JPS5931695A - Immunoassay unit - Google Patents

Abstract

PURPOSE:The titled device capable of determining an antigen or antibody in a liquid specimen simply and quickly, by separating previously a reagent layer from an enzymatic reaction layer, releasing an enzyme (or substrate) out of a cell, bringing the reagent layer into contact with the enzymatic reaction layer to carry out the enzymatic reaction. CONSTITUTION:When a blood specimen is dropped on the surface of the developing layer 1, the concentration becomes uniform, the specimen is sent to the reagent layer 3, an antigen (or antibody) in the specimen and an antigen (or antibody) bonded to the surface of a cell film involved in or immobilized to a reagent are subjected to a specific antigen-antibody reaction, a complement is activated, the cell film is dissolved and an enzyme (or substrate) is released out of the cell. The second reaction unit base 4B is raised, and the reagent layer 3 is superimposed on the enzymatic reaction layer 5. The enzyme (or substrate) is transferred to the enzymatic reaction layer 5, subjected to an enzymatic reaction with the substrate (or enzyme) in the enzymatic reaction layer 5, to give a reaction product. This reaction product is determined electrochemically by the electrode part 6 inserted into the electrode insertion hole 4E.

Description

[Detailed Description of the Invention] [Technical Field of the Invention] The present invention relates to the technical field of clinical testing devices that measure various components in samples collected from living organisms.

[Technical background of the invention and its problems]

One method of clinical chemical analysis is the dry chemistry method.

In the dry chemistry method, a sample is dropped onto F paper impregnated with at least one reagent or a dry film coated with the reagent, and after a reaction between the reagent and the sample, color develops on the part of the film or F paper onto which the sample has been dropped. rC is used to analyze components in multiple samples by applying a reagent to develop a color and measuring its absorbance.

However, this dry chemistry method requires a coloring mechanism to develop color in the reaction area for the reaction between the reagent and the sample, and also requires a backing mechanism to detect the specific wavelength that falls on the colored area. There are problems in that the ground must be suppressed and an optical system is required for absorbance measurement, making the apparatus complicated.

In addition, in clinical chemistry analysis, as analysis methods that apply enzymes, there are a solution method in which the enzyme is used in a solution state and an immobilized enzyme method in which the enzyme is bound to an insoluble phase and used repeatedly.

However, the solution method has the disadvantage of short enzyme life, and the immobilized enzyme method has no economic advantage, and although the enzyme life is longer than that of the solution method, it is still insufficient. There is a drawback.

(3) Furthermore, in clinical chemistry analysis, as an immunoanalysis method,
There are radioimmunoassay methods and esozyme immunoassay methods, but both require a long time, from several hours to several tens of hours, before sufficient detection is possible.

[Purpose of the invention]

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide an immunoassay unit with a simple configuration that can easily and quickly quantify antigens or antibodies in a liquid sample.

[Summary of the invention]

To achieve the above object, the present invention comprises an enzyme reaction layer supporting a substrate or an enzyme and having a detection means for detecting an enzyme reaction product, and a microcapsule surface which is subjected to lytic action by complement activity. A reagent layer that holds a reagent that binds an antigen or an antibody and has microcapsules containing therein an enzyme or a substrate that reacts with the substrate or enzyme supported on the enzyme reaction layer, and that can be superimposed on the enzyme reaction layer. and (4) a developing layer that is superimposed on the reagent layer and that develops the dropped sample.

[Embodiments of the invention]

An embodiment of the invention will be described with reference to the drawings. FIG. 1 is an explanatory diagram showing an embodiment of the present invention.

In FIG. 1, numeral 3 denotes a reagent layer formed by supporting a reagent on a carrier, which is formed on the first reaction unit substrate 4A, which also includes a defective conductor.

