JPH09236602A - Immunoagglutination reaction reagent and manufacture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunoagglutination reaction reagent which restricts non-specific reaction along with higher sensitivity by using casein treated in an alkaline solution as blocking agent. SOLUTION: This immunoagglutination reaction reagent has casein treated by alkali supported on a reagent. Otherwise, the immunoagglutination reaction reagent supporting an antibody or an antigen on a carrier undergoes a blocking treatment by casein treated by alkali to produce a reagent. Or a blocking agent comprising the casein treated by alkali is produced. Thus, a highly sensitive immunoagglutination reaction reagent with a limited non-specific reaction can be produced simply.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reagent for measuring components in a test liquid by causing an agglutination reaction immunologically by utilizing an antigen-antibody reaction, which does not cause a non-specific agglutination reaction and has high sensitivity. And a method for producing the same.

[0002]

2. Description of the Related Art As a method for measuring a specific component in a test liquid, there are many immunological measuring reagents utilizing an antigen-antibody reaction. Among immunological measurement reagents, immunoaggregation reaction reagents that utilize agglutination reactions are widely used because of the simple measurement operation.

In the production of an immunoagglutination reaction reagent, a method of supporting a protein as a blocking agent on a carrier in addition to an antibody or an antigen for a specific component to be measured and suppressing a nonspecific agglutination reaction is usually used. As the protein of the blocking agent, bovine serum albumin (hereinafter abbreviated as BSA) is generally used, but casein or gelatin may be used.

Regarding the immunoreagent using casein as a blocking agent, Japanese Patent Laid-Open No. 6-313766 discloses that casein has a higher stability in the enzyme immunoassay (hereinafter abbreviated as EIA) than BSA, and is highly stable. It is disclosed that an antibody-immobilized carrier can be obtained. Further, JP-A-6-194366 describes that a carrier that suppresses non-specific reaction can be prepared by using casein that has been heat-treated at 100 ° C. for 30 minutes at pH 7.2 in the EIA system. In addition, Japanese Unexamined Patent Application Publication No.
36353 has a molecular weight of about 1,000 using digestive enzymes.
It discloses that casein decomposed to ˜26,000 is added to the reaction solution for immunoassay to suppress non-specific reaction.

In the immunoassay utilizing the agglutination reaction of the carrier, in JP-A-63-16266, 3-5 times amount of 30% hydrochloric acid was added to casein, and hydrolysis was carried out at 80-100 ° C. for about 40 hours. A substance is present in the measurement solution to suppress non-specific reactions.

[0006]

As described above, it is known to use BSA or casein as a blocking agent for an immunoagglutination reaction reagent.

However, when BSA is used as a blocking agent, there is a problem that BSA does not sufficiently suppress the non-specific reaction depending on the analyte such as serum, plasma or urine. On the other hand, when casein is used, the non-specific reaction is suppressed, but there is a problem that satisfactory sensitivity cannot be obtained because the agglutinability of the carrier decreases.

Further, the method of hydrolyzing casein with acid or by neutralizing it with heat requires a high temperature of 80 ° C. or higher, and the operation is complicated and dangerous. Japanese Patent Laid-Open No. 63-16
In the method of hydrolyzing hydrochloric acid of No. 266, it is necessary to carry out a reaction at a high temperature of 80 to 100 ° C. for about 40 hours under the condition of 30% hydrochloric acid. Further, after completion of the decomposition, hydrochloric acid is removed by concentration under reduced pressure, which makes the operation complicated. And it was dangerous.

Therefore, there has been a demand for a blocking agent which can suppress a non-specific reaction, obtain sufficient sensitivity, and can be prepared simply and by an operation at a low temperature.

[0010]

Means for Solving the Problems As a result of intensive studies on a blocking agent in order to solve the above problems, the present inventors have shown that casein treated in an alkaline solution (hereinafter sometimes abbreviated as alkali-treated casein) It was found that an immunoagglutination reaction reagent that suppresses non-specific reaction and can obtain high sensitivity can be obtained by using as a blocking agent.

That is, the present invention is the immunoaggregation reaction reagent in which an antibody or an antigen is carried on a carrier, and casein which has been treated with an alkali is carried on the reagent.

