JPH0424660B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for fluorescent immunoassay of various antigens or antibodies leaking from a fluorescent immunoassay element using dry chemistry. 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 of these is the immunochemical quantitative method, which is based on the specific binding of antibodies and antigens and their reaction Ab (antibody) + Ag (antigen).
It takes advantage of the fact that Ab (antigen-antibody complex) follows the law of mass action. Immunochemical quantitative methods were developed in 1958 by Berson and
Since Yallow succeeded in measuring insulin in serum using insulin labeled with radioactive iodine ( ¹³¹ I) and an anti-insulin antibody, radioimmunoassay (radioimmunoassay) has been developed.
RIA) are 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 the scanning 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 tests cannot be performed in regular clinical laboratories is a major drawback. Therefore, it is a more practical method for clinical testing.
There was a need for a method that would not lead to environmental pollution. For this reason, methods that replace radioactive isotopes with other labels have begun to attract attention. Examples include labeling substances such as enzymes, bacteriophages, metal and organometallic complexes, coenzymes, enzyme substrates, enzyme-hindering substances, cycling reactants, organic prosthetic groups, chemiluminescent reactants, and fluorescent molecules. can be mentioned. Among these, those currently used 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 fluid sample volumes (eg, 0.1 to 1.0 ml) are required compared to conventional chemical or blood analysis, which typically uses 10 to 200 micron fluid samples. 2. The specific binding reaction of the test mixture requires a large amount of time (eg several hours to overnight). 3. After the reaction is complete, physical separation of the antigen-antibody binding complex and unbound material (referred to as B/F separation) is required. 4. Many steps are required to complete an immunoassay reaction, and each step must be performed separately. (For example, steps such as sample addition, incubation, separation, label quantification, etc.) 5. Difficult to adapt to automated systems. An analytical system using dry chemistry that can overcome these drawbacks is disclosed in JP-A-55-90859. In this method, for example, a structural layer made of a polymer bead polymer (referred to as a registration layer) and a structural layer made of a colored polymer bead polymer immobilized with antibodies are sequentially applied onto 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, in this method, A) detection of unbound labeled antigen migrated to the registration layer (F
or B) determining the amount of unlabeled antigen in a fluid sample by detection of bound labeled antigen bound to immobilized antibody (referred to as B detection), but by detection of either F or B. , which determines the amount of unlabeled antigen, has the disadvantage that it may necessarily contain large errors. This is particularly noticeable when detecting components that are present in extremely small amounts. In other words, the detection results for trace amounts of components include errors due to variations in the amount of labeled antigen, non-specific bond formation, variations in coating film thickness, or variations in fluorescence measurement conditions (fluctuations in fluorescence intensity due to variations in light source brightness, etc.). This has the disadvantage of lowering the reliability of quantitative values. As a result of extensive studies aimed at overcoming the above-mentioned drawbacks, the present inventors have developed a material that is thermally stable and does not swell to non-tested fluids on a liquid-impermeable, light-transparent support. A detection layer consisting of a particle conjugate having interconnected voids formed by bonding of organic polymer particles, a shielding layer, and an antigen-antibody reaction layer containing an immobilized antibody (or antigen). The antigen (or antibody) to be analyzed is added to the immobilized antibody (or antigen) in the antigen-antibody reaction layer of the fluorescent immunoassay element, in the presence of a fluorescently labeled antigen (or fluorescently labeled antibody). The amount of antigen in the test fluid (or This objective could be achieved using a fluorescence immunoassay method, which is characterized by quantifying the amount of antibodies. The present invention will be explained in detail below. In the present invention, labeled antigen-antibody complex B and unreacted labeled antigen F are simultaneously measured in immunoassay to determine the amount of unlabeled antigen. 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 habten), 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 the antigen-antibody reaction layer. The specimen can uniformly permeate from the antigen-antibody reaction layer 4 side through the flow paths of the interconnecting voids of 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, a labeled antigen is added directly to a sample (containing unlabeled antigen), and then the sample containing the labeled antigen is applied to a fluorescence immunoassay element for analysis.
Adding labeled antigen and specimen separately to a fluorescence immunoassay device (e.g., adding label antigen immediately before or after addition of sample; adding label antigen to a fluorescence immunoassay device, followed by drying, and adding sample) rewet the immunofluorescence analyzer).
The labeled antigen is incorporated into the fluorescent immunoassay element so that analysis can be initiated by simply applying the analyte. For example, a labeled antigen can be incorporated into the shielding layer and detection layer of a fluorescent immunoassay device, or into the antigen-antibody reaction layer of a fluorescent immunoassay device containing immobilized antibodies. 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 fluorescence is measured from the support side of the fluorescence immunoassay element, the amount of unreacted labeled antigen (F) will be measured, and if measured from the antigen-antibody reaction layer side, the amount of labeled antigen that has reacted (B) will be measured.
(Actually, unreacted labeled antigen and other non-specific binding products also exist, but this can be corrected from the F value.) 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 it is 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. Also a detection layer,
The antigen-antibody reaction layer may contain 0.1-25% by weight of highly reflective components such as pigments (e.g. TiO ₂ or BaSO ₄ ), depending on the channel diameter of the interconnecting voids and the balance with fluorescence detection. It is also possible to
