JP2013227557A - Latex particle for diagnostic reagent and method of manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a latex particle for a diagnostic reagent which is obtained by the soap-free polymerization of an aromatic vinyl monomer and has monodispersity with excellent particle size distribution and manufacture reproducibility, and to provide a method of manufacturing the latex particle.SOLUTION: A latex particle for a diagnostic reagent comprises an aromatic vinyl polymer containing a group 2 metal, and the content of the group 2 metal in the latex particle is 0.005-0.05 wt.%. Then, a method of manufacturing the latex particle for the diagnostic reagent is obtained by performing soap-free polymerization of an aromatic vinyl monomer in the presence of a metal sulfate of the group 2 metal, phosphate of alkali metal and hydroxide of alkali metal.

Description

  The present invention relates to latex particles for diagnostic reagents and a method for producing the same.

  Aromatic vinyl polymer particles such as polystyrene latex particles are suitably used as a diagnostic reagent carrier. As latex particles as a diagnostic reagent carrier, particles produced by emulsion polymerization and having an average particle size of 0.1 to 0.5 μm are generally used. When preparing latex particles having a larger particle size, it is necessary to increase the particle size by changing the polymerization conditions. For example, techniques such as (1) reducing the amount of emulsifier, (2) reducing the amount of polymerization initiator, and (3) adding an inorganic electrolyte during emulsion polymerization have been proposed.

  In the methods (1) and (2), stability at the time of polymerization is reduced, aggregates are generated, and latex particles having good dispersibility are hardly obtained. Moreover, there is a drawback that the particle size distribution of the obtained latex particles is widened. As a method of (3), for example, Patent Document 1 discloses a method of obtaining latex particles having a large particle size of 0.2 μm or more by emulsion polymerization in the presence of an inorganic electrolyte. Patent Document 2 discloses a method for producing 1.0 to 10 μm monodisperse vinyl polymer particles by emulsion polymerization in the presence of a divalent metal sulfate.

  However, the maximum particle size value disclosed in Patent Document 1 is about 0.3 μm. Moreover, since the particle | grains exceeding 1.0 micrometer disclosed by patent document 2 have a large sedimentation speed, the measurement of the aggregation degree at the time of using as a diagnostic reagent becomes difficult, and cannot be used as a diagnostic reagent support | carrier. Furthermore, since the methods of Patent Document 1 and Patent Document 2 are production methods using an emulsifier, the latex particles obtained after polymerization and the emulsifier remaining in the latex solution use the latex particles as a diagnostic reagent carrier. Cause disturbing reactions.

  For example, since a part of the emulsifier used at the time of polymerization is adsorbed or chemically bonded to the surface of the latex particle, it becomes difficult to sensitize the latex particle with an antigen or an antibody. Further, the emulsifier that is present in the latex solution in a free state may cause the latex particles to aggregate when sensitizing the latex particles with antigens or antibodies. Further, even when sensitized latex particles are obtained, non-specific reactions are likely to occur. The emulsifier contained in the latex particle surface and latex solution can be removed by an ion exchange method or a dialysis method, but these steps may deteriorate the stability of the latex particle and make it impossible to use as a diagnostic reagent.

  In addition, methods for producing latex particles as industrial materials such as thermoplastic resin particles, heat-resistant resin particles, toner material particles, and paper composition particles have been proposed. However, these production methods often use an emulsifier as in Patent Documents 1 and 2, and particles having a special composition, functional group, or structure that are inappropriate for use as a diagnostic reagent. Since it is a production method and a product cannot be obtained in the state of a latex solution in which particles are dispersed, it is unsuitable as a method for producing a diagnostic reagent carrier.

  As a method for producing latex particles as a diagnostic reagent carrier, there is a production method by a soap-free polymerization method without using an emulsifier. Patent Document 3 discloses a styrene soap-free polymerization method carried out in the presence of a divalent metal oxide or hydroxide. According to this method, latex particles having an average particle size value of 0.1 to 1.5 μm can be obtained. However, the method of Patent Document 3 is poor in reproducibility of polymerization, and the operation is complicated because it is necessary to adjust the pH of the reaction system and perform heating under alkaline conditions after the completion of the polymerization reaction.

