JPH0420859A - Immunological agglutination reagent - Google Patents

Abstract

PURPOSE:To improve measuring sensitivity by incorporating polyvinylpyrrolidone into a measuring reagent which is formed by containing insoluble carrier particles obtained by fixing an antigen or a antibody and a buffer. CONSTITUTION:In a measuring reagent which is formed by containing insoluble carrier particles obtained by fixing antigen or antibody and buffer, polyvinylpyrrolidone is incorporated. As the polyvinylpyrrolidone, the material whose K value in the Fikentscher's viscosity formula that is the index of the viscosity is 20 or above has high sensitivity improving effect and the material is suitable. When the K value is 50 or above, the material is further suitable. With respect to the using amount of the polyvinylpyrrolidone, the suitable concentration of the polyvinylpyrrolidone with respect to the total liquid amount in measurement is about 0.01 - 5%. As the buffer, phosphoric acid buffer, glycine/sodium hydroxide buffer and the like are suitable.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a measurement reagent based on an immunological agglutination reaction.

[Prior art and its problems] Conventionally, insoluble carrier particles (hereinafter referred to as immobilized carrier particles) in which antigens or antibodies are immobilized by physical adsorption or the formation of covalent bonds, and specimens such as serum or urine have been used. An immunological measurement reagent is known that measures the corresponding antibody or antigen in a sample by observing the agglutination reaction or agglutination inhibition reaction based on the antigen-antibody reaction between the corresponding antibody or antigen in the sample. . This measurement method using immobilized carrier particles can quickly, accurately and easily measure relatively small amounts of antibodies or antigens contained in a sample, and can be carried out on the spot within a few minutes. diagnosis is possible and widely used.

However, in medical settings that use immunoagglutination reagents, there is a need to measure smaller amounts of antibodies or antigens than ever before in order to achieve earlier diagnosis and early treatment. There is.

Conventionally, it is known that adding polyethylene glycol to the reagent for immunological agglutination reaction improves the measurement sensitivity of the reagent (Japanese Unexamined Patent Publication No. 58-47256). However, the specificity of the measurement is poor due to the reaction between the polyethylene blade coal and the immobilized carrier particles. Furthermore, a technique for adding polyvinylpyrrolidone in the dimensional immunodiffusion method that does not use insoluble carrier particles is already known (Japanese Patent Application Laid-Open No. 58-58468). , the addition of polyvinylpyrrolidone sharpens the sedimentation ring, and does not mention anything about the effect of improving sensitivity.

[Means for Solving the Problem 1] As a result of intensive research to obtain an immunological agglutination reaction reagent capable of achieving the above object, the present inventors have developed a measurement reagent comprising immobilized carrier particles and a buffer solution. The inventors have discovered that when polyvinylpyrrolidone is included, specificity is not impaired in clinical diagnosis and sensitivity is improved, and the present invention has been completed.

That is, the present invention is a measurement reagent comprising insoluble carrier particles on which an antigen or antibody is immobilized and a buffer solution, and is an immunological agglutination reaction reagent characterized by containing polyvinylpyrrolidone.

In the present invention, polyvinylpyrrolidone is contained in a measurement reagent comprising immobilized carrier particles and a buffer solution. The measurement reagent does not need to be a single liquid, and may be composed of multiple liquids and solids. If the measurement reagent is a single liquid, the sample and measurement reagent are mixed and used, When the measurement reagent is composed of a plurality of liquids and solid substances, each component of the reagent and the specimen are mixed and used according to a certain operating procedure. When the measurement reagent is composed of a plurality of liquids and solids, components containing polyvinylpyrrolidone may be selected so that the properties of the reagent do not change during storage after adding polyvinylpyrrolidone. If the measurement reagent is a single solution, prepare two solutions: a solution in which immobilized carrier particles are dispersed and a buffer solution containing polyvinylpyrrolidone, and add 1 solution immediately before use.
It is preferable to use it by mixing it with a liquid because non-specific reaction between the immobilized carrier particles and polyvinylpyrrolidone during storage can be completely prevented. Furthermore, it is also possible to use the dried immobilized carrier particles and the solid polyvinylpyrrolidone separately as one component of the measurement reagent.

The polyvinylpyrrolidone used in the present invention can be any industrially synthesized polyvinylpyrrolidone with a molecular weight of about 10,000 to 360,000. A value of 20 or more is preferable because it has a high sensitivity improvement effect, and a K value of 5
A value of 0 or more is more preferable.

In the present invention, polyvinylpyrrolidone can be used as a component of the reagent as a solid, but in order to simplify the measurement operation, it is preferable to dissolve it in M buffer before use. Various buffers can be used without limitation, including phosphate buffer, glycine-sodium hydroxide buffer, Tris-hydrochloric acid buffer,
M fresh solutions such as ammonium chloride-ammonia buffer are preferably used.

