JPS5915861A - Material for immune analysis - Google Patents

Abstract

PURPOSE:To obtain an accurate measured value from the difference coloring intensities or fluorescent intensities of a porous medium carrying a substance peculiarily bonded to the immunologically active substance in a liquid specimen and a medium not carrying the same, by providing both media on one liquid impermeable support. CONSTITUTION:An immune reaction region 1 fomed by supporting a substance peculiarily bonding to an immunologically active substance such as an antigen, an antibody or hapten by a porous medium (membrane filter, glass fiber filter paper) having a mutually communicated void structure and a compensation region 2 comprising the aforementioned porous medium not carrying the immunologically active substance are adhered to a liquid impermeable support such as polyester to form an analytical material. The regions 1, 2 may be provided either to one surface or both surfaces of the support. After this analytical material is immersed in a test tube in which, for example, a living body liquid specimen is accommodated to be reacted, it is reacted with a labeled antibody and the activity of the bonded labeled antibody is measured by an optical method. The measured value of the region 2 is subtracted from the measured value of the region 1 to obtain an accurate quantitative analytical value by erasing the variation in the measured value due to non-peculiar bonding.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a material for an immunological assay, and more particularly to a material for a quantitative immunoassay.

Various analytical methods have been developed to detect all substances contained in biological fluid samples in minute amounts. This analytical method is mainly based on the principle of immune reaction.

Examples of substances that can stimulate an immune reaction include antigens, antibodies, -butene antigens, complements, etc. For example, immunologically active substances present in serum (hereinafter abbreviated as antigens for convenience)
A specific binding reaction (i.e., immune reaction) between a substance that specifically binds to the immunoactive substance (hereinafter abbreviated as an antibody for convenience) is involved.As a measurement method using the sound principle described above, Various methods have been developed, but immunoassays (hereinafter referred to as immunoassays) are known as the most accurate.

Immunoassay was invented in 1958 by Berso
Since N. and Yellow succeeded in measuring insulin in serum using bovine insulin labeled with radioactive iodine and an anti-insulin antibody gold in the serum of diabetic patients, radioimmunoassay (radioimmunoassay) has been developed. Immunoassay (hereinafter abbreviated as R-A) is widely used.

Since then, various types of labeling compounds other than radioactive isotopes have been developed. Other sensitive compounds include, for example, enzymes, enzyme substrates, coenzymes, enzyme inhibitors, bacteriophages, circulating reactants, metal and organometallic complexes,
Examples include organic prosthetic group chemiluminescent reactants, fluorescent molecules, and the like.

One of the important technical issues in the above immunoassay VC11 is the separation of the substance that stimulated binding (hereinafter abbreviated as B) and the substance that did not (hereinafter abbreviated as F).
This will be abbreviated as B/F separation below.

One means for this separation is an insolubilizing reagent. In other words, in immunoassays, an insolubilizing reagent is used in the form of an antigen or antibody that specifically binds to the substance to be measured through an immune reaction. However, its performance greatly influences the quality of analytical results.Initially, cellulose, cross-linked dextran,
Natural polymeric substances or synthetic polymeric substances, such as cross-linked acrylamide gels, were used3.Since these carriers are fine particles, when separating reagents that have specifically bound to the analyte from the liquid during the analysis process, Usually, centrifugation is required, and the separated reagents also need to be thoroughly washed, so they have the disadvantage of being laborious operations. In order to avoid this, methods have emerged in which a whole carrier of plastic or glass molded into an appropriate size and shape is used, and a method which uses the circular surface of a test tube as a support.

Molded carriers such as those described above have a smaller specific surface area than fine particles, and therefore support a smaller amount of antibody or antigen, which inevitably leads to variations in the amount, narrowing the measurement range, and reducing reproducibility. It also has the disadvantage of being easily deteriorated.

In order to overcome all of these drawbacks, an immobilized carrier with a large QC5 specific surface area and good operability is important, but as the specific surface area increases, non-specific adsorption reactions tend to increase. For this reason, there is a drawback that the background of the calibration curve increases and the accuracy decreases.

The object of the present invention is to eliminate all of the above-mentioned drawbacks and to provide all materials for immunoassays that have the same measurement accuracy as above.

