JPS58221167A - Element for fluoresence immunity analysis - Google Patents

Abstract

PURPOSE:To make possible a highly accurate analysis of trace components in a fluid, by forming in the order of a detecting layer, a fluorescence shading layer and an antigen-antibody reaction layer joined to each other with organic polymer particles which is unswelling to the fluid to be inspected on a liquid impermeable, light transmissible supporting body. CONSTITUTION:An element for fluorescence immunity analysis is made by forming in the order of a detecting layer 2, a fluorescence shading layer 3 and an antigen-antibody reaction layer 4 having a mutually communicating space on each layer by joining particles by dispersing uniformly organic polymer particles (a ternary polymer of styrene-n-butyl methacrylate-glycidyl methacrylate etc.) which is unswelling to a fluid to be inspected and combining by chemically reactive radicals of the particles with each other on a liquid impermeable, light transmissible supporting body 1. An antibody (an antigen) is preliminarily immobilized immovably and a mixture mixed with the fluid to be inspected (containing the antigen or the antibody) with a fixed quantity of a fluorescence dystuffs labelled antigen(antibody), is dropped on the layer 4 and is reacted with the layer 4. Hereafter, the fluorescence of the layers 4 or that of the layer 2, is measured from each side of the layer 4 and the supporting body and the combined labelled antigen and the uncombined labelled antigen, are measured and then, the quantitative analysis of a trace quantity of the antigen(antibody) in the fluid to be inspected is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical element for measuring various antigens or antibodies by fluorescence immunoassay.

Various analytical methods have been developed for the detection and quantification of antibodies, antigens, and other substances present in small amounts in fluids (eg, body fluids). The most accurate method among them is the immunochemical quantitative method, which is based on the fact that antibodies and antigens specifically bind to each other.
(Anti-w,) # It takes advantage of the fact that Ag/Ab (antigen/antibody complex) follows the law of mass action.

The immunochemical quantitative method was developed in 1958 by Berson and Ya.
Since the successful measurement of insulin in serum using insulin labeled with radioactive iodine (III) and anti-insulin antibodies in the serum of diabetic patients, radioimmunoassay methods (Radioimmunoa, SeiRI) have been developed.
A) is widely used.

However, although this RIA method has high sensitivity, it has various major drawbacks due to the use of radioactive isotopes, such as the risk of injury to the human body due to radioactivity, the need for special facilities to prevent the spread of radiation, and operational problems. is complicated and takes a long time to measure, the beta and gamma ray counters required for measurement are expensive, and in the case of isotopes with short half-lives, labeled antigens (antibodies) cannot be stored for long periods of time. Although the number of tests is increasing, the fact that regular clinical laboratories cannot perform the tests is a major bottleneck.

For this reason, a more practical method for clinical testing and a method that does not lead to environmental pollution has been sought.

2 - For this reason, methods that replace radioactive isotopes with other labels have begun to attract attention. Examples include labeled substances such as enzymes, bacteriophages, metal and organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors, cycling reactants, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules. Can be mentioned. Among these, those currently in actual use for measurement include enzyme immunoassay (EIA) using enzymes and fluorescence immunoassay (FIA') using fluorescent molecules.

However, the above-mentioned immunoassay methods that are currently in use have various drawbacks as shown below.

1) Relatively large volumes (
For example, a fluid sample of 0.1 to 1,0 IIJ) is required.

2) A large amount of time (eg, several hours to overnight) is required for the specific binding reaction of the test mixture.

3) After the reaction, physical separation (referred to as separation) of the antigen-antibody binding complex and the unbound substance is required.

4) Many steps are required to complete an immunoassay reaction, and each step must be performed separately. (e.g. sample addition, incubation, separation, labeling (
5) Difficult to adapt to automated systems.

An analysis system using dry chemistry that can overcome these drawbacks was developed in Japanese Patent Application Laid-Open No. 55-9.
No. 0859. In this method, for example, a structural layer made of a polymeric bead polymer (referred to as a registrello layer) and a structural layer made of a colored polymeric bead polymer immobilized with antibodies are sequentially coated on a flexible plastic support. By applying a certain amount of a sample mixed with an unlabeled antigen (test fluid) and a certain amount of fluorescently labeled antigen to the analytical element, the unlabeled antigen and labeled antigen competitively bind to the antibody and migrate to the registration layer. The antigen in a fluid sample is quantified by measuring the amount of unreacted labeled antigen.

