JPH0682128B2 - Latex reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to a latex reagent having excellent storage stability.

[Problems to be Solved by Prior Art and Invention]

In immunological tests utilizing the antigen-antibody reaction, the agglutination reaction is used together with the precipitation reaction, the complement fixation reaction, or as a reaction which is significantly simpler and more sensitive than these reactions. The agglutination reaction is a reaction that detects free cells and antigens localized on the surface of bacterial membranes, and anti-serum with high specificity can be obtained by the progress of purification technology for antigens and antibodies, and thus highly specific antibodies can be obtained. Blood cell particles, bentonite particles,
Application range in clinical tests, such as immobilizing on particle carriers such as kaolin particles and latex particles, reacting corresponding antigens to cause aggregation, and directly or indirectly measuring the aggregation to test the antigen concentration Is remarkably expanding.

Moreover, in recent years, with the progress of purification technology of antigens, development of highly specific antibodies, and further development of quantitative analysis technology, high sensitivity, non-specific agglutination reaction does not occur, and more excellent storage stability There is a demand for the development of a diagnostic reagent having properties such as swelling.

However, the requirement for the diagnostic reagent to improve the stability during storage while maintaining the rapidity and sensitivity during immunological agglutination reaction is a request to satisfy both contradictory properties. Is very difficult. That is,
Diagnostic reagents with excessive colloidal chemical qualities have excellent storage stability, but many immunological agglutination reactions do not have sufficient speed and sensitivity, and conversely, immunological agglutination reactions In order to increase the speed and agility of
It cannot be used as a diagnostic reagent because it tends to non-specifically spontaneously aggregate and has poor storage stability. Therefore, it can be said that currently known diagnostic reagents sacrifice any of the above properties.

[Means for solving problems]

The present inventors have systematically studied to develop a reagent having excellent storage stability and rapid and sensitive immunological agglutination reaction. In addition to the results, it was found that these contradictory properties can be exhibited by controlling the latex particle size and the particle size of the latex reagent in which the latex particles are sensitized with an immunoactive substance, and the present invention has been completed. Then I came to propose here.

That is, the present invention is a latex reagent having an average particle size (D) of 0.1 to 0.5 μm obtained by sensitizing latex particles with an immunoactive substance, and the latex reagent is a latex reagent for the average particle size (d) of latex particles. Average particle size (D)
The latex reagent has a ratio (D / d) of 1.3 to 3.0.

The type of latex particles used in the present invention is not particularly limited, and known ones can be used. The most commonly used latex particles are exemplified by the following formula [I], (However, R ₁ is a hydrogen atom or an alkyl group, and R ₂ is a halogen atom, a substituted or unsubstituted phenyl group, an alkoxycarbonyl group, an acyloxy group, an alkoxy group, or a cyano group.) It is a latex particle composed of a high-molecular polymer containing a system monomer unit. Here, the substituent of the phenyl group is not particularly limited, and examples thereof include a halogen atom, a haloalkyl group, and an alkyl group. Among such hydrophobic vinyl-based monomer units, R ₂
A hydrophobic vinyl monomer unit in which is a substituted or unsubstituted phenyl group, chlorine atom, or alkoxycarbonyl group is preferable. Examples of the monomer that gives such a monomer unit include styrene, vinyltoluene, chloromethylstyrene, chlorostyrene, vinyl chloride, vinyl bromide, methyl methacrylate, ethyl methacrylate, propyl methacrylate, vinyl acetate. , Ethyl vinyl ether, propyl vinyl ether, butyl vinyl ether, acrylonitrile, methacrylonitrile, etc. are preferably used, and styrene, vinyltoluene, chloromethylstyrene, vinyl chloride, methyl methacrylate, ethyl methacrylate, etc. are particularly preferably used. In addition, the following formula [II] on the surface of the latex particles composed of high molecular weight polymer (However, R is a hydrogen atom or an alkyl group.) Those having a monomer unit represented by are preferably used in the present invention. Examples of the monomer that gives the monomer unit represented by the formula [II] include glycidyl acrylate and glycidyl methacrylate.

Latex particles composed of only one monomer unit represented by the above formula [I] or [II], or a monomer unit represented by the above formula [I] and [II] Latex particles composed of both are particularly preferably used in the present invention.