Briefly, the present invention consists of a porous medium having an interconnecting pore structure carrying a substance that specifically binds to an immunologically active substance in a fluid sample on a liquid-impermeable support. An immunoassay material with an immunoreactive area is different only in that it does not support the entire substance.
The present invention relates to a material for immunoassay, characterized in that a correction area made of the same medium as that of the immunoreaction area is provided on the same side or on the opposite side of the support as the immunoreaction area.

The area of the present invention is not limited to % as long as it is porous and has an interconnected void structure. Examples of materials with such a structure include cloth, nonwoven fabric,
Glass fiber paper, paper, membrane filters, etc. can be used.

Furthermore, the porous media described in U.S. Pat.
Fibrous structure development layer described in each specification of No. 1, patent application No. 55-179
613, 55-179614, particle combinations described in each specification, Japanese Patent Application Nos. 56-155788, 57-650
Examples include particle combinations such as No. 5 consisting of particle units having a hydrophobic core and a hydrophilic shell. The above % application 1986-1
The particle units of No. 55788 and No. 57-6505 can further have ionizing radiation or photocrosslinkable functional groups introduced onto their surfaces. These functional groups are
No. 56-5762 and No. 56-5762 have detailed descriptions.

Similarly, [a part or all of the reagent can be contained in the hydrophilic core.] Preferably, these contain reagents related to the detection reaction.

All of the above porous materials are suitable for use, but include mechanical strength, processability, ice-cutting properties, swelling properties, etc. Preferable ones from the viewpoint of species include membrane filters and European Patent No. 1:5156, Japanese Patent Application No. 1983-
No. 82572, No. 55-179613, No. 55-17
'7S14, 56-155788 and 57-6
No. 505, there are items listed in each specification.

At least one of the areas of porous material contains all of the antigen or antibody. As a method for supporting the antigen or antibody, for example, a method using physical adsorption or a method using chemical bonding can be used.

In the physical adsorption method, the antigen or antibody is dissolved in clean water or an appropriate buffer solution, and the entire particle unit or particle unit whole particle conjugate of the present invention is immersed therein for adsorption. At this time, an appropriate buffer solution of about 0.01 to 1M can be used. Further, the concentration of the substance to be adsorbed is in the range of 0.001 to 1.0 yen, and the particle unit or particle combination of the present invention whose surface has been sufficiently cleaned is immersed in the substance to be adsorbed.

The temperature for the above adsorption is preferably room temperature or lower, and the time is preferably 10 to 100 hours. The obtained particle units or particle combinations are separated and washed with a buffer solution,
It is preferable to remove all the antigen or antibody that was not absorbed by adsorption + c6.

In methods using chemical bonding, it may be possible to bond antigens or antibodies to functional groups on the surface of the particle units of the invention using rK contacts or multifunctional reagents. These methods are
For example, the enzyme immobilization technique described in ``Immobilized Enzyme'' (edited by Chibata, 197s, published by Mu Kodansha) VC can be applied. - Examples include diazotization, amidation, alkylation and glutaraldehyde, hexamethylene diisonanate.

Naturally, it is possible to bind the antigen or antibody to the particle unit of the present invention and then create a particle conjugate, or to create a particle conjugate in advance and then bind the antigen or antibody. I'll go.

Furthermore, the particle unit VC of the present invention can carry a protein that is not involved in the immune reaction as a measuring element, if necessary, for the purpose of completely eliminating non-specific reactions in the immune reaction. Typical examples of these include mammalian normal serum proteins, albumin, gelatin, and decomposition products thereof.

As these supporting methods, physical adsorption methods and chemical bonding methods can be used as appropriate in the same manner as described above.

Furthermore, the immunoassay material of the present invention is provided with a correction area similar to the above-mentioned immune reaction area.

It is essential that the above-mentioned correction area uses the same material as the above-mentioned area, has the same film thickness, and has the same surface area. This correction area is exactly the same as the immunoreactive area with whole antigen or antibody described above, except that it does not contain whole antigen or antibody. The purpose of establishing these correction areas is to enable specific binding of the main antigen and antibody during immune reactions and to enable analysis. It progresses quite a bit. It has been found that the provision of the above-mentioned correction area is effective in correcting the increase in blank value due to this non-specific reaction. That is, the correction area is a correction area for non-specific reactions with respect to the immune reaction area.

This correction area may be on the same side of the support as the immunoreaction area, or may be on the opposite side, as long as it is uniformly immersed in the fluid sample to be analyzed. .

This point will be specifically explained with reference to the accompanying drawings.