Another aspect of the present invention is a method for producing an immune agglutination reagent, which comprises subjecting an immune agglutination reagent carrying an antibody or an antigen to a carrier to a blocking treatment with an alkali-treated casein.

Another aspect of the present invention is a blocking agent for an immunoagglutination reaction reagent, which is composed of an alkali-treated casein.

In the present invention, the above-mentioned means makes it possible to easily produce an immunoaggregation reaction reagent having high sensitivity and less nonspecific reaction. The reason is not clear, but the product obtained by partially hydrolyzing casein under alkaline conditions does not reduce the cohesiveness of the carrier,
It is presumed to suppress the non-specific reaction.

As the carrier used in the present invention, any known carrier can be used without particular limitation, as long as it can carry an antibody or an antigen and the carrier after carrying is aggregated when an antigen-antibody reaction occurs.

Examples of carriers that can be preferably used include polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, and acrylic. Fine particles of organic polymer such as acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, latex such as polyvinyl acetate acrylate, or inorganic oxidation such as silica, silica-alumina and alumina Examples thereof include inorganic particles in which a functional group is introduced by subjecting a substance or the inorganic oxide to a silane coupling treatment and the like, and particles of biological origin such as human O blood cells and sheep red blood cells.

The particle size of the above-mentioned carrier is not particularly limited, but the average particle size is 0.05 to 10 μm from the viewpoint of the easiness of aggregation after the antigen-antibody reaction and the easiness of discrimination of aggregation.
Preference is given to using the carriers mentioned above. The amount of carrier used may be appropriately determined depending on the type of antibody or antigen,
From the viewpoint of loading efficiency and operability, it is preferable to use it so that the concentration in the solution at the time of measurement is 0.001 to 15 wt%, and it is generally used in the form of a suspension.

The antibody or antigen carried on the carrier in the present invention is not particularly limited as long as it causes an antigen-antibody reaction with the antigen or antibody to be tested. Examples of the antigens or antibodies preferably used in the present invention include β2-microglobulin (hereinafter abbreviated as β2-m), anti-β2-m antibody, fibrinogen, anti-fibrinogen antibody, fibrin degradation product (FDP), anti-antibody. FDP antibody, albumin, anti-albumin antibody, human immunoglobulin G (IgG), anti-human I
gG antibody, C-reactive protein (CRP), anti-CRP antibody, ferritin, anti-ferritin antibody, α-fetoprotein (AFP), anti-AFP antibody, insulin, anti-insulin antibody, Treponema pallidum (TP), streptolysin O (SLO) ) And hepatitis B virus (HBV).

As a method for supporting an antibody (or an antigen) on a carrier, known methods can be used without particular limitation. As a basic supporting method, there are a physical adsorption method and a chemical bonding method, and the physical adsorption method is preferably used from the viewpoint of the convenience of the supporting operation.

The immunoagglutination reagent of the present invention is a reagent which utilizes a phenomenon in which an antibody or an antigen is carried on a carrier and brought into contact with the antigen or the antibody in a sample to cause the carrier to agglutinate with an immune reaction. Examples of the qualitative reagent include a latex agglutination reagent and a microtiter reagent, and examples of the quantification reagent include a latex quantification reagent that optically measures the degree of agglutination.

The casein used in the present invention is not particularly limited, but those purified from milk are preferable in terms of economy and purity.

The condition for the alkali treatment of casein is not particularly limited as long as it is a condition under which casein is gently hydrolyzed, and the pH, temperature and time during the treatment can be appropriately selected.

The preferred pH during the treatment is 9 to 14,
A pH of 12 to 14 is more preferable in that it does not require a high temperature during the treatment and the treatment time is short. Further, the method of setting the alkaline conditions is not particularly limited, but it is preferable to use an aqueous solution of an alkaline compound such as sodium hydroxide or potassium hydroxide from the viewpoint of easy operation and economical efficiency. The treatment temperature is preferably selected in the range of 4 ° C to 100 ° C, and is determined in consideration of pH and time during the treatment. The range of 4 ° C to 60 ° C is more preferable from the viewpoint of operability and safety during processing. The treatment time is appropriately determined according to the pH and temperature during the treatment. However, if too much alkali treatment is performed, the effect as a blocking agent becomes weaker,
Agglutination tends to occur during the production of immunoaggregation reaction reagents.