This enhances light scattering within the layer and makes effective use of the excitation light. Moreover, the film layer of each layer is 20 μm to 800 μm,
Preferably it is 50 μm to 500 μm. As an example of the particle combination of the present invention, Japanese Patent Application No.
As described in the specifications of No. 179613 and No. 55-179614, particles of non-swellable, impermeable, and thermally stable organic polymers are used for the specimen, and reactive groups on the surface of the particle units are used. 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. These particle combinations have interconnecting voids formed by filling and aligning particles, and can easily accommodate macromolecules involved in immune reactions and cause reactions. Further, other examples of the particle combination of the present invention include Japanese Patent Application No. 56-155788. The above-mentioned particle combination has a two-layer shell consisting of a central core made of an organic polymer particle that is hydrophobic and non-swellable to a fluid sample, and a shell made of a hydrophilic polymer that surrounds the central 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. Needless to say, 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 covalent conjugation can be used. This method is a method in which antibodies (or antigens) are covalently supported on 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, see Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai, eds. Enzyme Immunoassay, p.30-60 (Kodansha, Tokyo 1978)
can be referred to. 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. Fluorescent encapsulants include Waterton Red B pigment (manufactured by EI DuPont-Moers), Permanent Purple (manufactured by GAF), Sol First Methyl Violet (manufactured by Shearwin-Williams), and India First Blue (manufactured by Harmon Colors). ), Regal 300 (manufactured by Gabotu),
Examples include non-fluorescent pigments and dyes such as Monolight Blue (manufactured by ICI) and Variophorst 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, cellulose acetate,
These include polymeric materials such as polyethylene terephthalate, polycarbonate and polyvinyl compounds (eg polystyrene), glass, and the like. A 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 emission within the element is transmitted from within the element to the external detector through the support, 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 luminescence as the support material. The particles used in the present invention are those in which a particle bond is produced by chemically bonding the reactive groups contained in the particles together when the liquid carrier of the dispersion is removed, or through a low-molecular compound, or by using 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 carriers 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 organic 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) Support this stable dispersion; and (3) removing the liquid carrier while causing chemical bonding between the reactive groups of the particles at a temperature below the thermal stability temperature of the organic polymer particles. 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. In order to produce such a stable dispersion,
Many methods can be used alone or in combination. 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:
There are nonionic surfactants such as Triton x-100 (octyl phenoxy polyethoxy ethanol manufactured by Rohm and Haas) and Surf Actant 10G (nyl phenoxy polyglycidol manufactured by Olean). The above surfactants can be used in widely selected amounts, but based on the weight of the polymer particles,
10 weight percent to 0.005 weight percent, preferably 6 weight percent to 0.05 weight percent may be used. Still other methods include sonication, physical mixing, and physical agitation of the particles and a liquid carrier, and pH adjustment. 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, each layer can be formed separately and then laminated prior to use, or it can be stored as an individual member that is laminated at the time of fluid contact as an element. It is. 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 individually 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 the constituent layers of the analytical element either 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. The analytical element having the particle combination layer of the present invention can be coated by various coating 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 using the methods described in US Pat. No. 2,761,791 and US Pat. No. 8,370,095. EXAMPLES Below, examples will be shown to explain the present invention in more detail, but the present invention is not limited thereto. Example 1 10 parts by weight of particles of poly(styrene-co-n-butyl methacrylate-co-glycidyl methacrylate) (75:15:10% by weight) with an average particle size of 21 μm were mixed with the nonionic surfactant Zeonyl FSN (EI DuPont). (manufactured by Wako Pure Chemical Industries, Ltd.) 0.1 part by weight, bovine serum albumin (Wako Pure Chemical Industries, Ltd.)
The particles were suspended in a phosphate buffer (PH 7.6) solution containing 0.1 parts by weight and the suspension was coated at a coating weight of 1.4 g/dm on a subbed polystyrene support (180 μm) exhibiting low level fluorescence. Controlled and applied to ² ,
A detection layer with interconnecting voids was provided. A shielding layer and an antigen-antibody reaction layer were formed on this detection layer by coating in the same manner to prepare a fluorescence immunoassay element (1) for measuring human α-fetoprotein. The shielding layer and antigen-antibody reaction layer are as follows. Shielding layer: Poly(styrene-co-n-butyl methacrylate-co-glycidyl methacrylate) (75:15:10) containing 2% by weight of Wotton Red B pigment (manufactured by EI DuPont Mores) dispersed therein.
red particles with an average particle size of 21 μm (applied amount 0.7 g/d)
^m2 ). Antigen-antibody reaction layer Particles (coating amount: 1.4 g/dm ² ) prepared by adsorbing rabbit anti-human α-fetoprotein antibody (manufactured by Dako) to the particles used for the detection layer. For this analytical element (1), 10μ of normal adult serum from which α-fetoprotein was removed using an immunoadsorbent was labeled with FITC label (fluorescein isocytocyanate) α-feto as a label antigen. Test sera containing 1 x 10 ^-7 M protein and various α-fetoproteins ranging from 0 to 1 x 10 ^-6 mol as unlabeled antigen were prepared and each was applied to the analytical element (1) and heated at 37°C 20 Incubate for minutes. Thereafter, using a reflection fluorometer with an excitation filter at 485 nm and an emission filter at 515 nm, we measured the results on the support side (F) and the antigen-antibody reaction layer side (B).
The fluorescence intensity was measured and the results shown in Table 1 were obtained.

