JP2996690B2 - Immunological agglutination reagent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an assay reagent based on immunological agglutination.

[Prior art and its problems] Conventionally, insoluble carrier particles (hereinafter abbreviated as immobilized carrier particles) in which an antigen or an antibody is immobilized by physical adsorption or formation of a covalent bond to insoluble carrier particles, and samples such as serum and urine An immunological measurement reagent for measuring a corresponding antibody or antigen in a sample by observing an agglutination reaction or an agglutination inhibition reaction based on an antigen-antibody reaction with a corresponding antibody or antigen in the sample is known. . Then, the measurement method using the immobilized carrier particles can quickly, relatively accurately and easily measure a relatively small amount of antibody or antigen contained in the sample, and can be measured within a few minutes. Diagnosis is possible and widely used.

However, from the medical practice using immunological agglutination reagents, it is required to measure a smaller amount of antibody or antigen than ever before in order to diagnose earlier and realize early treatment. I have.

It has been known that the addition of polyethylene glycol to a reagent for immunological agglutination reaction improves the measurement sensitivity of the reagent (JP-A-58-47256). There is a disadvantage that the specificity of the measurement is inferior due to the reaction between the polyethylene glycol and the immobilized carrier particles. Also, do not use insoluble carrier particles,
The technique of adding polyvinylpyrrolidone in the one-dimensional immunodiffusion method is already known (JP-A-58-58468).
This technology is designed for small-molecule antigens with unclear sedimentation rings.
The addition of polyvinylpyrrolidone sharpens the sedimentation ring, and does not mention any effect of improving sensitivity.

Furthermore, it is known to dissolve a stabilizer such as polyvinylpyrrolidone in advance in the reagent in which the immobilized carrier particles are dispersed in order to prevent sedimentation and aggregation of the immobilized carrier particles during storage of the reagent. Thus, even when polyvinylpyrrolidone is dissolved in the liquid from the beginning of preparation of the reagent, the polyvinylpyrrolidone acts slowly on the immobilized carrier particles, and the specificity of the measurement also increases with time. Problems that were being spoiled.

[Means for Solving the Problem] The present inventors have intensively studied to obtain an immunological agglutination reagent capable of achieving the above object, and at least as a result,
An insoluble carrier particle on which an antigen or an antibody is immobilized, a reagent comprising three components of polyvinylpyrrolidone and a buffer,
When the insoluble carrier particles are adjusted to a suspended state in a buffer in which polyvinylpyrrolidone is dissolved at the time of use, it has been found that specificity is not impaired in clinical diagnosis, and that sensitivity is improved. It was completed.

That is, the present invention relates to a reagent comprising at least an insoluble carrier particle on which an antigen or an antibody is immobilized, polyvinylpyrrolidone and a buffer, wherein the insoluble carrier particles are suspended in a buffer in which polyvinylpyrrolidone is dissolved at the time of use. It is an immunological agglutination reagent characterized by being adjusted to a state in which it has been prepared.

The measurement reagent of the present invention comprises insoluble carrier particles on which an antigen or an antibody is immobilized, polyvinylpyrrolidone, and a buffer. The measurement reagent is composed of a plurality of liquids and solids so that the above components are adjusted to a state in which insoluble carrier particles are suspended in a buffer in which polyvinylpyrrolidone is dissolved at the time of use. These plural liquids and solids are used by mixing each component of the reagent and the sample according to a certain operation procedure. That is, when the solid components of the reagent are separated into immobilized carrier particles and polyvinylpyrrolidone, these may be dissolved and suspended in a buffer solution as a liquid component. Of course, the order is not particularly limited.

Alternatively, after the immobilized carrier particles and polyvinylpyrrolidone are dry-mixed in advance, they can be mixed with a buffer solution.

In another embodiment, when the buffer solution in which polyvinylpyrrolidone is dissolved in advance and the immobilized carrier particles are separated, the buffer solution may be used by mixing the immobilized carrier particles.

Further, in the case where polyvinylpyrrolidone is separated into a buffer in which the immobilized carrier particles are suspended and polyvinylpyrrolidone, polyvinylpyrrolidone may be dissolved in the buffer before use.

In particular, if a liquid in which the immobilized carrier particles are dispersed and a buffer solution to which polyvinylpyrrolidone is added are prepared and mixed and used immediately before use, the non-specificity of the immobilized carrier particles and the polyvinylpyrrolidone during storage is reduced. This is preferable because the reaction can be completely prevented.