The amount of polyvinylpyrrolidone used should be determined based on the effect of improving sensitivity, the phenomenon of nonspecific coagulation of immobilized carrier particles, which reduces specificity, and the phenomenon of increasing reagent viscosity, which makes stirring difficult. The concentration of polyvinylpyrrolidone in the total liquid volume at the time of measurement should be 0.01 to 5%.
Particularly preferred is a range of .

In the present invention, immobilized carrier particles are prepared by immobilizing antigens or antibodies 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, and fine particles with an average particle diameter of about 10 μm or less are preferably used, as will be described in detail later.

Various types of particles used in antigen-antibody reactions are already known, and in the present invention, any of these known particles can be used without particular limitation. Particularly preferred examples include polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer obtained by emulsion polymerization. Fine particles of organic polymer substances such as organic polymer latex particles, or inorganic oxides such as urea, silica-alumina, and alumina, or inorganic materials in which functional groups are introduced into the inorganic oxides through operations such as silane coupling treatment. Particles include particles derived from living organisms such as human type ○ red blood cells and sheep red blood cells.

Regarding the particle size of the above-mentioned insoluble carrier particles, when the particle size is large, the change in particle size due to aggregation is large but the aggregation reaction rate is slow; when the particle size is small, Brownian motion is active and the aggregation reaction rate is fast but first-order. Since the particle size is small, the amount of change in particle size due to aggregation reaction is small. For this reason, the average particle diameter of the insoluble carrier particles used in the aggregation reaction is 10
Insoluble carrier particles having a size of about .mu.m or less, preferably 0-05 to 5.0 .mu.m are suitably used.

In the present invention, the antigen or antibody to be immobilized on the insoluble carrier particles is not particularly limited, and any known antigen or antibody can be used. Typical examples include human C-reactive protein (CRP), anti-CRP antibody, streptolysin
Anti-streptolysin 0 antibody, modified gamma globulin/rheumatoid factor, albumin/anti-albumin antibody, immunoglobulin G (IgG)/anti-IgG antibody, IgA/anti-Ig
A antibody, IgM/anti-IgA antibody, complement third component (C3)
・Anti-C3 antibody, C4 anti-C4 antibody, alphafetoprotein (AFP) ・anti-CRP antibody, carcinoembryonic antigen (CEA)
) Anti-CEA antibody, human placental lactogen (hPL)
Anti-CP antibody, human chorionic gonadotropin (hCG), anti-CE antibody, insulin Anti-insulin antibody, hepatitis B surface antigen (HBs) ・Anti-HBs antibody, Candid
a albicans ・Anti-Candida albi
Examples include known antigens or antibodies such as cans antibody, Trebonema pallidum antigen, rubella antigen, anti-rotavirus antibody, and anti-adenovirus antibody.

In the present invention, the method for immobilizing antigens or antibodies on insoluble carrier particles may be either physical adsorption or formation of chemical covalent bonds, and many methods have already been proposed for the formation of chemical covalent bonds. , the immobilization method can be selected from known methods according to the characteristics of the antibody to be immobilized. Generally, the antibody can be mixed with insoluble carrier particles in a dispersion medium in the presence of a cross-linking agent if necessary. As the agent, known agents such as glutaraldehyde and 1-ethyl-5-(3-dimethylaminopropyl)carbodiimide hydrochloride can be used.

The dispersion medium for immobilizing antigens or antibodies on insoluble carrier particles is not particularly limited and any known one may be used, but when the above crosslinking agent is used, the components in the dispersion medium may react with the crosslinking agent. It is necessary to use a dispersion medium that does not The concentration of insoluble carrier particles in the dispersion medium during immobilization is not particularly limited, but generally it is 0 at the time of mixing with the antigen or antibody.
, 05% by weight or more, preferably 0.2 to 2.0% by weight. The concentration of the antigen or antibody is also not particularly limited, and -i is selected so that it is 0.0005% by weight or more, preferably 0.002 to 0-2% by weight when mixed with the insoluble carrier particles. is preferable.

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

[Actions and Effects] The chemical properties of polyvinylpyrrolidone include the ability to adsorb various inorganic and organic compounds, form complexes, and significantly increase the solubility of iodine and dyes, as well as polymer electrolytes such as polyacrylic acid and proteins. 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 particle, and the insoluble carrier particle is considered to be a polyanion. It is also believed that immobilized antigens or antibodies are present, and that polyvinylpyrrolidone interacts with the antibodies or antigens in the specimen to mediate the agglutination reaction. It is thought that this brings the distance between the proteins closer, making it easier for interactions to occur, promoting the antigen-antibody reaction, and improving measurement sensitivity.