  In order to increase the particle size of latex particles as conventional diagnostic reagent carriers, for example, to obtain latex particles of 0.5 to 1.0 μm, a production method that satisfies the following conditions is required. That is, a method of obtaining latex particles by polymerizing an aromatic vinyl monomer, which is a material suitable for a diagnostic reagent carrier, a soap-free polymerization method without using an emulsifier, and a monodisperse with a good particle size distribution A latex particle having good properties, and good polymerization reproducibility and easy operation.

JP 59-22904 A JP-A 63-254104 JP-A-58-198508

  An object of the present invention is to provide latex particles for a diagnostic reagent containing a Group 2 metal in the particles. Another object of the present invention is to provide a method for producing latex particles for diagnostic reagents using a soap-free polymerization method. In particular, the diagnostic reagent latex particles having an average particle size of 0.5 to 1.0 μm and good monodispersity, and the diagnostic reagent latex particles having good polymerization reproducibility and easy operation. It aims to provide a method.

  The inventors of the present invention solved the above problems by incorporating latex group 2 metal into the latex particles. Moreover, the said subject was solved by performing soap free polymerization of an aromatic vinyl monomer in the dispersion medium which melt | dissolved the group 2 metal salt and specific salt. That is, the present invention is as follows.

[1] Latex particles for diagnostic reagents comprising an aromatic vinyl polymer containing a Group 2 metal, wherein the content of the Group 2 metal in the latex particles is 0.005 to 0.05 wt. % Diagnostic reagent latex particles.
[2] The latex particle for diagnostic reagent according to [1], wherein the Group 2 metal is magnesium.
[3] The latex particle for diagnostic reagent according to [1] or [2], wherein the average particle size of the latex particle for diagnostic reagent is 0.5 to 1.0 μm.
[4] A soap-free polymerization of an aromatic vinyl monomer in the presence of a Group 2 metal sulfate, an alkali metal phosphate and an alkali metal hydroxide [1] to [3] The method for producing latex particles for a diagnostic reagent according to any one of [3].
[5] Step 1 of dissolving Group 2 metal sulfate, alkali metal phosphate and alkali metal hydroxide in dispersion medium, Step 2 of adding aromatic vinyl monomer to dispersion medium, polymerization The method for producing latex particles for a diagnostic reagent according to any one of [1] to [3], comprising a step 3 of performing soap-free polymerization in the presence of an initiator.

  The latex particle for diagnostic reagent of the present invention is a monodisperse latex particle for diagnostic reagent having an average particle size of 0.5 to 1.0 μm, and a latex reagent having excellent measurement performance can be prepared. Moreover, according to the method for producing diagnostic reagent latex particles of the present invention, the diagnostic reagent latex particles can be produced with good reproducibility by a simple operation without impairing the monodispersity of the particle size distribution.

Calibration curve showing the relationship between the amount of HBs antibody and absorbance

  The present invention is latex particles for diagnostic reagents comprising an aromatic vinyl polymer containing a Group 2 metal. The Group 2 metal is a metal of Group 2 of the Periodic Table, specifically, magnesium, calcium, strontium, and barium. Preferred Group 2 metals are magnesium and calcium, and a more preferred Group 2 metal is magnesium.

  The term “latex particles contain a Group 2 metal” in the present invention means that the Group 2 metal is present on the surface of the latex particles or inside the particles, and the Group 2 metal is an ordinary diagnostic reagent. It is present on the surface or inside of the latex particle in a state where it is not liberated, detached or exfoliated in the environment of the production process and the use process.

  The latex particles for diagnostic reagents of the present invention have a content in the above-mentioned Group 2 metal latex particles of 0.005 to 0.05% by weight. When the content of the Group 2 metal is less than 0.005% by weight and when the content of the Group 2 metal exceeds 0.05% by weight, the average particle size value is 0.5 to 1.0 μm. It may be difficult to form monodisperse particles, and reagent performance may be reduced. The preferred Group 2 metal content is 0.007 to 0.04% by weight.

  The average particle size of the diagnostic reagent latex particles of the present invention is preferably such that the lower limit is 0.5 μm and the upper limit is 1.0 μm. When the average particle size value is less than 0.5 μm and when the average particle size value exceeds 1.0 μm, the reagent performance may deteriorate. More preferably, the lower limit of the average particle size value is 0.55 μm and the upper limit is 0.95 μm.