The polyvinylpyrrolidone used in the present invention can be used without limitation, and has a molecular weight of about 10,000 to 360,000 which is industrially synthesized. However, the K value in the viscosity equation of Fikenture which is an index of the viscosity of polyvinylpyrrolidone can be used. Those having a value of 20 or more are preferable because they have a high sensitivity improving effect, and those having a K value of 50 or more are more preferable.

In the present invention, polyvinylpyrrolidone can be used as a component of the reagent as it is in a solid state, but in order to easily perform a measurement operation, an embodiment in which polyvinylpyrrolidone is dissolved in a buffer solution in advance and used is preferable. As the buffer used in the present invention, various buffers can be used without limitation, but buffers such as phosphate buffer, glycine-sodium hydroxide buffer, Tris-hydrochloride buffer, ammonium chloride-ammonia buffer, and the like can be used. Liquid is preferably used.

The amount of polyvinylpyrrolidone used has the effect of improving sensitivity, the phenomenon that the immobilized carrier particles are non-specifically aggregated and the specificity is reduced, and the phenomenon that the viscosity of the reagent increases and stirring becomes difficult. Although it may be determined in consideration of the above, it is particularly preferable that the concentration of polyvinylpyrrolidone is in the range of 0.01 to 5% with respect to the total liquid volume at the time of measurement.

In the present invention, the immobilized carrier particles are prepared by immobilizing an antigen or an antibody on insoluble carrier particles.

The insoluble carrier particles are insoluble carrier particles that are substantially insoluble in the liquid medium used for immobilization, storage and measurement. Fine particles having an average particle diameter of about 10 μm or less, which will be described in detail later, are preferably used.

As these particles, various ones already used for an antigen-antibody reaction are known, and in the present invention, these known fine particles can be used without any particular limitation. Particularly preferred examples include, for example, polystyrene,
Styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-fine particles of organic polymer latex particles such as organic polymer latex particles obtained by an emulsion polymerization method such as ethylene glycol dimethacrylate copolymer, Alternatively, silica, silica-alumina, inorganic particles such as alumina or inorganic particles obtained by introducing a functional group into the inorganic oxide by an operation such as silane coupling treatment, and also human O-type erythrocytes and biological origin such as sheep erythrocytes Particles.

Regarding the particle size of the insoluble carrier particles, when the particle size is large, the amount of change in the particle size due to aggregation is large but the agglutination reaction speed is slow, and when the particle size is small, the Brownian motion is active and the agglutination reaction speed is fast but the primary Since the particle diameter is small, the amount of change in the particle diameter due to the aggregation reaction is small. For this purpose, the average particle size of the insoluble carrier particles used in the agglutination reaction is 10
Insoluble carrier particles of about μm or less, preferably 0.05 to 5.0 μm, are suitably used.

In the present invention, the antigen or antibody immobilized on the insoluble carrier particles is not particularly limited, and any known antigen or antibody can be used. Representative examples include, for example, human C-reactive protein (CRP) / anti-CRP antibody, streptolysin O / anti-streptolysin O antibody, denatured gamma globulin / rheumatic factor, albumin / anti-albumin antibody, immunoglobulin G (IgG), anti-IgG antibody, IgA, anti-IgA antibody, IgM, anti-I
gM antibody, third component of complement (C3), anti-C3 antibody, C4, anti-C4 antibody, alpha fetoprotein (AFP), anti-AFP antibody, carcinoembryonic antigen (CEA), anti-CEA antibody, human placental lactogen (hPL)・ Anti-hPL antibody, human chorionic gonadotropin (hC
G) anti-hCG antibody, insulin anti-insulin antibody,
Hepatitis B surface antigen (HBs) / anti-HBs antibody, Candida albica
ns / anti-Candida albicans antibody, treponema syphilis antigen, rubella antigen, anti-rotavirus antibody, anti-adenovirus antibody,
And other known antigens and antibodies.

In the present invention, the method of immobilizing the antigen or antibody on the insoluble carrier particles may be either physical adsorption or formation of a chemical covalent bond. Many methods for forming a chemical covalent bond have already been proposed, and an immobilization method may be selected from known methods according to the characteristics of the antibody to be immobilized.
Generally, the antibody may be mixed with the insoluble carrier particles in a dispersion medium in the presence of a crosslinking agent, if necessary. Known crosslinking agents such as glutaraldehyde and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used.