However, with this method, there is no cash register storage.

B) detection of unbound labeled antigen that has migrated to the immobilized antibody (referred to as F detection); or B) detection of bound labeled antigen bound to immobilized antibodies (referred to as B detection). However, since the amount of unlabeled antigen is determined by detecting either F or B, it has the disadvantage that it may inevitably contain a large error. This is particularly noticeable when detecting components that are present in extremely small amounts.

In other words, the detection results of extremely trace components are due to variations in the amount of labeled antigen,
It has the disadvantage that it includes errors due to non-specific bond formation, variations in coating film thickness, or fluctuations in fluorescence measurement conditions (such as fluctuations in fluorescence intensity due to fluctuations in light source brightness). It makes the value unreliable.

As a result of intensive studies aimed at overcoming the above-mentioned drawbacks, the present inventors have found that a liquid-impermeable, light-transparent support that is thermally stable and does not swell with the test fluid. 5- A trace amount of acid in a fluid, characterized in that a detection layer, a shielding layer, and an antigen-antibody reaction layer are formed of a particle combination having interconnecting voids formed by bonding of organic polymer particles of 5- We were able to achieve this goal using a fluorescent immunoassay device.

The present invention will be explained in detail below.

In the present invention, the amount of unlabeled antigen is determined by simultaneously measuring labeled antigen-antibody complex () and unreacted labeled antigen (2) in immunoassay.

A test fluid (hereinafter referred to as a specimen) according to the present invention is a fluid containing a measurement target (an immunoactive substance such as an antigen, an antibody, or a hapten), and is a body fluid such as blood, serum, or urine.

FIG. 1 shows a sectional view of an example of an embodiment of the present invention.

1 is a support, 2 is a detection layer, 3 is a shielding layer, and 4 is an antigen-
This is an antibody reaction layer. The specimen can permeate uniformly from the antigen-antibody reaction layer 4 side through the channels of interconnecting voids in each layer 4.3 and 1.

Since the shielding layer 3 exists and the support 1 is transparent, the states of the detection layer 2 and the antigen-antibody reaction layer 4 can be observed separately.

In the present invention, any necessary auxiliary layer may be provided.

First, the principle of measurement using the fluorescence immunoassay element of the present invention will be explained.

A specimen to be analyzed for an unknown antigen is brought into contact with a fluorescent immunoassay element in the presence of a labeled antigen. A labeled antigen can be associated with a fluorescent immunoassay device in one of several ways, including the following.

That is, (1) a labeled antigen is directly added to a specimen (containing an unlabeled antigen), and then the specimen containing the labeled antigen is applied to a fluorescence immunoassay element for analysis.

■Adding the labeled antigen and specimen individually to the fluorescent immunoassay element (for example, (a) Adding the labeled antigen immediately before or after adding the sample and ←) Adding the labeled antigen to the fluorescent immunoassay element, and then drying and re-wet the fluorescence immunoassay element upon addition of the sample).

(2) Incorporating a labeled antigen into a fluorescent immunoassay element so that analysis can be initiated by simply applying the specimen. For example, a labeled antigen may be added to the shielding layer and detection layer of a fluorescent immunoassay device, or an antigen-labeled antigen may be added to the shielding layer and detection layer of a fluorescent immunoassay device containing an immobilized antibody.
Can be incorporated into the antibody reaction layer. In any case, when incorporating a labeled antigen into a fluorescent immunoassay device, care must be taken to keep the labeled antigen separate from the immobilized antibody and to avoid premature binding of the labeled antigen to the antibody.

When a specimen is contacted with a fluorescent immunoassay device in the presence of a co-labeled antigen as described above, the labeled and unlabeled antigens (which are present in the specimen and are the unknowns to be measured) are present within the device. competitively binds to the immobilized antibody present in the antigen-antibody reaction layer.

As a result, if the fluorescence is measured from the support side of the fluorescence immunoassay element, the amount of unreacted label antigen (■) is measured, but if it is measured from the antigen-antibody reaction layer side, the amount of reacted label antigen is 0 (actually, the amount of unreacted label antigen, Although functional valve-specific binding products also exist, F
It is possible to correct from the value of . ) can be measured.