When the latex particles are used as a raw material for an adsorptive latex reagent, the content of the monomer unit represented by the formula [II] is 0 to 20 mol% in the latex particles, preferably 0.05 to 15 mol. % Is preferred. Also,
When the latex particles are used as a raw material of a covalent bond type latex reagent, the content of the monomer unit represented by the formula (II) is 20 to 100 mol% in the latex particles, preferably 30 to 99 mol. It is preferable to select from the range of%.

When latex particles are used as a raw material for a diagnostic latex reagent, hydrophilic vinyl monomer units are present in the latex particles in a range that does not adversely affect the properties of the latex reagent, for example, 20 mol% or less. May be included. Examples of the monomer that provides the hydrophilic vinyl-based monomer unit include methacrylic acid, acrylic acid, styrene sulfonic acid, sodium styrene sulfonate, 2-hydroxyethyl (meth) acrylate, vinylpyrrolidone,
Examples thereof include polyethylene glycol (meth) acrylic acid ester. Further, if necessary, a crosslinkable monomer such as divinylbenzene, ethylene glycol dimethacrylate, diethylene glycol methacrylate, and bisphenol A diglycidyl ether can be preferably used.

The polymerization method for obtaining latex particles composed of the polymer used in the present invention is not particularly limited, and a known method is preferably adopted. For example, a method of emulsion polymerization using a water-soluble radical initiator in an aqueous medium in the presence of an anionic surfactant or a nonionic surfactant, a water-soluble radical initiation in an aqueous medium without using a surfactant. Method of heterogeneous polymerization using an agent, partially saponified polyvinyl alcohol, suspension polymerization in the presence of a protective colloid such as folinylpyrrolidone, in an organic solvent in which vinyl monomers dissolve but the polymer does not dissolve A method such as precipitation polymerization is adopted.

The average particle size of the latex particles used in the present invention is not particularly limited, but generally 0.05 to 0.38μ, preferably 0.07 to 0.
Those in the range of 3μ are preferably used. Furthermore, it is desirable that the latex particles have a small particle diameter dispersion value because the reproducibility is good. For example, the dispersion value of the particle diameter is preferably 10% or less, more preferably 5% or less. The dispersion value is a value obtained by dividing the standard deviation by the average particle diameter and multiplying by 100, and the unit is expressed in%.

The latex particles thus obtained can be used as a diagnostic reagent for immunological reaction by obtaining a latex reagent by sensitizing an immunologically active substance. The immunologically active substance is not particularly limited, and may be, for example, any of various antigens and antibodies that can be used in agglutination reaction, agglutination inhibition reaction and the like. To show a typical example,
For example, denatured gamma globulin, rheumatoid factor, antinuclear factor, human albumin, anti-human albumin antibody, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), streptolysin O, anti-streptolysin O Antibody, C-reactive protein, anti-C-reactive protein antibody, alpha-fetoprotein (AFP), anti-AFP antibody, carcinoembryonic antigen (CEA), anti-CEA antibody, human placental lactogen (HPL), anti-HPL antibody, Human chorionic gonadotropin (HCG), anti-HCG antibody, anti-estrogen antibody, anti-insulin antibody, hepatitis B surface antigen (HBS), anti-HBS antibody, syphilis tryponema surface antigen, rubella antigen, complement component C _1q , anti-complement Known _immunoactive substances such as component C _1q antibody can be mentioned.

The latex reagent of the present invention is obtained by sensitizing the latex particles with an immunoactive substance. The latex particles are generally well dispersible and non-aggregating. However, when an immunoactive substance is sensitized, the latex particles are aggregated, and the latex reagent exists as aggregated particles. The latex reagent provided by the present invention needs to have a particle diameter (D) in the range of 0.1 to 0.5 μm. If the particle size of the latex reagent is smaller than the above range, the latex reagent is remarkably stable and is not sufficient in terms of rapidity and sensitivity required in the immunological agglutination reaction.
On the contrary, when the particle size of the latex reagent is larger than the above range, the reagent may not be a stable reagent because of strong cohesiveness.