The dispersion medium for immobilizing the antigen or antibody on the insoluble carrier particles is not particularly limited, and a known dispersion medium is used.When the above-described crosslinking agent is used, the components in the dispersion medium react with the crosslinking agent. It is necessary to use a dispersion medium that does not. The concentration of the insoluble carrier particles in the dispersion medium during immobilization is not particularly limited, but generally, at the time of mixing with the antigen or the antibody.
It is suitable to select 0.05% by weight or more, preferably 0.2 to 2.0% by weight. The concentration of the antigen or antibody is not particularly limited, but it is generally preferred that the concentration is 0.0005% by weight or more, and preferably 0.002 to 0.2% by weight when mixed with the insoluble carrier particles.

In the present invention, the immunological measurement method using the immobilized carrier particles, that is, an agglutination reaction based on an antigen-antibody reaction between the antigen or antibody on the antibody-immobilized carrier particles and the corresponding antibody or antigen in the subject, etc. Alternatively, as a method for observing the aggregation inhibition reaction, known methods such as visual observation and optical measurement can be used without particular limitation.

[Actions and Effects] As the chemical properties of polyvinylpyrrolidone, various inorganic and organic compounds are adsorbed, complexes are formed, for example, the solubility of iodine and dyes is remarkably increased, and polyelectrolytes such as polyacrylic acid and proteins are used. It is known to interact with.

In the present invention, an anion such as a fragment of a polymerization initiator is present on the surface of the insoluble carrier particles, and the insoluble carrier particles are considered as a kind of polyanion. Further, it is considered that an immobilized antigen or antibody exists, and polyvinylpyrrolidone interacts with the antibody or antigen in the sample to mediate agglutination. Therefore, it is considered that the distance between the proteins is reduced, the interaction is likely to occur, the antigen-antibody reaction is promoted, and the measurement sensitivity is improved. On the other hand, it can be inferred from the fact that the solubility of iodine and the dye is remarkably increased, which is considered to contribute to the stabilization of the dispersion of the immobilized carrier particles.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Examples 1 to 3 (1) Preparation of Immobilized Carrier Particles After affinity purification of an anti-Candida albicans antibody against Candida albicans, a fungus causing vaginal candidiasis, according to a conventional method, the purified antibody was subjected to 1/60 molar phosphate buffering. Dialyzed against a liquid (hereinafter abbreviated as PB, pH 6.3), added with PB and added at a concentration of 0.25m
A g / ml antibody solution was prepared. As Example 1, the average particle diameter
0.728μm polystyrene latex particles (carrier particles)
Was diluted with PB to prepare a dispersion having a latex concentration of 1% by weight, and an equal volume was added to the antibody solution, followed by stirring and mixing immediately.
After standing for 2 hours, bovine serum albumin (freeze-dried product) was added to 0.
One part by weight was added and the mixture was allowed to stand for another 2 hours. Then, the mixture is centrifuged, and the obtained precipitate (immobilized carrier particles) is mixed with the above antibody solution.
Suspension by adding double volume of PB (particle concentration: about 0.5% by weight)
Then, a dispersion of antibody-immobilized carrier particles on which an anti-Candida albicans affinity-purified antibody was immobilized, that is, a Candida reagent was obtained.

(2) Agglutination Test Candida albicans c-a-111 bacteria concentration per 1ml was diluted with stock solution of antigen was prepared (Chiba obtained from eukaryotic microbes Center) in a cell concentration 10 ⁸ cells / ml in PB 25 × 10 ⁵ pcs, 12.5
× 10 ⁵ , 6.3 × 10 ⁵ , 3.2 × 10 ⁵ , 1.6 × 10 ⁵ , 0.8 ×
10 ^5, 0.4 × 10 ⁵ was 0.2 × 10 ^5, liquid antigen was prepared. On the other hand, polyvinylpyrrolidone K60 (molecular weight 160,000)
The concentration of polyvinylpyrrolidone K60 is 10%, 1.0%, 0.10% in PB
Prepare a solution, mix each with the above antigen solution in equal amounts,
Antigen diluents containing polyvinylpyrrolidone K60 at a concentration of 5.0%, 0.50%, 0.050%, and a bacterial concentration of 12.5 × 10 ⁵ to 0.1 × 10 ⁵ were prepared, and were set as Examples 1, 2, and 3, respectively. . Also, polyvinylpyrrolidone to evaluate negative images
A solution containing K60 at a concentration of 5.0%, 0.50%, and 0.050% and containing no antigen was prepared. One drop of the Candida reagent obtained in (1) is dropped on a glass plate, and one drop of the above-mentioned Candida albicans antigen diluent is added to each drop of the Candida albicans, mixed with a toothpick, and immediately, Hirasawa Seisakusho Co., Ltd. The glass plate was horizontally rotated at a speed of 120 rotations per minute for 2 minutes by a Teher-type stirrer to stir. The agglutination state after stirring was visually determined, and the minimum concentration of the antigen in which the agglutination was clearly observed in the antigen diluent was used as the sensitivity of the Candida reagent to the antigen. The results are shown in Table 1.