FIGS. 1 to 3 are schematic cross-sectional views showing embodiments of the immunoassay material of the present invention.

In each figure, numeral 1 means an immunoreaction area, 2Fi correction area, and 3 a support.

As is clear from the drawings, the correction area can be placed in any position in the material of the invention with respect to the immunoreactive area on the support.

As is clear from the above description, if a non-immune protein for eliminating non-specific reactions is carried in the immune reaction zone, the above-mentioned protein must be carried in the correction zone as well.

Various kinds of liquid-impermeable supports can be effectively used in the present invention. Examples include Niha, cellulose acetate, polyethylene terephthalate, polycarbonate 1.
, polystyrene, organic polymeric substances such as polyvinyl chloride, glass, metal, and paper (eg, resin-treated paper, synthetic paper, etc.).These supports can be appropriately selected depending on the mode of detection reaction. Moreover, it is also possible to color using various dyes and pigments, if necessary.

For example, in order to generate a white background for measuring visible or ultraviolet absorption by reflection, it is possible to include a white pigment such as titanium dioxide, baricum sulfate, etc. In addition, in the case of fluorescence photometry, a pigment or dye that absorbs in the wavelength range of fluorescence emitted from a fluorescent substance may be contained in order to suppress undesired fluorescence.

In this case, for example, Wachtung
Red B pigment ■ (Ift, ■, DuPont)
, Permanent Burple ■ (Ghv Company), Trufast Methyl Violet ■ (Sherwine Williams Company), Regal 300 ■ (Cabot Company), and the like.

Further, in the case of scintillation Li measurement, it is also possible to contain a radiation sealant such as lead.

Furthermore, it is possible to laminate a layer containing the pigment or dye mentioned above in one hydrophobic polymer on the support.

The pigment or dye is about 0.0% of the weight of the support or layer.
It is possible to contain about 5 to 50%.

The particle conjugates forming each section of the immunoassay material of the present invention can be constructed using a variety of conventional methods. One of the preferred methods includes the following steps.

(1) Dispersing the particle units of the present invention in a liquid carrier that does not completely dissolve the particles to prepare a stable dispersion free of agglomerates; (2) applying the dispersion to a support; (3) ) removing the liquid carrier while allowing the particle units to bond together at a suitable temperature.

Each area of the immunoassay material of the present invention can be supported by various coating methods, such as dip coating, air knife coating, curtain coating, and extrusion coating using a hopper (see U.S. Pat. No. 2,681,294). Can be applied to the body. If desired, two or more layers may be applied simultaneously (see US Pat. No. FF 2,761,791 and UK Pat. No. 8,57095).

If the regions of the material of the invention already have a porous structure, they can be adhered to a support using a suitable adhesive.

The immunoassay material of the present invention can be used in various immunoassays in which conventional immunoreaction areas can be used.

For example, it can be used in known measurement methods such as RA, enzyme immunoassay, and fluorescence immunoassay.

Moreover, various formats are known for each measurement method, and for example, a competitive method, a Sand-Deutsch method, a two-antibody method, etc. can be used.

It should be noted that it is self-evident that the operation for detection differs depending on the labeled compound. [For details on the actual implementation of these immunoassays, see Radioimmunoassay J (published by Kodansha, 1974) edited by Minoru Irie, edited by Eiji Ishikawa and two others. "#
Immunoassay J (see Igaku Shoin Publishing, 1978).

The immunoassay material of the present invention may have two or more immunoreactive areas carrying different antibodies or antigens as described above. By modifying this and performing the detection reaction in different reaction systems, it is also possible to perform two or more types of antigen-antibody reactions simultaneously.

The immunoassay material of the present invention can be used to analyze biological fluid samples such as serum, urine, and lymph that can be analyzed using conventional immune reaction areas, and there are no particular restrictions on the field of use. . It can be applied to measurement targets suitable for immunoassays such as habten, antigens, and antibodies.

For example, α-1-fetoprotein, carcinoembryonic antigen (
A wide range of drugs, including serum proteins such as ammu), immunoglobulin 01mu, and M (enzygogG, 1gmu, enzygogM), hormones such as insulin and growth hormone, steroid hormones, viruses such as HB antigen, and drugs such as theophylline. It can be used in various fields.

The material for immunoassay of the present invention is easy to manufacture, has a small background, has a wide measurement range, and can be used to perform immunoassay analysis with extremely good reproducibility.

