JP4151771B2 - Immunological agglutination reagent - Google Patents

Description

【００２５】
（２） 抗梅毒トレポネーマ（ＴＰ）抗体の測定
（ａ） ＴＰ抗原感作ラテックスの作成
平均粒径０．４μｍのポリスチレンラテックスを５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）に１Ｗ／Ｖ％の濃度で分散し、梅毒菌体破砕物（蛋白濃度０．２５ｍｇ／ｍｌ）を等量混合し、４℃で一晩ゆっくり振とうした。さらに、上記溶液の半量の同緩衝液に溶解した５％ＢＳＡ溶液を加え、室温で４時間ゆっくり振とうしブロッキングした。その後、０．０１％のノニルフェノール系界面活性剤を含む０．１％ＢＳＡ含有５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）にて遠心洗浄を３回行い、最終的にラテックス濃度０．１５％となるように０．１％ＢＳＡ含有５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）に分散し、ＴＰ感作ラテックスを得た。
【００２６】
（ｂ） 検体希釈用緩衝液
検体希釈用緩衝液として、（１）ＡＦＰの測定で用いた緩衝液を使用した。
（ｃ）抗ＴＰ抗体の測定
（１）ＡＦＰの測定と同じように日立７１５０型自動分析装置を用いて測定した。
この結果を以下に示す。

【００２７】
（３）＜結果＞
実施例で示した通り、感作粒子の表面の抗原または抗体と、検体中の対応する抗体または抗原との間で、モルフォリノアクリルアミドあるいはＮ−ビニルアシルアミド重合体が相互に作用して凝集反応を起こしやすくし、その結果として抗原と抗体の反応が促進され、測定感度が向上したものと考えら、比較例の水溶液高分子と比べて優れた効果がある。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an agglutination reagent in a measurement reagent based on an immunological agglutination reaction utilizing an antigen-antibody reaction.
[0002]
[Prior art]
Conventionally, insoluble carrier particles (hereinafter abbreviated as sensitized particles) in which an antigen or antibody is sensitized by physical adsorption or covalent bonding have been used as a measurement reagent utilizing an antigen-antibody reaction. The corresponding antibody or antigen in the sample is measured by observing the agglutination reaction or the anti-aggregation reaction based on the antigen-antibody reaction between the sensitized particles and the corresponding antibody or antigen in the sample such as serum or urine. Reagents are known. This measurement method using sensitized particles is widely used because it can rapidly measure a small amount of antibody or antigen contained in a specimen with high accuracy and simplicity.
[0003]
[Problems to be solved by the invention]
However, in order to diagnose diseases at an earlier stage and start treatment at an earlier stage from medical sites using immunological agglutination reagents, it is required to measure a smaller amount of antibodies or antigens than ever before. ing.
It is known that the addition of nonionic water-soluble polymers such as polyethylene glycol, dextran, or polyvinylpyrrolidone to conventional immunological agglutination reagents improves the measurement sensitivity of the reagent and exhibits a reaction promoting effect. ing. However, if a water-soluble polymer with a large molecular weight is added, the viscosity of the reagent will increase and mixing with the specimen will not proceed quickly, or if the measurement with the reagent probe of the automatic measuring machine or the pipetting of the method will be constant. There is not. Furthermore, these water-soluble polymers react with proteins in the specimen, or sensitized particles aggregate together, resulting in inferior measurement specificity.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to obtain an immunological agglutination reaction reagent capable of eliminating the above-mentioned drawbacks, the present inventors have found that morpholinoacrylamide or N-vinylacyl in a measurement reagent comprising sensitized particles and a buffer solution. It has been found that the inclusion of an amide polymer does not impair the specificity of the antigen-antibody reaction in clinical diagnosis and the sensitivity is improved, and the present invention has been completed.