(3) Specificity test In place of Candida albicans ca-111, 6 species of fungi of other genera (obtained from Eukaryotic Microorganism Center, Chiba), and the presence of Candida reagent in the specimen when using Candida reagent Bacteria 14
Species (obtained from RIKEN) diluted 1m with PB
A bacterial cell fluid was prepared so that the bacterial concentration per l became 100 × 10 ⁵ cells. An aggregation test was performed in the same manner as the sensitivity test. The agglutination state is determined with the naked eye. If no agglutination is observed (-), it is difficult to determine the presence or absence of agglutination (±), or if agglutination is clearly observed (+), the above 20 types are determined. Table 1 shows the ratio of occurrence of (+).

Comparative Examples 1 to 7 Comparative Example 1 was performed in the same manner as in Example 1 except that the diluted solution of polyvinylpyrrolidone K60 was mixed and the antigen diluted solution was replaced with PB only. Mix a diluent of polyethylene glycol 6000 instead of a diluent of polyvinylpyrrolidone K60, and mix
6000 at 5.0%, 0.50%, 0.050%
Prepare an antigen diluent of 12.5 × 10 ⁵ to 0.1 × 10 ⁵
These were Comparative Examples 2, 3, and 4, respectively. Similarly, antigen diluents containing dextran (molecular weight of 60,000 to 90,000) at concentrations of 5.0%, 0.50%, and 0.050% and having a bacterial concentration of 12.5 × 10 ⁵ to 0.1 × 10 ⁵ were prepared. 5, 6, and 7. A sensitivity test and a specificity test were performed in the same manner as in Examples 1 to 3, and the obtained results were The results are shown in Table 1.

As shown in Table 1, in Examples 1 to 3 in which polyvinylpyrrolidone K60 was added, the sensitivity was improved by 2 to 16 times as compared with Comparative Example 1 in which no additive was added, and a negative image and specificity were not reduced. Reagent properties. On the other hand, in Comparative Examples 2 to 7 in which polyethylene glycol 6000 or dextran was added, the sensitivity was improved when the addition concentration was low, but the negative image and specificity did not include Examples 1 to 3 and the additives. Inferior to Comparative Example 1. When the addition amount was further increased, both the negative image and the positive image aggregated non-specifically, making measurement impossible.

Examples 4 to 6 In place of polyvinylpyrrolidone K60 in Example 1, when an antigen diluent was prepared so that polyvinylpyrrolidone K90 (molecular weight: 360,000) became 2% at the time of measurement (Example 4), polyvinylpyrrolidone K25 ( When an antigen diluent was prepared so as to be 4% at the time of measurement (molecular weight 24,500) (Example 5), and when an antigen diluent was prepared such that polyvinylpyrrolidone K15 (molecular weight 10,000) was 8% at the time of measurement (implementation). The sensitivity and specificity of Example 6) were evaluated in the same manner as in Example 1. The results are shown in Table 2.

As shown in Table 2, the sensitivity of the reagent was clearly improved in Examples 4 and 5 as compared with Comparative Example 1 containing no additive, but Example 6 having a K value of 15 was used. In the case of (1), there was a case where aggregation was observed for a sample with an antigen concentration of 3.2 × 10 ⁵ / ml and a case where it was difficult to determine the presence or absence of aggregation, and the sensitivity was only slightly increased. When the addition concentration of Example 4 was increased to 4% and the addition concentration of Example 5 was increased to 8%, the reagent became viscous, and it was necessary to mix the reagent and the sample vigorously.

