JPH03183952A - Production of solid phase body of immune body - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing a highly sensitive immobilized immune substance for immunological test reagents that are frequently used in testing, diagnosis, etc. . In particular, the present invention relates to an improvement in the method for producing an immobilized immune substance by entrapping immobilization. In addition, the immune body as used in the present invention refers to an antibody, an antigen, or a hapten.

[Prior art/problems to be solved by the invention] Detection and measurement of trace components (e.g., hormones) and drugs in biological samples contained in body fluids such as serum and urine are extremely important clinically. Conventionally, this has been done by various methods.

Among such methods, an immunoassay method that utilizes antigens (or antibodies) that are test substances in body fluids and antibodies (or antigens) that have specific binding properties for these is known. ing. Among them, radioimmunoassays (R [8) using radioactive isotopes, enzyme-labeled antibodies and antigens, etc.] are used to determine the presence or absence of a reaction between antigen and antibody and the extent of the reaction.
Enzyme-labeled immunoassay (EIA) using enzyme-labeled immunobodies
enzyme-linked immunoassay (EI), but especially in recent years enzyme-linked immunoassay (EI
A) is widely used because of its high sensitivity, stability of measurement, simplicity, safety, etc.

These methods make it possible to measure trace amounts of antigenic substances, which were previously impossible to measure, by taking advantage of the highly specific and sensitive nature of antigen-antibody reactions. . These methods were first developed as methods for measuring insulin in the blood, and over the next few years they have been developed as methods for measuring polypeptide hormones, which can themselves serve as antigens. Furthermore, it has become possible to create antibodies that react with small molecules, such as albumin or other large molecules, by binding them to albumin or other large molecules, even if they cannot act as antigens on their own. Currently, it is also used to detect trace substances such as small molecule peptide hormones, thyroid hormones, and steroid hormones.

Such immunoassays usually require separation of the bound type (type B) produced as a result of the antigen-antibody reaction and the unbound free type (type F), so-called B/F separation. Such B/F separation methods include immunological methods such as the two-antibody method, electrophoresis,
Physicochemical methods such as chromatography dialysis, partition methods using polyethylene glycol, alcohol, dioxane, etc., methods using chemical adsorbents such as ion exchange resins and Sephadex, solid phase methods using plastic, Sepharose, and porous glass pieces. etc. are used.

In particular, according to this solid phase method, an antibody (or antigen) is immobilized on the surface of a solid phase carrier, and after the antigen (or antibody) to be measured is bound to the immobilized antibody (or antigen), a reaction is performed. By washing away antigens (or antibodies) that are not involved, type B antigens or antibodies can be separated on a carrier as a solid phase.

In the so-called heterogeneous noncompetitive method most commonly used in enzyme-labeled immunoassay (ETA), the solid phase B
type antigen (or antibody) and further enzyme-labeled antibody (or antigen)
After acting, the activity of the B-type labeled complex is measured, and the amount of antigen (or amount of antibody) to be measured is determined.

Therefore, the method of immobilizing antibodies or antigens on carriers is one of the important techniques in immunoassays. Although methods have been known for some time, many problems still exist. For example, in the covalent bond method, the binding yield of the immune body is usually about 20 to 85%, and the remaining immune body is not utilized, and the activity of the immune body is deactivated during the binding process by the covalent bond method. , or often inhibit it. Furthermore, the covalent bonding method requires a high degree of skill to operate, is complicated, and has unstable reproducibility.

In the physical adsorption method, the immobilization rate of the immune body is generally low, and depending on the carrier, the immune body may be released from the carrier, and on the other hand, foreign proteins may be adsorbed, changing the reactivity of the immune body. Sometimes.

Attempts have also been made to entrap and solidify immune bodies with high molecular weight polymers or natural polymers. This method is relatively simple to operate, and when no crosslinking agent is used, there is relatively little inactivation of immune bodies.

Although there are only a few types, depending on the polymer, the sensitivity can be equal to or higher than other solid-phase methods.

Particularly in recent years, it has been found that entrapping solid phase formation using hydrophilic polymers having surfactant-like properties, such as polyvinyl alcohol (PVA) and hydroquinepropylmethylcellulose (HPMC), provides high sensitivity.