The reaction unit substrate 4A is provided with an opening 4C at a predetermined position, and by attaching an enzyme reaction layer 5, which will be described later, to the opening 4C, the reagent layer 3 and the enzyme reaction layer 5 are brought into contact. It is now possible to do so. On the other hand, a second reaction unit substrate 4 is located below the first reaction unit substrate 4A.
B is placed. A frame 4D that can be attached to the opening 4C is provided on the upper surface of the second reaction unit substrate 4B (the surface facing the first reaction unit 4A), and a substrate or an enzyme is contained in the frame 4D. The enzyme reaction layer 5 holding the reaction unit is solidified and formed, and the second reaction unit substrate 4
An electrode insertion hole 4E is opened at a predetermined position of B, and a detection means such as an electrode section 6 can be attached to this electrode insertion hole 4E. The first reaction unit substrate 4A and the second reaction unit substrate 4B are coupled by an appropriate link mechanism 4F, and the link mechanism 4F is driven by an appropriate drive means 4G for driving the link mechanism 4F. The structure is such that the enzyme reaction layer 5 formed on the second reaction unit substrate 4B and the reagent layer 3 formed on the first reaction unit substrate 4A can come into contact with each other through the reaction unit substrate 4B. When the electrode part 6 is attached to the electrode insertion hole 4E, the protruding electrode like the electrode part 6 is inserted into the enzyme reaction layer 5, and the product of the enzyme reaction proceeding in the enzyme reaction layer 5 is removed. can now be detected electrochemically. A lead wire 7 drawn out from the electrode section 6 is connected to an amplifier 8, and a detection signal 11 outputted from the electrode section 6 is connected to an amplifier 8.
1. configured to amplify the flow.

The detection current t;j: is further outputted to the calculation section 1 ( ) via the @ cable, and the concentration of the specific component in the sample is determined in the calculation section 10 .

The immunoanalysis unit will be described in further detail.

The phase in the reagent layer 3 includes the reagents described below, or
Any material can be used as long as it can be immobilized, such as polyacrylamide, polyurethane, &lJ vinyl alcohol, synthetic polymer glues having polar groups such as polyvinylpyrrolidone fat, polysaccharide glues such as agarose fat, and colladan. , gelatin protein glue, aluminum hydroxide, titanium hydroxide, metal hydroxide glue,
Bonded supports such as ion exchange resins, porous glass beads, plastic membranes, zeolite montmorillonite, etc., which have or have a polarity of amide groups on the surface due to surface treatment, etc. It will be done.

The reagent in the reagent layer 3 contains microcapsules that bind antibodies (-), bamboo antigens) to the surface of microcapsules that undergo lytic action due to complement activity, and contain enzymes (or substrates) inside. Buffers containing physiological concentrations of salts can be used.

As microcapsules capable of binding antibodies (or antigens) and accommodating enzymes (or substrates), red blood cells from animals such as sheep can be suitably used.

Furthermore, even if the red blood cells of other animals or animal cells other than red blood cells can bind antibodies (or antigens) to the cell membrane and contain enzymes (or substrates) within the cells, they can be used in this invention. Can be used as cells.

Furthermore, liposomes can also be used as artificial membranes.

The antibody (or antigen) that binds to the cell membrane is appropriately selected to induce a specific antigen-antibody reaction with the antigen (or antibody) in the sample. For example, when the antigen in the sample is α-fetoprotein, an α-fetoprotein antibody can be used.

Enzymes accommodated within cells include oxidoreductases such as oxalate oxidase, pyruvate oxidase, lactate oxidase, D-amino acid oxidase, and L-amino acid oxidase.
-Amino acid oxidase.

Monoamine oxidase, glucose oxidase, xanthine oxidase, diamine oxidase, alcohol oxidase, uricase, galactose oxidase, acyl coenzyme A oxidase, cholesterol oxidase, sarcosine oxidase, peroxidase, catalase, lactate dehydrogenase (LDH) ,
Glycerol-3-phosphate dehydrogenase, 3-hydroxybutyrate dehydrogenase, 3α steroid dehydrogenase, glucose-6-phosphate zohydrodanase,
Glucose dehydrogenase, malate dehydrogenase, incitrate dehydrogenase.

Hydrolytic enzymes such as glutamic acid decal xylase, aspartase, adenosine deaminase, nucleositase and ocytosine deaminase.

Creatine Φ amidinohydrolase, urease.

Acetoacetate decarboxylase, alkaline phosphatase, acid phosphatase, pyruvate tecarpoxylase, urokinase, phospholipase C, lipase, calxypeptidase, leucine aminopeptidase, amylase, cholinesterase, aldolase, zolasmin, trinosine, ligenuclease, deoxylynuclease, Grotease, glucoamylase, transferases such as pyribate kinase, myokinase.

Acyl-coenzyme A synthetase, ornithine carbamyltransferase, creatininase, asnocarboxylic acid aminotransferase.