Example 7 and Comparative Example 8 Anti-AFP against alpha fetoprotein (hereinafter AFP)
After affinity purification of the antibody according to a conventional method, the purified antibody was dialyzed against a 0.1 mol ammonium chloride-ammonia buffer (hereinafter abbreviated as AB, pH 8.2), AB was added, and the concentration was 0.25 mg / ml.
Was prepared. Polystyrene latex particles (carrier particles) having an average particle size of 0.232 μm were diluted with AB to prepare a dispersion having a latex concentration of 1% by weight, and an equal volume was added to the antibody solution, followed by stirring and mixing immediately. After standing for 2 hours, 0.1 part by weight of bovine serum albumin (lyophilized product) was added,
Let stand for hours. Next, centrifugation is performed, and the obtained precipitate (immobilized carrier particles) is suspended by adding AB (4 times the volume of the above antibody solution) (particle concentration: about 0.25% by weight) to immobilize the anti-AFP affinity purified antibody. A dispersion of the antibody-immobilized carrier particles thus obtained was obtained.

Example 7 shows that polyvinylpyrrolidone K60 was added to AB at 0.4.
% To prepare a serum dilution.

AFP was measured under the following measurement conditions by applying the above reagents to a Hitachi 7050 biochemical multi-item automatic analyzer. First, serum 20
μl was sampled into an optical cell and serum
Add 1 and stir. About 5 minutes after adding the serum diluent, 100 μl of the dispersion liquid of the antibody-immobilized carrier particles is added and stirred. The amount of change in absorbance at a wavelength of 600 nm in about 4 minutes between about 1 minute and 5 minutes after the addition of the antibody-immobilized carrier particles was automatically measured.

When the serum diluent was only AB containing no polyvinylpyrrolidone K60 in place of the serum diluent of Example 7 (Comparative Example 8), the absorbance change was 0.021 Abs for a sample having an AFP concentration of 2000 ng / ml. However, the reagent of Example 7 showed that the absorbance change amount was 0.152 Abs.
It showed twice the sensitivity. In addition, the amount of change in absorbance described above was 0.001 Abs. Or less in both Example 7 and Comparative Example 8 for a sample having an AFP concentration of 10 ng / ml or less.

Comparative Examples 9 and 10 In the preparation of (1) immobilized carrier particles in Examples 1 to 3, instead of adding and suspending twice the volume of PB of the antibody solution to the immobilized carrier particles, double the volume of the antibody solution. A Candida reagent containing polyvinylpyrrolidone was obtained in the same manner except that a 10% polyvinylpyrrolidone K60 (molecular weight: 160,000) PB solution was added and suspended. Then, this is kept at 10 ° C for 3 months and 6 months.
Stored for months. Next, in the (2) agglutination test of Examples 1 to 3, the same operation was performed except that an antigen diluent obtained by diluting the antigen solution only with PB was used, and this was mixed with the above-mentioned polyvinylpyrrolidone-containing Candida reagent after storage. The agglutination test was performed. Further, another bacterial cell fluid similar to that used in the (3) specificity test was prepared in place of Candida albicans, and an agglutination test was performed in the same manner as described above to perform a specificity test. The results are shown in Table 2.

Table 2 also shows, as a control test, a Candida reagent prepared in the same manner as in (1) Preparation of immobilized carrier particles in Examples 1 to 3, and then stored at 10 ° C. for 3 months and 6 months.
Using a polyvinylpyrrolidone-containing Candida reagent in which polyvinylpyrrolidone K60 was dissolved in a concentration of 2.5%, within one day after dissolving polyvinylpyrrolidone K60, the same procedure as in the above comparative example was carried out (2) Agglutination test and (3) The results of the specificity test are also shown.

As shown in Table 2, Comparative Examples 9 and 1 in which the Candita reagent was stored for a long time in a state containing polyvinylpyrrolidone K60
In the case of 0, the specificity of the measurement was significantly reduced as compared to the control test in which polyvinylpyrrolidone K60 was added to the Candita reagent immediately before use.

────────────────────────────────────────────────── ─── Continued on the front page (56) References JP-A-57-44854 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/543, 33/531

Claims (1)

(57) [Claims] 1. A reagent comprising at least three components of an insoluble carrier particle on which an antigen or an antibody is immobilized, polyvinylpyrrolidone and a buffer, wherein the insoluble carrier particles are suspended in a buffer in which polyvinylpyrrolidone is dissolved at the time of use. An immunological agglutination reagent, which is adjusted to a state.