Therefore, the purpose of the present invention is to provide a method for producing an immobilized immune substance that is more sensitive than the conventional method by improving the comprehensive immobilization method among the conventional immobilization methods for an immune substance. shall be.

[Means for Solving the Problems] In order to achieve the above object, the inventors of the present application have conducted various studies and found that when entrapping and immobilizing immune bodies on a carrier using a hydrophilic polymer, proteolytic decomposition Compounds (low molecular weight amino acid polymers) consisting of two or more amino acids bonded together by peptides, such as compounds and polypeptides, solid phase that allows for more sensitive measurements than conventional solid phase substances due to the presence of amino acids. The present invention has been developed based on the discovery that a compound can be obtained.

That is, the present invention uses a compound formed by peptide bonding of an amino acid and two or more amino acids (hereinafter referred to as This is a method for producing an immobilized immune substance, which is characterized in that immobilization is carried out in the presence of at least one compound selected from the group consisting of compounds (sometimes collectively referred to as amino acid compounds).

The immune body, hydrophilic polymer, and low molecular weight amino acid polymer are mixed and dissolved in an aqueous solution, then applied or impregnated onto a water-insoluble carrier, and dried.

In the present invention, the term "immune body" refers to various antigens or antibodies. Examples of antibodies include IgG, its Fab,
Any F (ab) 2 etc. may be used, and the antigen is not particularly limited, such as insulin, epidermal growth factor (ECF), thyroid stimulating hormone (TSH), etc.
, Follicle Stimulating Hormone (FSH) Luteinizing Hormone (
L I-(), adrenocorticotropic hormone (ACTH),
growth hormone (GH), mammary gland stimulating hormone (PRL),
Calcitonin (CT), for placental gonata, lopin (HC)
C,), placental lactogen, glucagon, secretin,
Examples include polypeptides such as gastrin, enzymes such as protease, phosphatase, and galactosidase, proteins or complexes thereof such as albumin and HBs antigen, ribopolysaccharides such as lipid A, nucleic acids, viruses, bacteria, and cells themselves. .

Examples of the hydrophilic polymer in the present invention include cellulose ether resins such as methylcellulose, ethylcellulose, hensylcellulose, cyanoethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxyethylcellulose, and aminoethylcellulose;
Examples include polyacrylic acid, polyvinyl alcohol (PVA), polyethylene glycol, and the like.

In the present invention, such hydrophilic polymers may be used alone or as a mixture of two or more.

In the present invention, the compound formed by peptide bonding of two or more amino acids (low molecular weight amino acid polymer) usually has 2 to 100 amino acids, preferably 4 to 70 amino acids, and more preferably 15 to 50 amino acids. A compound in which an acid is bonded to a peptide is suitable, and if considered in terms of molecular weight, it is 200 to 1.
Approximately 4000, preferably 600 to 9600, more preferably 2000 to 6000. Examples of such substances include protein decomposition products, oligopeptides, polypeptides, and the like.

Protein decomposition products are obtained by subjecting proteins to hydrolysis with acids or alkalis, decomposition with proteolytic enzymes, and the like. Conditions differ slightly depending on the type of protein, but the acids used include hydrochloric acid, sulfuric acid,
Examples include nitric acid, preferably pH 1.0 to 6.0.
, especially at pH 1.4 to 4.5, in a sealed tube/l to 110
°C1 especially 60-105 °C11-100 hours, especially 2
Those treated for ~30 hours are suitable. There are no particular restrictions on the protein, and examples thereof include casein, albumin, gelatin, actin, protamines, hemoglobin, myoglobin, γβ, α-globulin, and ossin.

Examples of oligopeptides and polypeptides include L-
Gluk saturated with L-tyrosine, glycyl L-glutamic acid with L-tyrosine, insulin, Vep I-n, poly L-
Examples include lysine (molecules ff 15,000 to 15,000), and -a is produced by a chemical synthesis method known per se.

There are no particular restrictions on amino acids; for example, acidic amino acids (
(e.g., aspartic acid, glutagic acid, etc.), basic amino acids (e.g., lysine, arginine, histidine, etc.), neutral amino acids (e.g., glycine, threonine, etc.),
The concept includes amino acid analogs such as azatryptophan, ethionine, fluorophenylanaline, and thienylalanine, as well as amino acid analogs such as azatryptophan, ethionine, fluorophenylanaline, and thienylalanine.