That is, the present invention relates to an immunological agglutination reagent comprising a morpholinoacrylamide or N-vinylacylamide polymer in an immunological measurement reagent comprising insoluble carrier particles sensitized with an antigen or an antibody. It is.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The molecular weight of the morpholinoacrylamide or N-vinylacylamide polymer of the present invention is preferably about 10,000 to 2,000,000, particularly preferably about 300,000 to 1,000,000 in the GPC method based on pullulan.
[0006]
The acyl group of the N-vinylacylamide used in the present invention is preferably a linear or branched acyl group having 1 to 5 carbon atoms, such as formyl group, acetyl group, propionyl group, isobutyryl group, etc. The group is particularly preferred. That is, N-vinylformamide and N-vinylacetamide are particularly preferable.
The morpholinoacrylamide or N-vinylacylamide polymer of the present invention is a morpholinoacrylamide or homopolymer of N-vinylacylamide, a copolymer of morpholinoacrylamide and N-vinylacylamide, or morpholinoacrylamide. Or the copolymer with the nonionic polymerizable monomer copolymerizable with N-vinylacylamide etc. are shown. The copolymerization ratio of morpholinoacrylamide and N-vinylacylamide can take any value from 1:99 mol% to 99: 1 mol%. Examples of copolymerizable nonionic polymerizable monomers include water-soluble monomers such as acrylamide, dimethylacrylamide, hydroxyethyl (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, N-vinylpyrrolidone, and methyl (meth) ) Hydrophobic monomers such as acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, styrene, vinyl acetate and acrylonitrile. The copolymerization ratio is usually from 1 to 90 mol%, preferably from 1 to 50 mol%, more preferably from 1 to 30 mol%, and hydrophobic monomers for water-soluble monomers with respect to morpholinoacrylamide or N-vinylacylamide. In general, it is 1 to 50 mol%, preferably 1 to 25 mol%, more preferably 1 to 10 mol%. A ternary or higher multi-component copolymer obtained by combining a plurality of these copolymerizable monomers can also be employed.
[0007]
Polymerization of these monomers can be achieved by employing general radical polymerization. That is, a monomer, a solvent, and an initiator are added to a stirrable reaction apparatus, and the polymerization is started by heating after nitrogen substitution, and the polymerization is completed by maintaining the temperature for a certain time. It is also good to obtain a polymer with a high degree of polymerization by raising the temperature as necessary to complete the polymerization. The obtained polymer is obtained as a solid by drying. If necessary, it can be pulverized into powder.
[0008]
As the solvent used in the polymerization, alcohols such as methanol, ethanol and isopropyl alcohol, organic solvents such as ethyl acetate, toluene, benzene and methyl ethyl ketone, or water are used. Moreover, these can also be mixed and used. Use of water is convenient because the polymer obtained can be used as it is.
[0009]
As the polymerization initiator, a peroxide type or azo type radical initiator generally used in radical polymerization is used. For example, inorganic peroxides such as potassium persulfate, ammonium persulfate and hydrogen peroxide, organic peroxide initiators such as benzoyl peroxide, t-butyl hydroperoxide, cumene peroxide, 2,2′-azo Such as bisisobutyronitrile, 2,2′-azobis (2-amidinopropane) dihydrochloride, dimethyl 2,2′-azobisbutyrate, dimethyl 2,2′-azobis (2-methylpropionate), etc. An azo-based initiator is used. Moreover, the redox initiator which combined the reducing agent with the peroxide-type initiator can also be employ | adopted.
[0010]
Although the temperature at the time of polymerization varies depending on the type of solvent and the type of initiator, it is preferable to employ a temperature around the 10-hour half-life temperature of the initiator. Generally, a temperature of 50 to 80 ° C. is employed.
The amount of morpholinoacrylamide or N-vinylacylamide polymer used may be such that the sensitized particles are non-specifically aggregated and the specificity is not lowered, and the concentration at the time of measurement is 0.01 to 5%. A range of 0.1% to 2% is particularly preferable.