The latex particle for diagnostic reagent of the present invention preferably has a coefficient of variation (CV value) of the particle size value of 5% or less. The coefficient of variation (CV value) of the particle size value can be obtained by photographing a latex particle image with a transmission electron microscope and analyzing the number of particles of 1000 or more with respect to the obtained photographed image.
CV value (%) = (standard deviation / average value) × 100

  The diagnostic reagent latex particles of the present invention are preferably diagnostic reagent latex particles comprising an aromatic vinyl polymer. The aromatic vinyl polymer is preferably a polymer obtained by polymerizing an aromatic vinyl monomer.

  Examples of the aromatic vinyl monomer constituting the diagnostic reagent latex particles of the present invention include styrene, methylstyrene, dimethylstyrene, ethylstyrene, isopropylstyrene, chlorostyrene, chloromethylstyrene, bromostyrene, vinyltoluene, vinylxylene, Non-crosslinkable aromatic vinyl monomers such as vinyl naphthalene, styrene sulfonic acid, styrene sulfonate, methyl styrene sulfonate, ethyl styrene sulfonate, divinyl benzene, trivinyl benzene, divinyl toluene, divinyl xylene, divinyl ethyl benzene And cross-linkable aromatic vinyl monomers such as divinylnaphthalene and divinylanthracene. A non-crosslinkable aromatic vinyl monomer is preferred. More preferred are styrene and styrene sulfonate. The diagnostic reagent latex particles of the present invention may be composed of two or more of the above aromatic vinyl monomers.

  As the monomer constituting the diagnostic reagent latex particles of the present invention, the above aromatic vinyl monomer and a monomer copolymerizable with the aromatic vinyl monomer (hereinafter referred to as “copolymer monomer”). May be described). As a comonomer, Preferably acrylic compounds or methacryl compounds, such as acrylic acid, methacrylic acid, and these esters, are mentioned. Two or more kinds of comonomer may be used. The upper limit of the preferable addition amount in the case of using a comonomer is 20 parts by weight with respect to 100 parts by weight of the aromatic vinyl monomer. When the amount exceeds 20 parts by weight, the reactivity when used as a diagnostic reagent may decrease. More preferably, it is comprised only from an aromatic vinyl monomer.

  The present invention includes a method for producing the above-mentioned “latex particles for a diagnostic reagent containing a Group 2 metal” (hereinafter also referred to as the method of the present invention). The method of the present invention includes a soap of an aromatic vinyl monomer in the presence of one or more Group 2 metal sulfates, one or more alkali metal phosphates, and one or more alkali metal hydroxides. This is a method for carrying out free polymerization (hereinafter, a group 2 metal sulfate, an alkali metal phosphate, and an alkali metal hydroxide may be simply referred to as “salts”).

  The Group 2 metal sulfate used in the method of the present invention is a Group 2 element sulfate, and specific examples thereof include magnesium sulfate, calcium sulfate, strontium sulfate, and barium sulfate. Magnesium sulfate and calcium sulfate are preferable, and magnesium sulfate is more preferable. Two or more kinds of Group 2 metal sulfates may be used.

  Examples of the alkali metal phosphate used in the method of the present invention include potassium phosphate, sodium phosphate, lithium phosphate, and their monohydrogen and dihydrogen salts. Preferred are potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate. Two or more alkali metal phosphates may be used.

  Examples of the alkali metal hydroxide used in the method of the present invention include potassium hydroxide, sodium hydroxide, lithium hydroxide and the like. Of these, potassium hydroxide and sodium hydroxide are preferred. Two or more alkali metal hydroxides may be used.

  The preferred lower limit of the total salt concentration is 0.5 mmol / L, and the upper limit is 50 mmol / L. When the average particle size is less than 0.5 mmol / L, the average particle size value decreases, and when it exceeds 50 mmol / L, the average particle size value increases and aggregates are likely to be generated. A more preferred lower limit is 1 mmol / L, and a more preferred upper limit is 20 mmol / L.