In the present invention, any carrier for immobilization that can be used in this field can be suitably used. For example, porous membranes made of water-insoluble polymers, glass fiber filters, nonwoven fabrics made of various synthetic fibers, beads made of various polymers, etc. are used. The carrier for solid phase formation is not limited to these, but among the above, porous membranes and nonwoven fabrics having a large specific surface area are preferably used.

Although not particularly limited, in the present invention, the concentration of the hydrophilic polymer in the hydrophilic polymer-immune preparation solution is preferably 0.1% by weight or more. Hydrophilic polymer - When the concentration of the hydrophilic polymer in the immune body preparation solution is less than 0.1% by weight, the hydrophilic polymer -
Even if a water-insoluble solid-phase carrier is coated or impregnated with an immune preparation solution and then dried, it is difficult to obtain high storage stability for the solid-phase immune body. Furthermore, it is desirable that the amount of the hydrophilic polymer in the hydrophilic polymer-immune preparation solution ranges from 0.5 to 2400 times, preferably from 2 to 800 times, the weight of the immunization body. . The amount of the amino acid compound to be used is preferably 0.01 to 500 times the weight of the immunized body.

According to the present invention, for example, the above-mentioned immune body, hydrophilic polymer, and amino acid compound are mixed in an aqueous solvent (for example, 0.1MB
Stir and dissolve in an ES buffer aqueous solution (pH 7, 2), etc.
The above-mentioned carrier is coated onto a suitable area with a capillary, pipette, metered dispensing device, etc., then preferably treated with alcohol, blocking agent, etc., and finally dried to provide a highly sensitive immune system. A phased product can be obtained.

Although the drying conditions are not particularly limited, it is usually preferable to dry at a temperature of 4 to 40'C and a humidity of 40 to 75% for about 1 to 48 hours.

After drying the hydrophilic polymer-immune preparation solution in this way, if it is sealed and stored, for example, with aluminum foil or resin film that does not allow moisture to pass through, the immobilized immune body can be stored more stably. can do.

Furthermore, in the production method of the present invention, the hydrophilic polymer-immune preparation solution is preferably dried by freeze-drying. Freeze-drying may be performed by a conventional method. By such freeze-drying, the immobilized immune body can be stored more stably.

When performing immunological measurements using immobilized immune bodies obtained by the above immobilization method, a solution containing a labeled immune body corresponding to the type of immobilized immune body is usually prepared. After adding the test liquid, the labeled immunobody mixed solution is added.

〔Example〕

The method for producing the immobilized immune substance according to the present invention will be explained below by showing Examples, Comparative Examples, and Test Examples, but the present invention is not limited to these Examples in any way.

Examples 1 to 13/Comparative Examples 1 to 5 For the purpose of investigating the effects of adding various protein decomposition products, polypeptides, or amino acids, the compounds shown in Table 1 were prepared by entrapping solid phase method using polyvinyl alcohol (PVA). Immobilized anti-HCG (human chorionic gonadotropin) antibodies were prepared.

As shown in Table 1, anti-HCG
Antibody (rabbit antiserum) 0.2 mg/ml, PVA
(PVA220, manufactured by Kuraray) 2.4% by weight and mixed in 0.1MBBS buffer (pH 7,2).
The mixture was dissolved to prepare a hydrophilic polymer-immune preparation solution.

20 .mu.l of these aqueous solutions were dropped and allowed to penetrate 15 mm from one end of glass fiber filter paper cut into 1010 mm x 70 strips.

The one end portion serves as a liquid suction port for the test liquid and reagent liquid during measurement. Next, the glass fiber filter paper was immersed in methanol at 23°C for 2 hours, air-dried at 15°C, and then immersed in a 1.8% milk casein, 2M sodium chloride aqueous solution at 4°C.
Soaked for an hour. Next, after immersing in 0.05 M Tris-HCL buffer (pH 7,4) at 23'C for 45 minutes,
It was air-dried at 15°C to obtain a solid phase.

[Test Example 1] A test was conducted using the solid phased product obtained by the above method.

