before and after the antigen-antibody reaction is measured. The antigen concentration in the test wave can be easily and quickly obtained based on the calibration curve. To date, there has been no example of immunoassay using changes in electrical conductivity as an index. In addition, the immunoassay cell of the present invention can be simply connected to an existing conductivity meter, so that the measurement operation does not require any special technique, and is easy to carry. Is also possible. Furthermore, if the immobilization method is constant, the immunoassay cell of the present invention has a small measurement variation, and can be used as a disposable cell.
なお、 前記実施例では電極表面に抗体を固定化して抗原濃度を測定したが、 電 極表面に抗原を固定化することにより抗体濃度を測定するように構成することも できる。  In the above embodiment, the antigen concentration was measured by immobilizing the antibody on the electrode surface. However, the antibody concentration may be measured by immobilizing the antigen on the electrode surface.

Claims

請求の範囲 The scope of the claims
1. 電気的に導通可能な一対の電極であって、 前記電極表面に被検物質に対して 反応する抗原又は抗体が固定化された一対の電極を該被検物質を含む被検波に 離隔して配置し、 1. A pair of electrodes that are electrically conductive and have a pair of electrodes on which an antigen or an antibody that reacts with a test substance is fixed on the electrode surface, is separated from a test wave containing the test substance. Place
前記電極表面に固定された前記抗原又は抗体が、 前記被検液中の被検物質と 抗原抗体反応複合体を形成するに十分な時間、 前記一対の電極を前記被検液中 に保持し、 次いで  The antigen or antibody immobilized on the electrode surface is a sufficient time to form an antigen-antibody reaction complex with the test substance in the test solution, holding the pair of electrodes in the test solution, Then
前記一対の電極間の電気伝導度を測定する、  Measuring the electrical conductivity between the pair of electrodes,
ことを特徴とする被検液中の被検物質の濃度を測定する免疫測定方法。  An immunoassay method for measuring the concentration of a test substance in a test solution, characterized in that:
2. 前記電極が、 白金、 金、 銀、 銅、 ニッケル、 鉄、 アルミニウム、 ステンレス、 炭素、 酸化錫、 酸化チタン、 酸化亜鉛、 酸化タングステン、 酸化イリジウム及 び酸化ロジウムからなる群から選択される材料によって構成される請求の範囲 1項記載の方法。  2. The electrode is made of a material selected from the group consisting of platinum, gold, silver, copper, nickel, iron, aluminum, stainless steel, carbon, tin oxide, titanium oxide, zinc oxide, tungsten oxide, iridium oxide, and rhodium oxide. The method according to claim 1, wherein the method comprises:
3. 前記電極が、 白金、 金、 銅又は炭素から構成される請求の範囲 2項記載の方 法。  3. The method according to claim 2, wherein the electrode is made of platinum, gold, copper, or carbon.
4. 前記抗体が、 コカイン、 メタンフェタミン、 アンフヱタミン、 マリファナ、 ヘロイン又はモルヒネに対する抗体である請求の範囲 1項記載の方法。  4. The method according to claim 1, wherein the antibody is an antibody against ***e, methamphetamine, amphotamine, marijuana, heroin, or morphine.
5. 前記抗体が、 多孔質電極材料の内部に保持されている請求の範囲 1項記載の 方法。  5. The method according to claim 1, wherein the antibody is retained inside a porous electrode material.
6. 前記多孔質電極材料が、 多孔質の白金、 金又は炭素である請求の範囲 5項記 載の方法。  6. The method according to claim 5, wherein the porous electrode material is porous platinum, gold or carbon.
7. 被検物質を含む被検液注入部を有するセル本体と、 表面に該被検物質と反応 する抗原又は抗体が固定化されかつ前記セル本体の被検液注入部に離間配置さ れた電気的に導通可能な一対の電極と、 前記一対の電極をこれら電極間の電気 伝導度を計測する計測器に接続する接続手段とを具備する免疫測定用セル。  7. A cell main body having a test liquid injection part containing a test substance, and an antigen or an antibody that reacts with the test substance fixed on the surface, and spaced apart from the test liquid injection part of the cell main body. An immunoassay cell comprising: a pair of electrically conductive electrodes; and connection means for connecting the pair of electrodes to a measuring instrument for measuring electrical conductivity between the electrodes.