On the other hand, it is inferred from the fact that it significantly increases the solubility of iodine and dyes that it also contributes to stabilizing the dispersion of immobilized carrier particles.

[Examples] Hereinafter, the present invention will be explained 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 Candida al.
anti-Candida albica against bi(ans)
After affinity purifying the ns antibody according to a conventional method, the purified antibody was added to a 1760 mol phosphate buffer (hereinafter abbreviated as PB).
Dialyzed at pH 6-3>, added PB to a concentration of 0.25 mg/
ml of antibody solution was prepared. As Example 1, polystyrene latex particles (carrier particles) with an average particle diameter of 0 to 728 μm were diluted with PB to prepare a dispersion liquid with a latex concentration of 1% by weight, and the same volume was added to the above antibody solution and immediately mixed by stirring. . After standing still for 2 hours, 0.1 part by weight of bovine serum albumin (lyophilized product) was added and left standing for another 2 hours.
After centrifugation, add twice the volume of PB as the above antibody solution to the resulting precipitate (immobilized carrier particles) and suspend (particle concentration: approx.
5ffijl) L, anti-Candida albican
A dispersion of antibody-immobilized carrier particles on which an affinity-purified antibody was immobilized, that is, a Candida reagent was obtained.

(2) Agglutination reaction test Candida albicans c-a-111 (
(obtained from Chiba University Eukaryotic Microbiology Center) was prepared to a bacterial concentration of 108 cells/ml and diluted with PB to make 10+
Bacteria concentration per 25 x 705 pieces, 12-5xl
O5 pieces, 63x10' pieces, 3-2x105 pieces, 1.6x
105 pieces, 0.8 x 105 pieces, 0-4X10” pieces, 0.
Prepare 2 x 10' antigen solutions. On the other hand, polyvinylpyrrolidone with a concentration of 60 (molecular weight 160,000) dissolved in PB and adjusted to a concentration of 10% is used as a stock solution, and the concentration of polyvinylpyrrolidone with 60 is 10%, 1. Or6. Prepare a 0.10% PB bath solution, mix with the above antigen solution in equal amounts, and add 60 to polyvinylpyrrolidone at 5.0% and 0.50%.
%, O-0 Contains at a concentration of 50%, and the bacterial concentration is 12.5x1
Prepare a diluted solution of 0' to 0.1 x 105 antigens,
Perform PAl, 2.3 and 50%, respectively, and also add 60 to 50% and 05 to polyvinylpyrrolidone to evaluate the negative image.
Solutions containing the antigen at concentrations of 0% and 0.050% and no antigen were prepared. Add one drop of the Candida reagent obtained in (1) onto a glass plate, and add the above Candida reagent to each drop of Candida reagent.
One drop of Ida albicans antigen dilution was added, mixed with a toothpick, and immediately stirred by horizontally rotating a glass plate at a speed of 120 revolutions per minute for 2 minutes using a Taber type stirrer manufactured by Hirasawa Seisakusho. The state of agglutination after stirring is visually determined, and the minimum concentration of the antigen in the antigen dilution solution where agglutination is clearly observed is determined as the sensitivity of the Candida reagent to the antigen.
Electrical formation is shown in Table 1.

(3) Specificity test In place of Candida albicans c-a-111, 6 strains of other fungi (obtained from the Chiba University Eukaryotic Microbiology Center), and also regular strains that are present in the specimen when using the Candida reagent Dilute 14 types of bacteria (obtained from RIKEN) with PB to obtain a bacterial concentration of 100 per ml.
The agglutination test was conducted in the same manner as the sensitivity test, with a bacterial cell solution prepared so that the number of cells was 105. Judging the state of aggregation with the naked eye,
'aa is not observed (-), it is difficult to determine the presence or absence of aggregation (±), and aggregation is clearly observed (10). The percentage of occurrence is shown in Table 1.

Comparisons S1 to 7 Comparative Example 1 was prepared by using only PB as the antigen dilution solution instead of mixing polyvinylpyrrolidone with 60 dilution solution and using it as the antigen dilution solution in Example 1. A diluted solution of polyethylene glycol 6000 was mixed with polyvinylpyrrolidone instead of a diluted solution of 60, and polyethylene glycol 6000 was mixed at 5.0%, 0.50%, and 0.050%.
Contains % concentration, bacteria concentration is 12.5x+05 to 0-
1×10 5 antigen dilutions were prepared and used as Comparative Examples 2, 3, and 4, respectively. Similarly, text run (molecular weight 6~
90,000) at concentrations of 5.0%, 0.50%, and 0.050%, and the bacterial concentration is 12.5 x 10' and 0.1 x 10'.
Comparative Example 5.6.7
Sensitivity tests and specificity tests were conducted in the same manner as in Examples 1 to 3, and the obtained results are shown in Table 1.