Another important requirement for the latex reagent of the present invention is that the ratio (D / d) of the particle diameter (D) of the latex reagent to the particle diameter (d) of the latex particles must be in the range of 1.3 to 3.0. Is. When the (D / d) is less than 1.3, it has a high storage stability, but it becomes difficult to detect a low concentration of antigen or antibody. That is, because electrostatic repulsion between latex particles sensitized with the corresponding antigen or antibody is too strong,
Inability to aggregate in the presence of low concentrations of antigen or antibody. Further, when the above (D / d) exceeds 3.0, it is indicated that large aggregates are formed during sensitization, which causes nonspecific aggregation during storage. Furthermore, since the size of the final aggregate in the presence of the antigen or antibody is fixed, it becomes difficult to detect a high concentration of the antigen or antibody.

The method for producing the latex reagent of the present invention is not particularly limited, but in general, the above properties can be selected depending on the latex particle size used as a raw material and the conditions for sensitizing an immunoactive substance. For example, the latex particle size may be selected from the range of 0.05 to 0.38 μm depending on the purpose of use as described above. Further, the sensitizing method such that the average particle diameter (D) of the latex reagent is in the range of (D / d) = 1.3 to 3.0, preferably 1.5 to 2.5 with respect to the average particle diameter (d) of the latex particles is as follows. Is preferred. For example, a stirring rod equipped with a motor is put into an aqueous suspension of latex particles, and while stirring the suspension, the immunoactive substance-containing liquid is quickly added, and stirring is continued as it is. The stirring speed varies depending on the size of the latex particles, the concentration of the suspension, and the concentration of the immunoactive substance-containing liquid,
100 to 500 rotations / minute, preferably 300 to 400 rotations / minute are suitably used. Also, when shaking the above, similarly 25 ~ 250
Times / minute, preferably 50 to 150 times / minute are suitably used. The sensitizing treatment is generally preferably carried out at a pH of about 6.0 to 8.6 and a temperature of about 4 to 37 ° C. In general, the immunologically active substance is used in a slightly excess amount over the saturated adsorption amount of latex particles, and the excess amount may be removed by centrifugation after sensitization. In general, the immunoactive substance is preferably selected so that the ratio of the antigen or antibody (mg) / surface area (m ² ) of the latex particles is in the range of 0.05 to 10 kg / m ² .

The latex reagent obtained as described above is washed with an aqueous solvent, if necessary, and then generally subjected to an immunological agglutination reaction in a state of being suspended in the aqueous solvent. Generally, the concentration of the latex reagent when suspended in an aqueous solvent is preferably selected from the range of 0.01 to 0.1% by weight.

In addition to the above properties, the latex reagent of the present invention has a zeta potential of -20 mV to 10 mV, preferably -10 mV to 0 mV, in a state of being suspended in the aqueous solvent, and also has an optical path length of 10 mm in absorbance. Most preferably, it is prepared, for example, by the above-mentioned method so as to be in the range of 0.5 to 1.5. These properties are suitable for obtaining a latex reagent having particularly good sensitivity.

[Action]

The latex reagent of the present invention having the above characteristics generally has an average of 2 to 3 latex particles aggregated and has an extremely high storage stability, but on the other hand, it has a high degree of sensitivity and agility because it is appropriately aggregated. is doing.

〔effect〕

INDUSTRIAL APPLICABILITY The latex reagent of the present invention is extremely stable in an aqueous medium and has excellent storage stability, and at the same time, it is extremely valuable as a latex reagent having a wide measurement range.

The examples below show that the latex reagent of the present invention has excellent storage stability, but the present invention is not limited to these examples.

In addition, the average particle size of the latex reagent in the examples is a laser rotary grading system automatic surface charge spectrum analyzer LASERZEE ^™ System3000 (PEN
KEM) (hereinafter abbreviated as particle analyzer).

Example 1 (1) Preparation of Reagent for Measuring Rheumatoid Factor A polystyrene latex particle having an average particle diameter of 0.178 μm was added to N
A suspension having a latex concentration of 1% by weight is prepared by diluting with 0.1 M borate buffer solution (hereinafter abbreviated as BBS) having a pH of 8.2 containing 0.05 M aCl. Next, heat-treat human IgG at 60 ° C for 10 minutes to BBS.
And dilute to 1 mg / ml. 1 volume of the heat-denatured human IgG was added while stirring 1 volume of the above latex suspension at 350 rpm, and then left at room temperature for 2 hours while stirring at the same speed. Then, after centrifugation and removal of the supernatant, the precipitate was redispersed in BBS containing 0.05% by weight of bovine serum albumin to adjust the latex concentration to 0.03% by weight to obtain a rheumatoid factor assay reagent. As a result of analysis with a particle analyzer, the average particle size of the latex reagent was 0.240 μm. The result of measuring the zeta potential with LASER ZEE Mode l501 (manufactured by PEN KEM) was −2.5 ± 0.2 mV (in BBS). The absorbance of the latex reagent is 1.25 (optical path length 10mm 580n
m) was.