[Table] As shown in Table 1, the immunoassay container according to the present invention is capable of detecting various concentrations of unlabeled α-fetoprotein contained in test serum, and is also quick, simple, and dry-type. Immunoanalysis could be performed quickly by fluorescence measurement. 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 Rabbit anti-human immunoglobulin G (γ Chain-specific antibody (manufactured by Dako)
Particles adsorbed with antibodies. A test containing FITC (fluorescein isocytocyanate)/human immunoglobulin G, 1×10 ^-7 M as a labeled antigen and 1×10 ^-7 M of unlabeled human immunoglobulin G as an unlabeled antigen in the analytical element (2). When serum was applied and the fluorescence intensity was measured in the same manner as in Example 1, the results shown in Table 2 were obtained.

[Table] The terms B+F should originally be the same, but in this example they are not constant. (This seems to be due to fluctuations in the light source) Therefore, F/B+F,
When calculating B/B+F for each, F/B
+F has a coefficient of variation of 3.4%, B/B+F has a coefficient of variation of 1.1
%was gotten. As described above, it is clear that measuring only F or B is likely to contain errors, and reproducibility can be improved by correcting by measuring both B and F according to the present invention.

[Brief explanation of drawings]

FIG. 1 shows one of the fluorescent immunoassay elements according to the present invention.
FIG. 3 is an example cross-sectional view. 1 is a support, 2 is a detection layer, 3 is a shielding layer, and 4 is an antigen-antibody reaction layer.

Claims (1)

[Claims] 1 Having interconnecting voids formed by bonding organic polymer particles that are thermally stable and non-swellable to the test fluid on a liquid-impermeable, light-transparent support. The immobilized antibody (or After causing a test fluid containing the antigen (or antibody) to be analyzed to act on the antigen (antigen) in the presence of a fluorescently labeled antigen (or fluorescently labeled antibody), the fluorescence intensity is measured from the support side of the analytical element. A fluorescence immunoassay method characterized in that the amount of antigen (or amount of antibody) in a test fluid is quantified by measuring the fluorescence intensity from the antigen-antibody reaction layer side.