Alanine aminotransferase, isomerization 9! Examples include glutamate racemase, alanine racemase, triose phosphate isomerase, and the like. It was,
When analyzing an enzyme component in a sample, the reagent in the reagent layer 3 is a substrate that specifically enzymatically reacts with the above-mentioned enzymes, such as L-aspartic acid, L-alanine, and α-ketogeltaric acid.

Pyruvate, L-lactic acid, L-leucinamide, Glu:r
-x -6-! J-acid, starch, acetylcholine.

Creatine, glutathione, etatoluamine, β-hydroxybutyric acid, ornithine, fructose bisphosphate, triglycerides, phosphoenolpyruvate, isocitric acid, malic acid, plus epsilon, dopamine, ascorbic acid, protein, and polynucleotides. It will be done. However, it is preferable that the substrate accommodated in the cell be a substance that does not easily permeate the cell membrane. For example, when the cell is a red blood cell or liposome, it may contain chloride ions, glycerol, hydrophobic substances such as phospholipids, or a substance that does not easily penetrate the cell membrane. Examples of substances that can be permeated by active transport include glucose amino acids. However, in this specification, the antigen-antibody reaction and the enzyme reaction are separated, so that they can be fully utilized as substrates.

1i of enzyme (or substrate) accommodated in microcapsules
It can be freely adjusted when adjusting the J1 micro force.
Mitsuru Yoshizawa; Biochemistry 53 (9), 1066 (1981))
.

Therefore, the concentration of the enzyme or substrate contained in the microcapsules can be adjusted as appropriate depending on the concentration range of the antigen (or antibody) to be measured. Usually an antibody (or antigen)
Since the amount of intracellular enzyme (or substrate) is greatly in excess of the amount, immunoassay measurements using the immunoassay unit of the present invention can be carried out in a short period of time.

Cells, such as sheep red blood cells, can be prepared by binding an antibody such as an α-fetoprotein antibody to the cell membrane and storing an enzyme such as glucose oxidase within the red blood cells, for example, as follows.

First, when animals such as rabbits, goats, mice, rats, etc. are injected with α-fetoprotein in a conventional manner, these animals are sensitized and produce α-fetoprotein antibodies within their bodies. After an appropriate period of time has elapsed after the injection, a predetermined amount of blood is collected from the animal, and the supernatant of the obtained blood is separated to obtain an antiserum containing anti-α-fetoprotein antibodies. Note that the antiserum may be further purified in order to improve the specificity of the antigen-antibody reaction.

On the other hand, υ red blood cells are suspended by collecting a predetermined amount of blood from another animal, such as a sheep, purifying it, and mixing it with a buffer solution (pH 7) containing salt at a concentration of 0.15M. Obtain the same solution. Since the red blood cells suspended in this tonic solution contain cell fluid, mitochondria, etc., the cell fluid is removed from the red blood cells by the standard dialysis method, and the enzyme glucoamylase is released into the red blood cells. to accommodate. The amount of glucoamylase to be contained can be determined as appropriate depending on the needs of the desired immunoassay method. Next, a suspension of sheep red blood cells and the above-mentioned anti-fetoprotein antibody containing α-fetoprotein antibody can be prepared. When mixed with serum, α-fetoprotein antibodies are adsorbed onto the cell membranes of sheep red blood cells, resulting in an isotope with red blood cells that bind α-fetoprotein antibodies to the thin 11i31 membranes. When it is difficult to adsorb antibodies, antigens (or antibodies) can be bound to the membrane surface by treating them with a divalent reagent such as glutaraldehyde or conotonic acid anhydride, or a peptide reagent layer such as Woodward reagent. I can do it.

To prepare the reagent layer 3, microcapsules can be encapsulated, for example, by a method using agar gel, which is commonly carried out in immunoassays. The above method is described in detail in Introduction to Immunochemical Experiments (Nao Matsuhashi et al., Gakkai Publishing Center (1981)) and Clinical Practice and Virology (Vol. 9, p. 479 (1981), Seiya Sato et al.).

The developing layer 1 transfers the sample dropped onto the upper surface of the developing layer 1 to the reagent layer 3.
This is for diffusing the sample so that it can be transferred at a uniform concentration to the reagent layer 3. For example, various types of barrels used in forming the reagent layer 3 can be used.