The measuring reagent of the present invention does not need to be one dispersion liquid, and may be composed of a plurality of solutions and dispersions. When the measurement reagent is a single dispersed liquid, it can be measured simply by mixing the sample and the measurement reagent. When the measurement reagent consists of multiple liquids, each part of the reagent and the sample can be mixed and used according to a certain operation procedure. . When the measurement reagent is divided into a plurality of components, morpholinoacrylamide or N-vinylacylamide polymer may be added to select the components so that the characteristics of the reagent do not change during storage. When making the measurement reagent into one dispersed liquid, prepare two liquids, a liquid in which sensitized particles are dispersed and a buffer solution to which morpholinoacrylamide or N-vinylacylamide polymer is added. Mixing with a liquid is preferable because nonspecific reaction between the sensitized carrier particles and morpholinoacrylamide or N-vinylacylamide polymer during storage can be completely prevented. It is also possible to use a dried product of sensitized particles and a solid morpholinoacrylamide or N-vinylacylamide polymer as components of separate measuring reagents.
[0011]
Subsequently, an immunological measurement method using the agglutination reagent of the present invention will be described. When the morpholinoacrylamide or N-vinylacylamide polymer used in the present invention is used in a buffer solution, various buffer solutions can be used. For example, a phosphate buffer solution, a glycine-sodium hydroxide buffer solution can be used. Tris-hydrochloric acid buffer solution, ammonium chloride-ammonia buffer solution or Good's buffer solution are preferable. The concentration and pH can be used without any particular limitation, but a concentration of 10 mM to 500 mM and a pH of 4 to 9 are preferred.
Sensitized particles are prepared by sensitizing an antigen or antibody to insoluble carrier particles.
[0012]
The insoluble carrier particles are particles that are insoluble in a liquid medium used for sensitization, storage and measurement, generally in a buffer solution, that is, insoluble in water.
Various fine particles already used for antigen-antibody reaction are known as these fine particles. In the present invention, these known fine particles are not particularly limited and can be used. Examples of those particularly preferably used include polystyrene, styrene-divinylbenzene copolymer, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl methacrylate copolymer, polyglycidyl methacrylate, and polyacrolein. Fine particles of organic polymer materials such as organic polymer latex obtained by a simple emulsion polymerization method, inorganic oxides such as silica, silica-alumina, alumina, or magnetic fine particles such as various iron oxides, or these inorganic oxides There are composite fine particles such as silane coupling treatment that introduce functional groups or inorganic fine particles coated with organic polymers. Biological particles such as human O-type red blood cells, sheep red blood cells, and chicken red blood cells There are particles of origin.
[0013]
Regarding the particle size of the insoluble carrier particles, when the particle size is large, the amount of change in the particle size associated with aggregation is large but the aggregation reaction rate is slow. Since the particle size is small, the amount of change in the particle size accompanying the aggregation reaction is small. For this purpose, insoluble carrier particles having an average particle diameter of 10 μm or less, preferably 0.05 to 5.0 μm, particularly preferably 0.1 to 1.0 μm, are used for the agglutination reaction.
[0014]
The antigen or antibody that sensitizes the insoluble carrier particles is not particularly limited, and known ones can be used. Representative examples include anti-human alphafetoprotein (AFP) antibody, anti-carcinoembryonic protein (CEA) antibody, anti-prostate specific antigen (PSA) antibody, hepatitis B surface antigen (HBs), anti-HBs antibody, anti-human. Reactive protein (CRP) antibody, anti-streptridine O antibody, anti-albumin antibody, anti-immunoglobulin G (IgG) antibody, anti-immunoglobulin A (IgA) antibody, anti-immunoglobulin M (IgM) antibody, anti-complement third Component (C3) antibody, anti-complement fourth component (C4) antibody, denatured gamma globulin, anti-human placental lactogen (hPL), anti-human chorionic gonadobin (hCG) antibody, insulin, anti-insulin antibody, syphilis treponema antigen, Known antigens or antibodies such as rubella antigen and anti-rotavirus antibody can be mentioned.