(2) Measurement method Absorbance was measured by a device in which a temperature controller and a magnetic stirrer were attached to the photometry section of Hitachi U-3200 type self-recording spectrophotometer. A cylindrical stirrer was placed in a glass optical cell having an optical path length of 10 mm, then 1980 μl of the rheumatoid factor measurement reagent obtained in (1) was dispensed, inserted into the photometry section, and kept at 37 ° C.

Next, 20 μl of the test liquid was added while stirring the rheumatoid factor measurement reagent with the stirring device. The measurement of the absorbance was started at the same time as the addition. The absorbance was measured using a light ray having a wavelength of 580 nm. The stirring was stopped 3 seconds after the addition of the test solution.

(3) Measurement of serum of known concentration Rheumatoid factor concentration of 1600 IU / ml Pooled serum of rheumatic patient serum was diluted with physiological saline to obtain rheumatoid factor concentration of 5,1
0,20,40,80,100,200,500,1000 IU / ml test solutions were obtained.

Under the measurement conditions of (2), the absorbance was measured 5 times for each of the above 9 types of test solutions and BBS.

Among the obtained absorbances, the difference between the absorbances at a fixed interval time was obtained from the absorbances 1 minute and 5 minutes after the addition of the test solution.

The difference in absorbance was similarly determined after the reagent obtained in (1) was stored at 4 ° C for 12 months. The results are shown in Table 1.

From the results in Table 1, the difference between the absorbance on the 7th day after preparation of the reagent and after storage at 4 ° C for 12 months is within the range of the coefficient of variation at all measured concentrations.

The average particle size of the reagent after storage at 4 ° C for 12 months was 0.240μ.
m, zeta potential was -2.3 ± 0.2 mV, and absorbance was 1.26.

Comparative Example 1 To 1 volume of the same latex suspension as used in Example 1, 1 volume of heat-denatured human IgG solution was added in a static state, and then left to stand for 2 hours. Thereafter, the same treatment as in Example 1 was performed. As a result of analysis with a particle analyzer, the average particle size of the latex reagent is 0.720 μm,
The absorbance was 2.35. The ratio (D / d) of the average particle size (d) of the latex particles to the average particle size (D) of the latex reagent was 4.0. The result of measuring pool serum of rheumatoid patient serum of known concentration in the same manner as in Example 1
Shown in the table.

As is clear from Table 2, 7 days after preparation of the reagent and 4 ° C, 12
The difference in absorbance after storage for a month is different. After reagent preparation 4
After 12 months, the average particle size was 0.931μm and the absorbance was 2.65.
Therefore, non-specific aggregation occurred during storage.

Comparative Example 2 To 1 volume of the same latex suspension as used in Example 1, 1/20 volume of nonionic surfactant (HLB = 15) was added, and the mixture was allowed to stand at room temperature for 1 hour. Next, 1 volume of human IgG solution 400 rpm
The mixture was quickly added with stirring for 4 minutes, and the stirring was continued at 400 rpm for 2 hours. Thereafter, the same treatment as in Example 1 was performed. As a result of analysis with a particle analyzer, the average particle size of the latex reagent was 0.196 μm, and the zeta potential was −35 m.
The absorbance was 0.47 at V. The ratio of the average particle diameter (D) of the latex reagent to the average particle diameter (d) of the latex reagent was (D / d) = 1.10. Table 3 shows the results obtained by measuring pooled sera of rheumatism patients with known concentrations in the same manner as in Example 1.

From the results in Table 3, there is a decision that storage stability is excellent, but no aggregation occurs at 5 IU / ml, which requires clinical measurement. The average particle size after 12 months at 4 ° C was 0.1.
The surface charge density was -37 mV and the absorbance was 0.48.