Note that the complement that dissolves cell membranes can be one that is contained in the blood of an animal; for example, guinea pig serum can be used as it is as a complement-containing liquid. In addition, the complement is added to the reagent layer 3 and the developing layer 1 in advance.
It may be added to either of the above, or it may be added later as a secondary reagent.

As the electrode part 6, electrochemical means can be adopted. For example, when the reaction product in the enzyme reaction layer 5 is an ion, an ion electrode is used, and when the reaction product is hydrogen peroxide, an ion electrode is used. Hydrogen electrodes can be used.

When using the immunoassay unit with the following two configurations, the antigen (or antibody) being screened can be analyzed in the following manner.

First, in the vl1 state, the reagent layer 3 and the enzyme reaction layer 5 are in a separated state. Therefore, when a sample, for example, a blood sample, is dropped onto the surface of the developing layer 1, the sample solution is diffused in the developing layer 1 and reaches the reagent layer 3 with a uniform concentration. Note that the blood sample may be serum, plasma, or other whole blood. After arrival, antigen (or antibody) during blood test
and the antibody (or antibody) bound to the membrane surface of the cell that is enclosed or immobilized in the reagent layer 3 induces a specific antigen-antibody reaction. Then, the complement that has been transferred with the blood sample from the development layer 1 or added to the reagent layer 3 is activated by the antigen-antibody reaction F, and the cell membrane is damaged by the cytolytic action of complement activation. When dissolved, the enzyme (or substrate) contained within the cell is released outside the cell.

Next, the driving means 4G drives the syringe mechanism 4F to raise the second reaction unit substrate 4B, bringing the upper surface of the enzyme reaction layer 5 and the lower surface of the reagent layer 3 into contact with each other, so that the syringe mechanism 4F is moved onto the enzyme reaction layer 5. The reagent layer 3 is superimposed.

Note that a semipermeable membrane diaphragm is arranged on the first reaction unit substrate 4B so that the reagent layer 3 does not leak from the opening 4C,
When the reagent layer 3 is formed on this diaphragm, the enzyme reaction layer 5 and the reagent layer 3 are brought into contact with each other via this diaphragm and overlapped. Then, the enzyme (or substrate) released into the reagent layer 3 moves to the enzyme reaction layer 5, and an enzyme reaction occurs with the substrate (or enzyme) held in the enzyme reaction layer 5 and the transferred enzyme (or substrate). and a reaction product is obtained. Then,
This reaction product is inserted into the electrode part 6 inserted into the electrode insertion hole 4E.
In the immunoassay unit with the above configuration, the reagent layer 3 holds a reagent having cells containing enzymes (or substrates) therein.
The enzyme reaction layer 6 holding the substrate (or enzyme) is separated in advance, and the enzyme (or substrate) is released outside the cell, and then the reagent layer 3 and the enzyme reaction layer 6 are brought into contact to initiate the enzyme reaction. Therefore, when a cell containing enzyme 3k (or group 'jl) and a substrate (or enzyme) coexist, the extracellular substrate (or enzyme) enters the cell and causes an enzymatic reaction within the cell. Even with a combination of enzyme and substrate, this immunoassay unit can perform an appropriate enzymatic reaction.

Furthermore, the present invention has been described in detail with respect to one embodiment of the present invention (this invention is not limited to the above-mentioned embodiment, and may be implemented with appropriate modifications within the scope of the gist of the invention). Not even.

In this invention, it is necessary to cause an antigen-antibody reaction in the reagent layer 3, and then move the enzyme (or substrate) released into the reagent layer 3 to cause an enzyme reaction in the enzyme reaction layer 5. In order to satisfy this need, as a second embodiment,
As shown in FIG. 2, a reagent layer 3 is superimposed on an enzyme reaction layer 5, and a semipermeable membrane 11 is formed that allows the enzyme (t or substrate) to pass through but not the substrate (!take enzyme). may be placed between the eyebrows. If the two layers are separated by such a diaphragm 11, when the substrate (or enzyme) is able to permeate the cell membrane, the substrate (or enzyme) will diffuse into the reagent layer 3 and will be absorbed within the cell before the antigen-antibody reaction. It is possible to prevent the enzymatic reaction from proceeding, and it is possible to always allow the enzymatic reaction to proceed after the antigen-antibody reaction.