As described above, the fluorescence immunoassay element of the present invention can simultaneously measure F and B. The amount of unlabeled antigen in a specimen can be determined with high accuracy using measured values F and B.

The average size of the particles forming the detection layer, shielding layer, and antigen-antibody reaction layer of the present invention is 1 to 350 μm, preferably 5 to 200 μm. Although particles of different sizes can be used in each layer, it is preferable to use particles of the same size. In addition, depending on the flow path diameter of the interconnecting void in the detection layer and the antigen-antibody reaction layer, and the balance with fluorescence detection,
．． 1-25% by weight of pigment (e.g. Tie, or Ba5
It is also possible to include highly reflective components such as O, etc.). This enhances light scattering within the layer,
Excitation light is effectively used.

In addition, the thickness of each layer is μ, m to 800 μm,
Preferably it is 500 μm.

As an example of the particle combination of the present invention, Japanese Patent Application No. 55-1796
As described in the specifications of No. 13 and No. 55-1179614, particles of non-swellable, impermeable, and thermally stable organic polymers are used for the specimen, and reactive groups on the surface of the particle unit There is a particle bonding body in which the particles are chemically bonded directly or through a low molecular weight compound at the contact portion between the particles.

9- These particle conjugates have interconnecting voids formed by the filling and alignment of particles, and can easily contain and cause reactions with macromolecules involved in immune reactions.

Furthermore, as another example of the particle combination of the present invention, for example,
No. 6-155788 is mentioned. The above-mentioned particle combination has a core-shell double-layer consisting of a hydrophobic, impermeable and non-swellable organic polymer particle as a core, and a shell of a hydrophilic polymer surrounding the core. It is composed of particle units having a structure, and these particle units form a structure by adhesion at their mutual joints. It is obvious that these, like the particle conjugates described above, have interconnecting voids that are effective for carrying out an immune reaction.

The antigen-antibody reaction layer of the present invention is a layer formed by the above-mentioned particle conjugate but containing immobilized antibodies or antigens. It is possible to immobilize the antibody (or antigen) on the surface of the polymer particles forming the particle conjugate according to the present invention while retaining immunological activity; for example, there is a method of supporting the antibody by physical adsorption.

-]〇- It goes without saying that it is possible to carry a wide range of amounts depending on the concentration of the antibody and the pH of the medium. As yet another method, a method known as a covalent bonding method can be used. This method is a method in which antibodies (or antigens) are covalently bonded to the particle surface, and is used to immobilize proteins such as enzymes on carriers.There are various methods depending on the reaction reagent used. can. For details thereof, reference may be made to "Enzyme Immunoassay", edited by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai, pp. 30-60 (Kodansha, Tokyo, 1978).

It is desirable that the antigen-antibody reaction layer contains a non-specific protein to prevent non-specific binding.

It also functions as a detection layer when measuring the fluorescence of the fluorescent label bound to the immobilized antigen-antibody complex.

The shielding layer according to the present invention is formed by the above-mentioned particle conjugate, but when measuring from the detection layer side in immunoassay, the shielding layer is formed from the fluorescent label bound to the immobilized antigen-antibody in the antigen-antibody reaction layer. When measuring from the antigen-antibody reaction layer side, this layer has the function of covering the fluorescence from the unreacted labeled antigen in the detection layer.

Such a function is achieved by including a pigment, dye, etc. (referred to as a fluorescent sealant) that absorbs the fluorescence from the labeled fluorescent substance in the labeled antigen.

As a fluorescent encapsulant, use Wo, Tonred B pigment■
(manufactured by EI DuPont Mores), Hermanent Purple ■ (GAFl + [), Trufast Methyl Violet ■ (manufactured by Sherwin-Williams), India 7 Earth F Blue ■ (manufactured by Harmon Colors), Regal 3
Examples include non-fluorescent pigments and dyes such as 00■ (manufactured by Gabot Co., Ltd. x Monolight Blue ■ (manufactured by ICI) or Vario Fast Blue (manufactured by BASF).

It is also desirable to include a non-specific protein to prevent non-specific binding.

The detection layer according to the present invention is formed by the above-mentioned particle conjugate, and is used to measure unreacted unlabeled antigen in immunoassay. When forming the particle conjugate of this layer, a non-specific protein (for example, bovine serum albumin, etc.) can be included in order to prevent non-specific adsorption not caused by an immune reaction.