As shown in Table 1, in Examples 1 to 3 in which 60 was added to polyvinylpyrrolidone, the sensitivity was improved by 2 to 16 times compared to Comparative Example 1 which did not contain the additive, and the negative image and specificity decreased. It showed good reagent properties. On the other hand, in Comparative Examples 2 to 7 in which polyethylene glycol 6000 or dextran was added, the sensitivity improved when the additive concentration was low, but the negative image and specificity were lower than those in Examples 1 to 3 and those containing no additives. It was inferior to Comparative Example 1. When the amount added was further increased, both negative and positive images became non-specifically ati, making measurement impossible.

Examples 4 to 6 Instead of 60 for polyvinylpyrrolidone in Example 1, 90 (molecular weight 360,000) was added to polyvinylpyrrolidone at the time of measurement.
% (Example 4),
When the antigen dilution solution was prepared so that polyvinylpyrrolidone contained 25 (molecular weight 24,500>4% at the time of measurement (Example 5)), polyvinylpyrrolidone contained 15 (molecules) 1000
Regarding the case (Example 6) in which the antigen dilution solution was prepared so that the concentration of 0) was 8% at the time of measurement, the sensitivity and specificity were evaluated in the same manner as in Example 1. The results are shown in Table 2.

As shown in Table 2, in Examples 4 and 5, the reagent sensitivity was clearly improved compared to Comparative Example 1, which did not contain any additives. In some cases, a& was shown for a sample with an antigen concentration of 3-2×10' antigens/ml, and in other cases it was difficult to determine the presence or absence of agglutination, and the sensitivity increased only slightly. The additive concentration in Example 4 was 4%, and the additive concentration in Example 5 was 8%.
When the amount of each sample was increased, the reagent became viscous and it was necessary to mix the reagent and sample vigorously.

Example 7 and Comparative Example 8 Anti-A against alpha-fetoprotein (hereinafter referred to as AFP)
After affinity purifying the FP antibody according to a conventional method, the purified antibody was dialyzed against 0-1 molar ammonium chloride-ammonia buffer (hereinafter abbreviated as AB, pH 8.2), and AB was added to the antibody at a concentration of 0.25 mg/ml. A liquid was prepared. Polystyrene late Nox particles with an average particle size of 0-232 μm (
carrier particles) were diluted with AB to give a latex concentration of 1% by weight.
A dispersion liquid was prepared, and an equal volume was added to the antibody solution described in 1 above, and the mixture was immediately stirred and mixed. After standing for 2 hours, 0.1 part by weight of bovine serum albumin (lyophilized product) was added and left standing for an additional 2 hours.Next, centrifugation was performed, and the resulting precipitate (immobilized carrier particles) was added with 4 parts by weight of the above antibody solution. Add twice the volume of AB and suspend (particle concentration:
(approximately 0.25% by weight) to obtain a dispersion of antibody-immobilized carrier particles on which an anti-AFP affinity-purified antibody is immobilized.

As Example 7, a diluted serum solution was prepared by adding 0-4% polyvinylpyrrolidone 60 to AB.

The above reagents were applied to a Hitachi 7050 biochemical multi-item automatic analyzer, and AFP was measured under the following measurement conditions. First, 20 μl of serum is sampled into an optical cell, and 300 μl of serum dilution is added and stirred.

Approximately 5 minutes after adding the serum dilution solution, 100 μl of a dispersion of antibody-immobilized carrier particles is added and stirred. The amount of change in absorbance at a wavelength of 600 nm was automatically measured for about 4 minutes between about 1 minute and 5 minutes after adding the antibody-immobilized carrier particles.

Implementation fg! In place of the serum dilution solution of 47, use only AB that does not contain 60 in the serum dilution solution or polyvinylpyrrolidone (
Comparative PA8) had the above change in absorbance of 0.152 Abs for the same sample with the ARP concentration of 2000 ng/mI, whereas the reagent of Example 7 had the change in absorbance of 0.152 Abs for the same sample. It showed about 7 times higher sensitivity. In addition, for a sample with an ARP concentration of 10 ng/ml or less, the above-mentioned absorbance change amount in both Example 7 and Comparative Example 8 was (L 0 OIAbs) or less.

Claims (1)

[Claims] An immunological agglutination reaction reagent comprising polyvinylpyrrolidone, the measurement reagent comprising insoluble carrier particles on which an antigen or antibody is immobilized and a buffer.