In addition, in order to satisfy the above-mentioned requirements, as a third embodiment, as shown in FIG. A magnetic diaphragm or shielding plate 13 may be interposed between the layers, and the shielding plate 13 may be pulled out from between the layers or inserted between the layers by a pull-out arm 4I driven by a drive means 4H.

In this way, when the antigen-antibody reaction is allowed to proceed in the reagent layer 3, the shielding plate 13 is interposed between both layers, and after the enzyme (or substrate) is released into the reagent layer 3, the shielding plate 13
By pulling out 3 from between both layers, the enzyme (also 1 substrate) diffused into the reagent layer 3 is transferred to the enzyme reaction layer 5, and the enzyme reaction is reliably performed after the antigen-antibody reaction, resulting in the same effect as in the previous example. I can play it.

In this invention, in order to quantify the antigen (or antibody) in the sample, it is necessary to analyze the products of the enzyme reaction that proceeded in the enzyme reaction layer 5. In order to satisfy this need, the method shown in FIGS. 3 and 4(A)'(B) uses the electrode part 6 attached to the electrode insertion hole 4E in contrast to the first embodiment. As shown, the reaction unit substrate 4
A lead Iti! has a pair of electrodes 6A arranged thereon, and also has electrodes 4 and a connecting portion 6B provided at the end of the reaction unit substrate 4 printed thereon. 6C, the stain electrode 6A may be buried in the reagent layer 3 by forming the reagent layer 3 on the reaction unit substrate 4.

Furthermore, as the extraction means, instead of using the electrochemical means as described above, optical detection means may be employed as in the fourth embodiment. That is, as shown in Figure 5,
A photoelectric detector 6D is disposed below the reaction unit substrate 4 made of a transparent material (separately from the surface on which the enzyme reaction layer 5 is formed (111I)), and a photoelectric detector 6D is placed in the enzyme reaction layer 5. A substance that emits light by reacting with a product such as H2O2 is impregnated with or supported on a substance such as luminol.In this way, the antigen (or anti-antigen) in the sample can be determined by quantifying the amount of luminol luminescence with the photoelectric detector 6D. element) can be quantified.

[Effects of the Invention] According to the present invention, although it has a simple configuration, immunoassays that conventionally required a long analysis time can be performed in just a few minutes to several tens of thousands of hours.
It can be done in minutes. In particular, the reagent layer carrying a reagent containing cells containing enzymes (or substrates) and the enzyme reaction layer are separated during the antigen-antibody reaction.
Even with substrates (or enzymes) that can permeate cell membranes, this immunoassay unit can be used to detect antigens (or antibodies) in samples.
can be quantified.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing one embodiment of this invention, and FIGS. 2, 3, 4 (4) (B), and 5 are explanatory diagrams showing other embodiments of this invention. . DESCRIPTION OF SYMBOLS 1... Development layer, 3... Reagent layer, 5... Enzyme reaction layer, 6... Polar part, 11... Diaphragm, 13... Impermeable diaphragm.

Claims (6)

[Claims] (1) An enzyme reaction layer that supports a substrate or an enzyme and has a detection means for detecting an enzyme reaction product; an antigen or antibody is bound to the surface of the microcapsule that is subjected to lytic action by complement activity; a reagent layer that holds a reagent having microcapsules containing an enzyme or substrate that reacts with the substrate or enzyme supported on the enzyme reaction layer, and that is superimposed on the enzyme reaction layer; An immunoassay unit characterized by having a developing layer for developing a dropped sample. (2) The immunoassay unit according to claim 1, wherein the microcapsules are sensitized red blood cells of animals. (3) The immunoassay unit according to claim 1, wherein the microcapsules are liposomes. (4) The enzyme reaction layer and the reagent layer are in a non-contact state in advance, and the enzyme reaction layer and the reagent layer are in contact with a reaction edge in the reagent layer.
The immunoassay unit according to any one of Items 1 to 3. (5) A non-contact state between the enzyme reaction layer and the reagent layer,
5. The immunoassay unit according to claim 4, characterized in that it is constructed by an inert diaphragm interposed between the enzyme reaction layer and the reagent layer. (6) The immunoassay unit according to claim 5, wherein the impermeable diaphragm can be freely inserted between the enzyme reaction layer and the reagent layer.