[0015]
The method for sensitizing an insoluble carrier particle with an antigen or an antibody may be either physical adsorption or chemical covalent bond formation. Sensitization by the physical adsorption method may be performed by mixing insoluble carrier particles and an antigen or antibody in an appropriate buffer, as is generally done, and the type, concentration, pH, antigen or antibody of the buffer. Optimum conditions such as the concentration of lysate, temperature at the time of sensitization, and time may be selected. Many methods have already been proposed for the formation of chemical covalent bonds, and a sensitizing method may be selected from known methods in accordance with the characteristics of the antigen or antibody to be sensitized. In general, an antibody may be mixed with insoluble carrier particles in a dispersion medium in the presence of a crosslinking agent as required. Known crosslinking agents such as glutaraldehyde and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used. In addition, after sensitizing the antigen or antibody, it is also common to sensitize immunologically inactive proteins such as bovine serum albumin, casein, gelatin, etc. to block the surface of the insoluble carrier particles without the antigen or antibody. Has been done.
[0016]
The dispersion medium for sensitizing the insoluble carrier particles with the antigen or antibody is not particularly limited and known ones are used, but when the above-mentioned crosslinking agent is used, the components in the dispersion medium are the crosslinking agent. It is necessary to use a dispersion medium that does not react with. The concentration of the insoluble carrier particles in the dispersion medium at the time of sensitization is not particularly limited, but is generally 0.05% by weight or more, preferably 0.2 to 2.0% when mixed with the antigen or antibody. It is preferable to select the weight%.
[0017]
In the present invention, an immunological measurement method using sensitized carrier particles, that is, aggregation based on an antigen-antibody reaction between an antigen or antibody on an antibody-sensitized carrier particle and a reacting antibody or antigen in a specimen. As a method for observing the reaction, known methods such as visual observation and optical measurement can be used without any particular limitation. Examples of the optical measurement method include a method based on a change in absorbance at a certain wavelength, turbidity or scattered light, or a change in turbidity due to all wavelengths of visible light.
[0018]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
(1) Measurement of AFP (a) Preparation of anti-AFP-sensitized latex Latex having an average particle size of 0.32 μm was dispersed in 50 mM glycine buffer (pH 9.5) at a concentration of 1 W / V%, and anti-AFP serum (goat) , IgG fraction) dissolved in the same buffer at a concentration of 0.25 mg / ml was mixed in an equal amount and allowed to stand at 4 ° C. overnight. Furthermore, 5% bovine serum albumin (BSA) solution dissolved in half the same amount of the above solution was added, and gently shaken at room temperature for 4 hours, and 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA. ), And was dispersed in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA to obtain a latex concentration of 0.25% to obtain an anti-AFP-sensitized latex. .
[0019]
(B) Sample Dilution Buffer Example 1 Polymorpholinoacrylamide polymorpholinoacrylamide aqueous solution was prepared by dissolving 80 g of morpholinoacrylamide and 20 mg of sodium hypophosphite in 240 ml of water, and using 80 mg of potassium persulfate as a polymerization catalyst. It was obtained by polymerization at 65 ° C. for 8 hours and at 80 ° C. for 2 hours by a conventional method. This polymer was dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%.
Example 2 Morpholinoacrylamide: acrylamide copolymer The morpholinoacrylamide: acrylamide copolymer was prepared by dispersing and dissolving 70 g of morpholinoacrylamide, 10 g of acrylamide and 20 mg of sodium hypophosphite in 240 ml of water. It was obtained in the same way. This copolymer was dissolved in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%.