HCG extracted from human urine was adjusted to 0.
The samples were dissolved in a 0.05 M ammonium bicarbonate aqueous solution (pH 7.6, containing 71.0% by weight and 0.9% by weight of bovine serum albumen), and this aqueous HCG solution was used as a test solution.

Separately, reagents for enzyme immunoassay were prepared.

The cleaning agent after B/F separation was 0.5% Tween 20.2
M sodium chloride, Tris-HCL buffer (pH 8,
2) was adjusted to an aqueous solution.

The washing reaction stop solution after the substrate/coloring solution reaction was 0.5% Tw.
An aqueous solution of een20.2M sodium chloride and citric acid-sodium hydroxide buffer (pH 5.0) was prepared.

The substrate/coloring solution was 1.2 mM 5-bromo-4-chloro-3-indolylphosphate, 0.16mM nitroblue tetrazolium, 1.0mM magnesium chloride, Tris-HCL buffer (pH 8,8 ) was adjusted to an aqueous solution.

The enzyme-labeled antibody is a mouse monoclonal anti-β antibody.
HCG antibody (manufactured by Oncogene Sciences, GFO3
), and alkaline phosphatase (derived from calf intestine, manufactured by Heringer Mannheim ■) was used as the enzyme. The conjugation of antibodies and enzymes is carried out using the well-known two-step glutaraldehyde method (^vraneas, S.).

and Ternyck, T., Immunochem.
A slightly modified method was used.

For measurement, a given amount of the test solution and each reagent solution are placed in each well on a glass plate in the order in which they are to be reacted. Test solution is 280 μl, labeled antibody solution 150 μl, washing solution 26
0 μl, substrate/coloring solution 150 μl, reaction stop solution 240 μl
Insert l in this order. Then, the solid phased body is made to suck the liquid from the liquid suction port in this order. However, allow a reaction time of 1 minute after absorbing the labeled antibody solution and 2 minutes after absorbing the 5 substrate/coloring solution. After absorbing the reaction stop solution, observe the color development within 30 seconds.

The results are shown in Table 1.

Unless otherwise specified, each additive is manufactured by Wako Pure Chemical Industries.

(Margin below) 6 * Measurement of serum collected 10 days or more after infection; no color develops even when looked at closely.

; Very pale blue color that is hard to see.

±; Pale but easily visible blue color.

+; Relatively strong coloring.

10: Strong and deep purple.

[Test Example 2] An anti-insulin antibody was immobilized on a solid phase and a test was conducted using this. Furthermore, a cellulose-based water-soluble polymer (hydroxypropyl methylcellulose) was used instead of PVA0.

Watori antiserum (anti-human insulin) was dialyzed against 0.1M acetate buffer (pH 5,8), and insoluble matter was removed by centrifugation, followed by 0.2% BSA (bovine serum albumin).
and 0.03 containing 0.1% sodium azide by weight.
In addition to M phosphate buffer (pi(7,4)), the antibody protein concentration was adjusted to 8.0 μ/ml.

0.5% by weight of this anti-human insulin solution at 200μ℃
In addition to 300μ of the same buffer as above containing 3.2% by weight of casamino acids or 1.0% glycine and 2.8% by weight of hydroxypropylmethyl 9 cellulose,
After mixing well, the mixture was allowed to stand at about 20°C for 1 day.

After that, solid phase formation and reagent preparation were carried out in the same manner as in Test Example 1, and the test was conducted. As a result, when casamino acids or glycine were added, the sensitivity increased compared to when they were not added or when casein was added (control and Comparative Examples 1 to 5). In addition, when the control had an insulin concentration of 0, non-specific color development occurred in 2 out of 7 measurements, but when casagonoic acid or glycine was added, no color development occurred even after the same number of measurements. No specific color development was observed.

〔Effect of the invention〕

According to the present invention, a highly sensitive immobilized immune body can be produced by a simple operation of making an amino acid compound present when entrapping and immobilizing an immune body with a hydrophilic polymer.

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Claims (1)

[Claims] When producing an immobilized immune body in which an immune body is entrapping immobilized on a carrier with a hydrophilic polymer, amino acids and 2
A method for producing a solid-phase immune substance, which comprises immobilizing it in the presence of at least one compound selected from the compounds formed by peptide bonding of the above amino acids.