  The method of the present invention is a production method in which soap-free polymerization of the aromatic vinyl monomer is performed in the presence of the salt. As used herein, the term “soap-free polymerization method” has the meaning generally understood in the field of the present invention. That is, when the monomer is polymerized, a polymerization initiator that is hardly soluble in the monomer and easily soluble in the dispersion medium is used without the presence of an emulsifier such as a surfactant capable of emulsifying the monomer. This is a known polymerization method.

  Examples of the polymerization initiator include persulfates, peroxides, azo compounds, and the like. Examples of the persulfate include potassium persulfate, sodium persulfate, and ammonium persulfate. Examples of the peroxide include hydrogen peroxide, t-butyl hydroperoxide, t-butyl peroxyneodecanoate, and t-butyl peroxypivalate. As the azo compound, 2,2′-azobis (2-methylpropaneamidine) dihydrochloride, 2,2′-azobis {2- [1- (2-hydroxyethyl) -2-imidazolin-2-yl] propane} Dihydrochloride, 2,2′-azobis [2- (2-imidazolin-2-yl) propane], 2,2′-azobis (1-imino-1-pyrrolidino-2-methylpropane) dihydrochloride, 2, 2 '-Azobis {2-methyl-N- [1,1-bis (hydroxymethyl) -2-hydroxyethyl] propionamide}, 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propion Amido], 4,4′-azobis-4-cyanovaleric acid, and salts thereof. A particularly preferred polymerization initiator is persulfate. The preferable lower limit of the concentration of the polymerization initiator is 0.1 mmol / L, and the upper limit is 1.0 mmol / L. When it is less than 0.1 mmol / L, the particle size becomes small, and when it exceeds 1.0 mmol / L, the polymerization reaction is too fast, making temperature control difficult.

  A more preferable production method of the method of the present invention includes a step 1 for dissolving salts in a dispersion medium, a step 2 for adding an aromatic vinyl monomer to the dispersion medium, and a step 3 for performing soap-free polymerization in the presence of a polymerization initiator. It is a manufacturing method including.

  In this production method, first, the above salts are dissolved in a dispersion medium (step 1). A preferred dispersion medium is water or a mixed solvent of water and a water-soluble organic solvent. More preferred is water. Examples of the method for dissolving salts include a method in which salts are simultaneously added to a dispersion medium and dissolved, and a method in which each salt is added in order and dissolved. More preferably, the alkali metal hydroxide is finally dissolved. In some cases, by dissolving the alkali metal hydroxide last, the group 2 metal sulfate and alkali metal phosphate can be dissolved without precipitation. After the salts are dissolved, an aromatic vinyl monomer is added to the dispersion medium (Step 2). There is no restriction | limiting in particular in the addition method, You may add all at once and may add sequentially by methods, such as dripping. Next, the polymerization initiator is added, and soap-free polymerization is performed in the presence of the polymerization initiator (step 3).

  As the polymerization reaction, known means for generating free radicals from the polymerization initiator can be used. Preferably, the polymerization reaction is performed by heating. The minimum of preferable polymerization temperature is 50 degreeC, and an upper limit is 90 degreeC. When the polymerization temperature is less than 50 ° C., the polymerization reaction may take a long time and the monodispersity of the particle size distribution may be impaired. When the polymerization temperature exceeds 90 ° C., aggregates may be generated. The emulsifier is not added through the above steps 1 to 3.

  The diagnostic reagent can be prepared by sensitizing the latex particle for diagnostic reagent of the present invention with a substance such as a known antibody, antigen or nucleic acid that specifically reacts with the substance to be measured. As the sensitization method, a known physical adsorption method or chemical bonding method can be used. Moreover, you may perform a well-known blocking process etc. as needed.

  As an antibody to be sensitized to latex particles, a monoclonal antibody or a polyclonal antibody can be used. In addition, as the type of antibody, immunoglobulins, antibody fragments such as Fab, Fab ', F (ab') 2 or Fv can be used.

  Examples of substances that can be measured by the diagnostic reagent obtained from the latex particles of the present invention include blood, serum, plasma, urine, spinal fluid, or known substances to be measured contained in biological samples such as cells or tissue disruption fluid. It is done.