Although there is no particular limitation after the alkali treatment, it is preferable to neutralize with an acid such as hydrochloric acid in consideration of the stability of the alkali-treated casein and the pH fluctuation during the preparation of the reagent.

The alkali-treated casein used in the present invention can be identified by analyzing a sample reduced with 2-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. The electrophoresis image shows that the molecular weight is about 32,000 when untreated casein is used as a sample, but the molecular weight is about 27,000 to about 3 in the case of alkali-treated casein.
It is 0000. It should be noted that if the alkali treatment is performed too much, the molecular weight of the components having 20,000 or less increases.

The blocking treatment with the alkali-treated casein as a blocking agent is carried on the carrier by adding the alkali-treated casein usually in a buffer solution after or simultaneously with supporting the antibody or antigen on the carrier. In a special case, the carrier may be preliminarily treated with alkali-treated casein before the antibody or the antigen is supported on the carrier. Other conditions for the blocking treatment are not particularly limited, and a conventionally known method is adopted. The amount of alkali-treated casein added during the blocking treatment varies depending on the immunoagglutination reagent used, but the carrier concentration during blocking is 1
It is preferable to add 0.01 to 2 mg with respect to mg.

In the present invention, as the alkaline treatment condition of casein, it is preferable to select and employ from the conditions of pH 12 to 14, temperature 4 ° C. to 60 ° C., treatment time 1 hour to 20 days. Specifically, pH 13, temperature 56 ℃
For 1 to 6 hours or at pH 13 and temperature of 25 ° C for 30 to 30
Treatment conditions include 60 hours, or pH 13, 4 ° C. for 10 to 20 days.

Next, as an example of the immunological agglutination reagent of the present invention, a reagent form consisting of two liquids (latex agent and second agent) of a latex quantification reagent will be shown. It is intended to be illustrative and not limiting of the invention.

Agent: A mixed solution containing the following (1), (2) and (3) as basic components (1) Buffer solution 20 to 1000 mM, pH 4 to 12 (2) Antibody (or antigen) and alkali-treated casein Supported carrier 0.01 to 0.5% (w / v; standard for Ax agent) (3) Sodium chloride 50 to 300 mM Otsuto: Mixed solution containing the following (4) and (5) as basic components (4) Buffer Liquid 20 to 1000 mM, pH 4 to 12 (5) Sodium chloride 50 to 300 mM Examples of the buffer solution include Tris buffer solution, phosphate buffer solution, glycine buffer solution, borate buffer solution and Good's buffer solution.

The following methods can be given as examples of the method for producing the above-mentioned upper shell agent.

First, the latex particles are added in an amount of 0.5 to 10% (w
/ V) 0.05% antibody (or antigen) in the suspended buffer
30 minutes after mixing a buffer solution containing ~ 100 mg / ml
Shake for 48 hours at 4 to 60 ° C. Next, add 0.05-200 mg / ml of alkali-treated casein, 3
After shaking under conditions of 0 to 48 hours at 4 to 60 ° C., the carrier and the solution are separated by centrifugation or the like to remove unbound antibody (or antigen). Finally, the carrier is dispersed in a buffer solution to give a former.

[0032]

EFFECT OF THE INVENTION By using casein treated with alkali as a blocking agent, non-specific reaction is suppressed, and an immunoaggregation reaction reagent having a higher aggregating property than the case of using untreated casein, that is, having a high sensitivity, is used. can do.

[0033]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to the scope described in these examples.

Examples 1 to 5 (1) Preparation of alkali-treated casein 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries, Ltd.) was added.
After dissolving in 10 ml of 1N sodium hydroxide aqueous solution, 2
The pH was adjusted to the conditions shown in Table 1 by adding N hydrochloric acid or 5N sodium hydroxide. Next, at the temperature and time shown in Table 1, the mixture was allowed to stand at 4 ° C., shaken at 25 ° C. and 56 ° C., and then adjusted to pH 7 by adding 2N hydrochloric acid. The obtained aqueous solution of alkali-treated casein was used for the following preparation.