[0020]
Example 3 Morpholinoacrylamide: vinyl acetate: N-vinylpyrrolidone copolymer Morpholinoacrylamide: vinyl acetate: N-vinylpyrrolidone copolymer was composed of 40 g of morpholinoacrylamide, 16.4 g of vinyl acetate, and N-vinylpyrrolidone. 36 g was dispersed and dissolved in 240 ml of water and obtained in the same manner as in the polymerization of Example 1. This copolymer was dissolved in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%.
Example 4 Morpholinoacrylamide: vinyl acetate: N-vinylformamide copolymer Morpholinoacrylamide: vinyl acetate: N-vinylformamide copolymer was composed of 40 g of morpholinoacrylamide, 16.4 g of vinyl acetate, N-vinylformamide. 36 g was dispersed and dissolved in 240 ml of water and obtained in the same manner as in the polymerization of Example 1. This copolymer was dissolved in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%.
[0021]
Example 5 Poly N-vinylformamide Poly N-vinylformamide (manufactured by Mitsubishi Chemical Corporation) was added to a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%. Dissolved.
Example 6 Poly N-vinylacetamide PolyN-vinylacetamide (manufactured by Showa Denko KK) was added to a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%. Dissolved.
[0022]
Comparative Example 1
50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA.
Comparative Example 2
Polyvinylpyrrolidone (K-90) was dissolved in a 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 0.5 W / V%.
Comparative Example 3
Dextran (average molecular weight 250,000) was dissolved in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1% BSA so as to have a concentration of 1.0 W / V%.
[0023]
(C) Measurement of AFP The latex agglutination method using sensitized latex may be performed by determining the amount of change in turbidity due to aggregation, that is, absorbance at a certain wavelength. Here, measurement was performed using a Hitachi 7150 type automatic analyzer widely used in biochemical examinations. The measurement parameters are shown.
Sample 15μL
Sample dilution buffer (R-1) 250 μL
Latex suspension (R-2) 80μL
Measurement wavelength 750nm
Metering point 2 points end (35-50 points)
In the Hitachi 7150 automatic analyzer, the sample dilution buffer is added immediately after sample dispensing, and the sample is diluted and mixed. Five minutes later, the latex suspension was added, and the amount of change in turbidity (ΔAbs. (750 nm)) from 35 points to 50 points was determined and used as the reaction amount.
The results are shown below.
[0024]

[0025]
(2) Measurement of anti-syphilis treponema (TP) antibody (a) Preparation of TP antigen-sensitized latex Polystyrene latex with an average particle size of 0.4 μm was added to 50 mM Tris-HCl buffer (pH 7.4) at a concentration of 1 W / V%. The mixture was dispersed, mixed with an equal amount of syphilis fungus crushed material (protein concentration: 0.25 mg / ml), and shaken slowly at 4 ° C. overnight. Further, a 5% BSA solution dissolved in half the same amount of the above solution was added, and the mixture was gently shaken at room temperature for 4 hours to block. Thereafter, centrifugal washing is performed three times with a 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.1% BSA containing 0.01% nonylphenol surfactant, and finally the latex concentration becomes 0.15%. As described above, the mixture was dispersed in a 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.1% BSA to obtain a TP-sensitized latex.
[0026]
(B) Sample dilution buffer As the sample dilution buffer, (1) the buffer used in the AFP measurement was used.
(C) Measurement of anti-TP antibody (1) It was measured using Hitachi 7150 type automatic analyzer in the same manner as the measurement of AFP.
The results are shown below.

[0027]
(3) <Result>
As shown in the Examples, the morpholinoacrylamide or N-vinylacylamide polymer interacts between the antigen or antibody on the surface of the sensitized particle and the corresponding antibody or antigen in the specimen to cause an agglutination reaction. As a result, it is considered that the reaction between the antigen and the antibody is promoted and the measurement sensitivity is improved.

Claims (1)

An immunoassay reagent comprising insoluble carrier particles sensitized with an antigen or an antibody, comprising a suspension of the sensitized particles and a buffer containing a morpholinoacrylamide or N-vinylacylamide polymer. An immunological agglutination reagent.