Hereinafter, the present invention will be specifically explained with reference to Examples and Application Examples, but the present invention is not limited thereto.

Example 1 (1) Synthesis of chromogen-containing particle unit 2.4-dichloro-5-methyl-6-[α-(2,4-
di-t-aminophenoxy)butyramide]phenol5. A solution of 1.5f of dibutyl phthalate and 1.0flC of ethyl acetate was added to deionized gelatin 9
．． In addition to an aqueous solution consisting of 9 tons of deionized water, 100 tons of deionized water, and alkanol IO (trade name, E, ■, manufactured by DuPont), an ultrasonic homogenizer USH-150N Model 25 [manufactured by Ultrasonic Industries Co., Ltd., provided that ultrasonic The oscillator is the company's UFX-
15ON25-5B type was used], a dispersion liquid was prepared by irradiating ultrasonic waves, and the dispersion wave sound was maintained at 40'c.

Furthermore, styrene-glycidyl methacrylate copolymer particles (weight ratio 9:1, average particle size 10 .mu.m) were suspended in the above dispersion.The suspension was maintained at 40.degree.

While stirring, add 10% acetic acid aqueous solution to this.
Adjust to H total 4, then 40℃ warm water (deionized water) f2
Added 50tnt. Further stirring is continued until the temperature reaches t-
The temperature was raised to 50°C, maintained at the same temperature for 1 hour, and then cooled to 5°C. 50 formalin aqueous solution 0
．． After adding 5@gi and then adding 10% sodium hydroxide aqueous solution while stirring to adjust the pH to 91c,
The temperature was raised to 50°C at a temperature increase rate of 1°%.
It was kept at °C for 30 minutes. This liquid was cooled to room temperature, filtered through a JKLs glass filter, washed with deionized water, and when the p liquid became neutral, it was taken out and dried. The average particle size was approximately 11 microns.

(2) Preparation of immune reaction zone A dispersion consisting of 51 of the above chromogen-containing particle units was prepared, and this was applied to a primed polyethylene terephthalate support with a total film thickness of 180 μm (containing 10% of titanium dioxide, a white pigment). ) was coated to a coating thickness of approximately 250 μm, and then dried at 40° C. for 1 hour to form a film.

This film was cut into t7×7mm pieces, immersed in a 1tiglutaraldehyde water bath solution at 57°C for 30 minutes, and then 0.01M
Wash several times with sodium phosphate buffer (pH 7,0),
0.0196 rabbit anti-human α-1-fetoprotein aqueous solution [mixed with 0.15 M sodium phosphate buffer (pH 7,6) K manufactured by Dakopack, Denmark] and incubated at 37°C for 1 hour. went. After reaction, 0
．． The film was washed with 0.1M sodium phosphate buffer (pH 7.0), immersed in 0.1M NaBH4 aqueous solution for 3 minutes, and treated with unreacted aldehyde foundation.

(3) Preparation of correction area Prepare the same coating solution as described in the preparation of the above-mentioned immunoreaction area, and apply it on a subbed polyethylene terephthalate (containing 10 titanium dioxide) support with a film thickness of 180 μm. The same coating thickness (approximately 250 μm) was applied, and after drying, the area was cut to 7×7 to form a correction area.゛(4
) Preparation of the material for immunoassay of the present invention The above-mentioned immunoreaction area and correction area were prepared on the material for immunoassay of the present invention by adhering the gold to the same position on the opposite side of a polyethylene terephthalate support with a film thickness of 200 μm.

Application example 1 (1) Peroxidase-labeled egret anti-human α-1
- Preparation of Fetoprotein Rabbit anti-human α-1-fetoprotein (Tenmark
Synthesis and separation were carried out in accordance with conventional methods using glutaraldehyde miberoxidase (Sigma Co., Ltd., USA) and glutaraldehyde miberoxidase (Sigma Co., Ltd., USA).

(2) Measurement standard for human α-1-fetoprotein Human α-1-fetoprotein (or Tenmark Dakovac) y, o, oIM sodium phosphate buffer (pH 7)
, 0, 0, 14 containing total bovine serum albumin, hereinafter abbreviated as buffer A), 25 ng, 100 ng, 40
Human α-1-fetoprotein standard solutions of 0 nH and 800 ng layers were prepared.