(2) Preparation of upper agent (anti-β2-m antibody-supporting latex suspension) Polystyrene particles having an average particle diameter of 0.12 μm were added to a pH of 8.
2m of a suspension having a latex concentration of 1% (w / v) by diluting with 6 of 0.02M glycine buffer (hereinafter abbreviated as GB).
1 was prepared. Then, 2 ml of a solution (2 mg / ml) obtained by diluting the anti-β2-m goat antibody with GB was added and mixed. After shaking at 37 ° C. for 16 hours, 1 ml of an aqueous solution containing the above-described alkali-treated casein was added, and the mixture was further shaken for 2 hours.
Then, by centrifugation, the resulting precipitate (anti-β2-m antibody-bearing latex) contains 8 ml of 0.1 M sodium chloride and 0.1% sodium azide, and a pH of 0.1 M
A Tris buffer (hereinafter, this mixed solution is abbreviated as TBS) was added and suspended to prepare an upper agent.

(3) Preparation of B-agent (buffer solution) TBS containing 1% PEG-6000 (Wako Pure Chemical Industries)
Was prepared as an ointment.

(4) Preparation of β2-m test solution A human β2-m antigen solution was diluted with TBS containing 0.1% (w / v) bovine serum albumin to 1000 ng / ml to prepare a test solution. did.

(5) Measuring method 15 μl of β2-m test liquid was added to 200 μl of the second agent in a glass cell, and the mixture was stirred and then allowed to stand at 37 ° C. for about 5 minutes. Next, 100 μl of the former was added and stirred, and the optical density change amount at a wavelength of 660 nm from 30 seconds to 200 seconds was measured. An automatic analyzer TBA-30R (Toshiba Medical) was used for the above operation.

(6) Relationship between Alkali Treatment Conditions and Optical Density Change A combination of an upper agent prepared by using alkali-treated casein treated under different pH, temperature and treatment time conditions as a blocking agent, and Otsu agent, The results of measuring the β2-m test liquid are shown in Table 1.

Comparative Example 1 (1) Preparation of Neutrally Treated Casein 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries, Ltd.)
After dissolving in 10 ml of 1N sodium hydroxide aqueous solution, hydrochloric acid was immediately added to adjust the pH to 7.0. 56 ° C
I shook it for 4 hours.

(2) The same operation as in Example 1 was carried out except that casein was prepared.

(3) Relationship between Neutral Treated Casein and Optical Density Change Amount of β2-m test solution was measured in Table 1 by using a combination of an upper agent and a second agent with neutralized casein as a blocking agent. Indicated.

Comparative Example 2 (1) Preparation of heat-denatured casein 10 g of 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries) was used.
10 mM phosphate buffer containing mM sodium chloride (pH
7.2). The solution was stirred at 100 ° C for 30 minutes and then cooled to 4 ° C.

(2) The procedure of Example 1 was repeated except that the casein was prepared.

(3) Relationship between Neutrally Treated Casein and Change in Optical Density Table 1 shows the results of measurement of β2-m test liquids using a combination of an upper agent and a second agent with heat-denatured casein as a blocking agent. It was

Comparative Example 3 (1) Preparation of untreated casein 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries, Ltd.) was added.
After dissolving in 10 ml of 1N sodium hydroxide aqueous solution, hydrochloric acid was immediately added to adjust the pH to 7.0.

(2) The same operation as in Example 1 was carried out except that the casein was prepared.

(3) Relationship between untreated casein and the amount of change in optical density The results of measurement of β2-m test liquids by combining an antifungal agent and an antifungal agent with untreated casein as a blocking agent are shown in Table 1. .

[0049]

[Table 1]

The change in optical density of the reagent using casein treated with alkali is ΔOD = 0.342 to 0.405.
The change in optical density of the reagent using casein treated with neutral was ΔOD = 0.162, and the change in optical density of the reagent using casein neutralized with heat was ΔOD = 0.1.
45, the amount of change in optical density of the reagent using untreated casein is 0.167, which is extremely large.