8. 前記電極が、 白金、 金、 銀、 銅、 ニッケル、 鉄、 アルミニウム、 ステンレス、 炭素、 酸化錫、 酸化チタン、 酸化亜鉛、 酸化タングステン、 酸化イリジウム及 び酸化ロジウムからなる群から選択される材料によって構成される請求の範囲 7項記載のセル。 8. The material wherein the electrode is selected from the group consisting of platinum, gold, silver, copper, nickel, iron, aluminum, stainless steel, carbon, tin oxide, titanium oxide, zinc oxide, tungsten oxide, iridium oxide, and rhodium oxide. Claims constituted by The cell according to item 7.
9. 前記電極が、 白金、 金、 銅又は炭素から構成される請求の範囲 8項記載のセ ル。  9. The cell according to claim 8, wherein said electrode is made of platinum, gold, copper or carbon.
10. 前記抗体が、 コカイン、 メタンフェタミン、 アンフェタミン、 マリファナ、 へロイン又はモルヒネに対する抗体である請求の範囲 7項記載のセル。  10. The cell according to claim 7, wherein the antibody is an antibody against ***e, methamphetamine, amphetamine, marijuana, heroin, or morphine.
11. 前記抗体が、 多孔質電極材料の内部に保持されている請求の範囲 7項記載の セル。  11. The cell according to claim 7, wherein the antibody is retained inside a porous electrode material.
12. 前記多孔質電極材料が、 多孔質の白金、 金又は炭素である請求の範囲 11項記 載のセル。  12. The cell according to claim 11, wherein the porous electrode material is porous platinum, gold, or carbon.
13. 前記一対の電極が、 相互に離隔して配置されている請求の範囲 7項記載のセ ル。  13. The cell according to claim 7, wherein the pair of electrodes are arranged apart from each other.
14. 前記一対の電極が、 同一平面上で離隔して配置されている請求の範囲 7項記 載のセル。  14. The cell according to claim 7, wherein the pair of electrodes are spaced apart on the same plane.
15. 前記電極が、 螺旋状に形成されている請求の範囲 7項記載のセル。  15. The cell according to claim 7, wherein the electrode is formed in a spiral shape.
16. 前記電極が、 平面状の絶縁体の両側に設けられている請求の範囲 7項記載の セル。  16. The cell according to claim 7, wherein the electrodes are provided on both sides of a planar insulator.
17. 前記一対の電極が、 交互に離隔して入り込んだ状態で配置されている請求の 範囲 14項記載のセル。  17. The cell according to claim 14, wherein the pair of electrodes are arranged so as to be alternately separated from each other.
18. 前記一対の電極が、 同心円状に配置されている請求の範囲 14項記載のセル。  18. The cell according to claim 14, wherein the pair of electrodes are arranged concentrically.
PCT/JP1994/000535 1993-03-31 1994-03-31 Immunoassay and immunoassay cell used therefor WO1994023287A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2757949A1 (en) * 1996-12-30 1998-07-03 Commissariat Energie Atomique MICROSYSTEM FOR BIOLOGICAL ANALYSIS AND ITS MANUFACTURING PROCESS
GB2347746A (en) * 1999-03-05 2000-09-13 Azur Env Ltd Detecting analytes, particularly nucleic acids, in a sample
WO2002012558A1 (en) * 2000-08-07 2002-02-14 Azur Environmental Ltd. Method of an apparatus for the detection of analytes
WO2002025274A1 (en) * 2000-09-20 2002-03-28 Yuko Seino Reaction detecting method, immunoreaction detecting method and device
CN108614021A (en) * 2018-05-15 2018-10-02 云南大学 A kind of electrochemical detection method of Capillary zone electropheresis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0224548A (en) * 1988-05-20 1990-01-26 General Electric Co (Ge) Electric detection of immunological reaction

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0224548A (en) * 1988-05-20 1990-01-26 General Electric Co (Ge) Electric detection of immunological reaction

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2757949A1 (en) * 1996-12-30 1998-07-03 Commissariat Energie Atomique MICROSYSTEM FOR BIOLOGICAL ANALYSIS AND ITS MANUFACTURING PROCESS
WO1998029740A1 (en) * 1996-12-30 1998-07-09 Commissariat A L'energie Atomique Micro system for biological analyses and method for making same
GB2347746A (en) * 1999-03-05 2000-09-13 Azur Env Ltd Detecting analytes, particularly nucleic acids, in a sample
WO2002012558A1 (en) * 2000-08-07 2002-02-14 Azur Environmental Ltd. Method of an apparatus for the detection of analytes
WO2002025274A1 (en) * 2000-09-20 2002-03-28 Yuko Seino Reaction detecting method, immunoreaction detecting method and device
CN108614021A (en) * 2018-05-15 2018-10-02 云南大学 A kind of electrochemical detection method of Capillary zone electropheresis

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