Dispense 20 μl of the standard wave sound of each concentration into 10 test tubes (10 WIIφ x 1004), and add buffer A.
3 times each were added, and the material for immunoassay of the present invention described in the above example was placed in each test tube, and cultured at 37°C for 1 hour. After incubation, this material was completely washed with buffer A, and further peroxidase-labeled rabbit anti-human α-1-fetoprotein solution (0.01M sodium phosphate buffer pH
7,0)3d was added, cultured for 1 hour, and the labeled antibody was fully reacted. After washing as above, 2q6 hydrogen peroxide and 1.6 x 10"-"M(7)N,N- ') Engineering fk
-3-mef 0.05 including 4-aminoaniline
M citric acid-0.1M phosphate buffer 6-addition, 67°
CT was incubated for 30 minutes, and then the material of the present invention was completely pulled up and immersed in a 1.5N sulfuric acid aqueous solution to completely stop the enzyme reaction. All red light (λmax=644 nm)
Then, the total reflection density of the immunoreactive area and the correction area was measured.

The results are shown in Table 1.

As is clear from the results shown in Table 1iC, the coefficient of variation is smaller by providing the entire correction area than by providing only the immune reaction area.

Example 2 (including Application Example 2) Using the immune reaction area and correction area prepared in Example 1,
They were cut into f 7 x 7 mm ICs.

The immunoreaction area and the correction area were divided into 180μ TiO2
It was adhered onto a polyethylene terephthalate-containing support in various manners as described below.

That is, for the above-mentioned polyethylene terephthalate support of 7 x 50 sheets, (1) a gold film with a gap of 1 dew was attached to the same side and adhered in parallel, (2) a gap of VC 30m was similarly provided and adhered to the same side. (3) Same VC30
All four types were prepared: (1) a material adhered to the opposite side with a gap of m, and (4) a material adhered to the same position on the opposite side, and were used as materials for immunoassays (1) to (4) of the present invention.

(Measurement of AIPF) 25 ng/mg, 10 Qng/, and 400 n
g of human α-1-fetoprotein standard solution at various concentrations,
4 Buffer A5 peroxidase-labeled anti-α-1-fetoprotein substrate solution All prepared in the same manner as in Application Example 1, and after performing the same operations as in Application Example 1, Sakura Photodensitometer PDA-65
Model [manufactured by Konishiroku Photo Industry Co., Ltd.] used, red light (λma
The reflection density of the immunoreactive and correction areas was measured at o = 644 nm). The results are shown in Table 2 below.

Table 2 As is clear from the results shown in Table 2, the immunoreactive area and correction area of the immunoassay material of the present invention show good results regardless of their relative positions.

As described above in detail, the material for immunoassay of the present invention has the remarkable effects of being free from the problems of the prior art and having the same reproducibility and analytical precision as above.

[Brief explanation of drawings]

1 to 3 are schematic cross-sectional views of embodiments of the immunoassay material of the present invention. 1: Immune reaction area 2: Correction area 3: Support Patent applicant Roku Konishi Photo Industry Co., Ltd. Agent Hirotoshi Nakamoto Akira Inoue / Figure 2 Figure 3 Procedural amendment (method) November 1982 May 4th, Commissioner of the Japan Patent Office, Mr. Wakasugi Wainu 1, Indication of the case, Patent Application No. 124535, filed in 1982
Name of No. Z invention Relationship with the case of persons making amendments to materials for immunoanalysis Patent applicant address 1-26-2 Nishi-Shinjuku, Shinjuku-ku, Tokyo Name (127) Konishiroku Photo Industry Co., Ltd. Representative Kawamoto Mr. Nobuhiko Nishi-Shinbashi Chuoka number 602 phone (a57) -5467
Name Patent Attorney (7850) Hiroshi Nakamoto (
(and 1 other person) 5. Date of amendment order: October 7, 1980 (Shipping date: October 26, 1982) & Target of amendment: Full text of the specification l Contents of the amendment: Engraving of the specification (no change in content)

Claims (1)

[Claims] 1. An immunoassay material in which an immunoreaction area is provided on a liquid-impermeable support, and is made of a porous medium having an interconnected pore structure that supports a substance that specifically binds to an immunoactive substance in a fluid sample. , a correction zone t consisting of the same medium as that of the nuclear immunoreaction zone, differing only in that it does not carry the substance, on the same side or opposite side of the support as the immunoreaction zone; An immunoassay material characterized by being provided with.