The magnitude of the change in optical density means that the cohesiveness is high or low, and the larger the quantity is, the better the cohesiveness is and the higher the sensitivity is. Therefore, it can be seen that the reagent of the present invention has improved sensitivity.

Nonspecific agglutination when measuring serum was not caused by any of the reagents of Examples 1-5 and Comparative Examples 1-3.

Examples 6 to 8 (1) Preparation of alkali-treated casein 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries, Ltd.) was added.
After dissolving in 10 ml of 1N sodium hydroxide aqueous solution, 1
The pH was adjusted to 12.0 by adding N hydrochloric acid. Then 4 ° C
40 days, shake at 37 ℃ for 4 hours, or 56 ℃
After shaking for 4 hours, the pH was adjusted to 7 with hydrochloric acid.
The obtained aqueous solution of alkali-treated casein was used for the following preparation.

(2) Preparation of an upper agent (antifibrinogen antibody-supporting latex suspension) Polystyrene particles having an average particle diameter of 0.12 μm were added to a pH of 8.
2m of a suspension having a latex concentration of 1% (w / v) by diluting with 6 of 0.02M glycine buffer (hereinafter abbreviated as GB).
1 was prepared. The anti-fibrinogen goat antibody was then used for GB
2 ml of the solution (2 mg / ml) diluted with was added and mixed. After shaking at 37 ° C. for 16 hours, 1 ml of an aqueous solution containing alkali-treated casein was added, and the mixture was further shaken for 2 hours. Then, by centrifugation, 8 ml of 0.1 M was added to the obtained precipitate (antifibrinogen antibody-bearing latex).
PH containing sodium chloride and 0.1% sodium azide
A 0.1 M Tris buffer solution (TBS) of 7.5 was added and suspended to prepare an upper agent.

(3) Preparation of B-agent (buffer solution) TBS containing 1% PEG-6000 (Wako Pure Chemical Industries)
Was prepared as an ointment.

(4) FDP test solution A serum sample having an FDP value of 40 μg / ml was used.

(5) Method of measurement 5 μl of the test substance was added to 300 μl of the second agent in a glass cell, and the mixture was stirred and then allowed to stand at 37 ° C. for about 5 minutes. Next, 1 instep
00 μl was added and stirred, and the optical density change amount at a wavelength of 660 nm from 30 seconds to 200 seconds was measured.

(6) Relationship between Alkali Treatment Conditions and Optical Density Change A combination of an upper agent prepared by using alkali-treated casein treated under different pH, temperature and treatment time conditions as a blocking agent, and Otsu agent, The results of measuring the FDP test liquid are shown in Table 2.

Comparative Example 4 (1) Preparation of untreated casein 0.1 g of commercially available casein (milky, Wako Pure Chemical Industries) was used.
After dissolving in 10 ml of 1N sodium hydroxide aqueous solution, hydrochloric acid was immediately added to adjust the pH to 7.0.

(2) The same operation as in Example 7 was carried out except that casein was prepared.

(3) Relationship between Untreated Casein and Optical Density Change Amount Table 2 shows the results of measuring the FDP test liquids using a combination of an upper agent and an Otsu agent with untreated casein as a blocking agent.

[0062]

[Table 2]

The change in optical density of the reagent using alkali-treated casein was ΔOD = 0.373 to 0.579, and the change in optical density of the reagent using untreated casein was 0.171. On the other hand, it can be seen that it is extremely large, and in the end it has excellent cohesiveness and sensitivity. In addition, non-specific aggregation when serum was measured did not occur in any of the reagents of Examples 6 to 8 and Comparative Example 4.

Claims (3)

[Claims] 1. An immunoaggregation reaction reagent in which an antibody or an antigen is carried on a carrier, wherein the alkali-treated casein is carried on the reagent. 2. A method for producing an immune agglutination reagent, which comprises subjecting an immune agglutination reagent carrying an antibody or an antigen to a carrier to a blocking treatment with an alkali-treated casein. 3. A blocking agent for an immunoagglutination reaction reagent, which comprises alkali-treated casein.