The support for the fluorescent immunoassay element of the present invention may be of any type as long as it is liquid-impermeable and light-transmissive, but useful support materials include cellulose acetate, polyethylene terephthalate, polycarbonate, and polyvinyl compounds ( Examples include polymeric materials such as polystyrene, glass, etc.

The preferred support for a given device is one that is compatible with the mode of result detection. In the case of the analytical element of the present invention, the fluorescence measurement detection method is such that the fluorescence emitted within the element is transmitted from within the element through the support to the external detector, so it is transparent to excitation light and fluorescence, and has a low level of It is desirable to use a material that exhibits only background emission as the support material.

When the liquid carrier of the dispersion is removed, the particles used in the present invention can be bonded by 13-chemical bonding of the reactive groups contained in the particles or through a low molecular weight compound and an adhesive layer. However, it is useful to have a catalyst for chemical bonding, such as an acid-alkali, present in the dispersion.

Among acid catalysts, it is particularly useful to use volatile acid catalysts (eg, acetic acid, etc.) and others. The liquid carrier is preferably removed at a temperature below the thermal stability temperature of the organic polymer particles, but preferably at a temperature of 10 to 70°C.

The liquid carrier of the dispersion can be an aqueous liquid. However, other liquid carriers can also be used, such as various organic liquids, provided that the particles are insoluble in the carrier and thus retain their particulate character.

Typical liquid liquids other than water include water-miscible organic solvents, aqueous mixtures of water and water-miscible organic solvents, and suitable water-immiscible organic solvents. Water-miscible organic solvents include lower alcohols (ie, alcohols in which the alkyl group has 1 to 4 carbon atoms), acetone, and tetrahydrofuran. Water-immiscible solvents include lower alkyl esters such as ethyl acetate, and halogenated organic solvents such as halogenated hydrocarbons such as chloroform, methylene chloride, and carbon tetrachloride.

The detection layer, shielding layer, and antigen-antibody reaction layer made of the particle conjugate according to the present invention can be manufactured using various methods. One of the preferred methods is the following process.

(1) Prepare a stable dispersion by dispersing the thermostable organic polymer particles containing reactive groups used in the present invention in a liquid carrier that does not dissolve the particles.2) Use this stable dispersion as a support. and (3) removing the liquid carrier while causing chemical bonding between the reactive groups of the particles at a temperature below the thermally stable moisture content of the organic polymer particles.

1. A stable dispersion means that the particles are present in the carrier without forming agglomerates.

Dispersions useful for making particle conjugate layers must be stable for a sufficient period of time to apply the dispersion onto a support.

Many methods can be used alone or in combination to produce such stable dispersions. For example, one useful method is to add surfactants to the liquid carrier to promote distribution and stabilization of the particles in the dispersion.

Examples of typical surfactants that can be used include Triton ■
There are nonionic surfactants such as Nylphenoxypolyglycidol (manufactured by Olean).

The surfactants can be used in widely selected amounts, but range from 10% to 0.005% by weight, preferably from 6% to 0.05% by weight, based on the weight of the polymer particles. I can do things. Still other methods include sonication, physical mixing, and physical agitation of the particles and liquid carrier to adjust the pH. These methods are even more useful when combined with the methods described above.

In the production of the fluorescent immunoassay element of the present invention, the individual layers can be formed separately and then laminated prior to use, or they can be stored as individual members that are laminated at the time of fluid contact as an element. be. Layers formed as individual parts can be spread to form a film if the dispersion forming the layer is spreadable. The surface on which the separately formed layers are spread is chosen to have such characteristics that the layers can be physically peeled off when dry. However, if adjacent layers are desired, the complication of multiple peeling and lamination steps can be avoided by appropriately allocating and aligning the constituent layers of the analytical element on the surface of the individual layers or on the element support. A convenient method may be to form the films and laminate them closely or closely together.

Analytical elements having a particle combination layer of the present invention can be coated by various methods such as dip coating, air knife coating, curtain coating, or extrusion coating using a hopper as described in U.S. Pat. No. 2,681,294. If desired, two or more layers can be applied simultaneously by the methods described in U.S. Pat. No. 2,761,79117- and British Patent No. 837,095. You can also do it.