Example 2 Polystyrene latex particles having an average particle diameter of 0.134 μm were mixed with N
0.1M glycine buffer (pHS 8.2, containing 0.05M aCl)
Is abbreviated) to prepare a suspension having a latex concentration of 1% by weight. 1 volume of the heat-denatured human IgG solution was added to 1 volume of the latex suspension in the same manner as in Example 1, and then 2 times with stirring.
Left for hours. Thereafter, the same treatment as in Example 1 was performed. As a result of analysis with a particle analyzer, the average particle size of the latex reagent was 0.201 μm, and the zeta potential was −3.4 ± 0.
At 2 mV (in GBS) the absorbance was 1.03. As in Example 1, pooled sera of rheumatoid patient sera of known concentrations were measured. The results are shown in Table 4.

From the results shown in Table 4, the difference between the absorbance on the 7th day after preparation of the reagent and after storage at 4 ° C for 12 months is within the range of the coefficient of variation at all measured concentrations.

The average particle size of the reagent after storage at 4 ℃ for 12 months was 0.205μ.
At m, the zeta potential was -3.3 ± 0.1 mV and the absorbance was 1.06.

Example 3 (1) Preparation of reagent for measuring C-reactive protein Polystyrene latex particles having an average diameter of 0.123 μm were diluted with an aluminum chloride-ammonia buffer (pH = 0.8) to prepare a suspension having a latex concentration of 1% by weight. To do. Then, an anti-CRP goat fractionated by salting out from an anti-CRP serum obtained by immunizing a goat with C-reactive protein (hereinafter abbreviated as CRP)
Ammonium chloride-ammonia buffer (pH =
0.8) to prepare a solution with a protein concentration of 2 mg / ml. 1 volume of the anti-CRP goat IgG fraction solution was added to 1 volume of the above latex suspension in the same manner as in Example 1, and the mixture was reacted at 37 ° C. for 2 hours.
Then, after centrifugation and removal of the supernatant, the precipitate was redispersed in ammonium chloride-ammonia buffer (pH = 0.8) containing bovine serum albumin at a concentration of 0.05% by weight to adjust the latex concentration to 0.05% by weight. A CRP measurement reagent was obtained.

As a result of analysis with a particle analyzer, the average particle size of the latex reagent was 0.213 μm. The zeta potential was -0.2 ± 0.2 mV (in ammonium chloride-ammonia buffer), and the absorbance was 1.30.

(2) Measurement method Measurement was performed in the same manner as in Example 1 except that 1990 μl of the reagent and 10 μl of the test liquid were used.

(3) Measurement of serum of known concentration CRP concentration of 0.10, 0.25, 0.50 was obtained by diluting a purified CRP solution with a CRP concentration of 240 mg / dl with CRP-free serum that had been treated with CRP to make it substantially CRP-free. , 1.0,2.5,5.0,10,15,20,3
A test solution of 0, 40, 60 mg / dl was obtained.

Under the measurement conditions of (2), the absorbance was measured 5 times for each of the 12 test liquids and the ammonium chloride-ammonia buffer.

Among the obtained absorbances, the difference in the absorbance for a certain interval time was obtained from the absorbances 1 minute and 5 minutes after the addition of the test solution. The difference in absorbance was similarly determined after storing the reagent obtained in (1) at 4 ° C. for 12 months. The results are shown in Table 5.

From the results in Table 5, the difference between the absorbance on the 7th day after preparation of the reagent and after storage at 4 ° C for 12 months is within the range of the coefficient of variation at all measured concentrations.

The average particle size of the reagent after storage at 4 ℃ for 12 months was 0.214μ.
At m, the zeta potential was -2.1 ± 0.3 mV and the absorbance was 1.31.

Claims (3)

[Claims] 1. A latex reagent having an average particle size (D) of 0.1 to 0.5 μm obtained by sensitizing latex particles with an immunoactive substance, wherein the latex reagent is a latex reagent with respect to the average particle size (d) of latex particles. A latex reagent having a ratio (D / d) of average particle diameter (D) in the range of 1.3 to 3.0. 2. The latex reagent has a zeta potential of -20 mV-10.
The latex reagent according to claim 1, which is in the range of mV. 3. The latex reagent according to claim 1, wherein the absorbance of the latex reagent is in the range of 0.5 to 1.5 in a cell having an optical path length of 10 mm.