Hereinafter, the present invention will be described in more detail. Examples will be shown, but the present invention is not limited to these in any way.

Example 1 Poly(styrene-two-n-butyl methacrylate-co-glycidyl methacrylate)) with an average particle size of 21 μm
(75:15:10% by weight) particles were mixed with the nonionic surfactant Zeonyl FSN (0.1 part by weight of EI DuPont), 0.1 part by weight of bovine serum albumin (Wako Pure Chemical Industries, Ltd.).
．． The particles were suspended in a phosphoric acid buffer (pH 7.6) solution containing 1 part by weight and the suspension was deposited on a subbed polystyrene support (180 μm) exhibiting low level fluorescence at a coating weight of 1.4 μm for the particles. ．． ! The coating was controlled at i'/dm' to provide a detection layer with interconnecting voids. A shielding layer and an antigen-antibody reaction layer were similarly formed on this detection layer by coating, and a fluorescent immunoassay element (1) for measuring human α-fetoprotein was formed.
)It was created.

The shielding layer and antigen-antibody reaction layer are as follows.

18- Shielding layer made of poly (styrene-two-n-butyl methacrylate-co-glycidyl methacrylate)) (75:15:10) containing 2% by weight of Wotton-Red B pigment (manufactured by EI DuPont Thomas) dispersed therein. Average particle size 21μ
m red particles (coating amount 0.11/drr?).

Antigen-antibody reaction layer Particles in which rabbit anti-human α-7 etho7'Rtein antibody (manufactured by Dako Co., Ltd.) was adsorbed to the particles used in the detection layer (Coating II 1)
．． 4 Il/dm).

This analytical element (1) was subjected to FTT as a labeled antigen against 10μ of normal adult serum from which α-7 ethobrotein was removed using an immunosorbent.
C-labeled (fluorescein isothiocyanate) α-fetoprotein lX10”M and O as unlabeled antigen.
Test sera containing various α-fetoproteins ranging from 10 −6 mol to I
) and incubated at 37°C. Then, using a reflection fluorometer with an excitation filter at 485 nm and an emission filter at 515 nm, the support side [F],
The fluorescence intensity was measured from the antigen-antibody reaction layer side 0, and the results shown in Table 1 were obtained.

Table 1 As shown in Table 1, the immunoassay container of the present invention is capable of detecting various concentrations of unlabeled α-7 ethoprotein contained in test serum, and is also capable of detecting unlabeled α-7 ethoprotein using a quick, simple, dry fluorescence method. The measurement enabled rapid immunoanalysis.

Example 2 An analytical element (2) was prepared under exactly the same conditions as in Example 1, except that the particles in the antigen-antibody reaction layer having a particle conjugate were different.

The particles of the above analysis element are as follows.

Analytical element (2) Antigen-antibody reaction layer (poly(styrene-butyl methacrylate-co-glycidyl methacrylate)) with an average diameter of 21 μm
Particles in which rabbit anti-human immunoglobulin G (r chain specific antibody manufactured by Dako Co., Ltd.) antibody was adsorbed to particles of (75:・15110% by weight).

FITC (y-fluorescein isothiocyanate)/human immunoglobulin G, IX]0''M as a labeled antigen and lXl0M as an unlabeled antigen were added to the analytical element (2).
A test serum containing unlabeled human immunoglobulin G of Not yet. (This seems to be due to fluctuations in the light source.) Therefore, when calculating F/B 10 F and Fl/B + F respectively, F/B 10 F has a coefficient of variation of 3.4%.
A coefficient of variation of 1.1% was obtained at IEI/B10F.

In this way, if only F or B is measured, there is a high possibility that the section manager will be included, and it is clear that reproducibility can be improved by correcting by measuring both B and F according to the present invention.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view of an example of the fluorescent immunoassay element of the present invention -η-. 1 is a support, 2 is a detection layer, 3 is a shielding layer, and 4 is an antigen-antibody reaction layer. Agent Yoshimi Kuwahara 23-

Claims (1)

[Claims] A particle assembly having interconnecting voids formed by bonding thermally stable and non-swellable organic polymer particles to a fluid to be tested on a liquid-impermeable, light-transparent support. 1. A fluorescence immunoassay element for trace components in a fluid, comprising a detection layer, a shielding layer, and an antigen-antibody reaction layer.