Example 1
Soap-free polymerization using two types of aromatic vinyl monomers was carried out in the presence of the salts used in the present invention.
To a 2 L separable flask equipped with a temperature sensor, a stirrer, a nitrogen introduction tube, a dropping funnel, and a reflux condenser, 1200 g of pure water (dispersion medium) was charged. While stirring the dispersion medium, 6 mL of 0.1 mol / L magnesium sulfate heptahydrate (Group 2 metal sulfate: Wako Pure Chemicals special grade) aqueous solution, 0.1 mol / L potassium dihydrogen phosphate (Alkali metal phosphate: Wako Pure Chemicals special grade) 15 mL aqueous solution, 0.1 mol / L potassium hydroxide (alkali metal hydroxide: Wako Pure Chemicals special grade) aqueous solution 10 mL was added to the dispersion medium in this order. Dissolved (total salt concentration 2.52 mmol / L). Nitrogen gas was passed through the separable flask and the temperature was raised. When the temperature reached 75 ° C., 150 g of styrene (aromatic vinyl monomer: special grade manufactured by Wako Pure Chemical Industries), sodium styrenesulfonate (aromatic vinyl monomer) Body: 0.20 g manufactured by Tosoh) and 0.20 g potassium persulfate (polymerization initiator: manufactured by Merck) were added to the dispersion medium, and a polymerization reaction was performed at 75 ° C. for 40 hours.

The average particle size value and particle size distribution of the latex particles were measured. The obtained polymer (latex containing latex particles) was collected, and a latex particle image was taken with a transmission electron microscope (JEM1010 type, manufactured by JEOL). The obtained photographed image was subjected to image analysis of 1000 or more particles using Image Hyper II (manufactured by Digimo), and the average particle diameter value and the coefficient of variation (CV value) of the particle diameter value were measured. The average particle size value was 0.58 μm, and the CV value of the particle size value was 4.9%. It was confirmed that latex particles having a CV value of 5% or less, which is an index of monodispersibility, and a monodisperse particle size distribution were obtained. Table 1 shows the concentrations of the salts used, the average particle size, and the coefficient of variation of the particle size distribution.

  Next, the content of Group 2 metal (magnesium) in the latex particles was measured. 2 mL of the obtained polymer (latex containing latex particles) was centrifuged (8000 rpm × 10 minutes), and the supernatant was removed. The precipitate was redispersed in 2 mL of an aqueous solution containing a Group 2 metal (0.1 mol / L magnesium sulfate heptahydrate) at the same concentration as during polymerization. The supernatant was removed by centrifugation again. 2 mL of pure water was added to the sediment, redispersed and centrifuged. The addition of pure water, redispersion and centrifugation were repeated a total of 3 times. After 2 mL of pure water was added to the sediment and redispersed, the total amount of the dispersion was dried at 95 ° C. for 2 hours. 1 g of the dried latex particles was heated, carbonized, treated with nitric acid, and the magnesium content (% by weight) was determined by ICP-AES (high frequency inductively coupled plasma emission spectroscopy) (detection limit 0.005%). The obtained results are shown in Table 1.

(Examples 2 to 4)
A polymerization reaction was carried out in the same manner as in Example 1 except that the concentration of the salt used in Example 1 was changed, and Examples 2 to 4 were made. Table 1 shows the concentrations of the salts used, the average particle diameter value, the CV value of the particle diameter value, and the Group 2 metal content. In either case, latex particles having an average particle size of 0.58 to 0.92 μm and a monodisperse particle size distribution were obtained.

(Comparative Examples 1-7)
Comparative Examples 1 to 4 were carried out by carrying out a polymerization reaction in the same manner as in Example 2 except that not all or some of the salts used in Example 2 were used. Further, a polymerization reaction was carried out in the same manner as in Example 4 except that some of the salts used in Example 4 were not used, and Comparative Examples 5 to 7 were obtained. Table 1 shows the types, concentrations, average particle diameter values, and CV values of the particle diameter values used. Comparative Example 1 is a system containing no salts, Comparative Example 2 is a system using only Group 2 metal sulfate and alkali metal phosphate, and Comparative Example 3 is Group 2 metal sulfate and alkali. The system using only metal hydroxide and Comparative Example 4 were carried out in a system using only alkali metal phosphate and alkali metal hydroxide. Comparative Example 5 was a system using only a Group 2 metal sulfate, Comparative Example 6 was a system using only an alkali metal phosphate, and Comparative Example 7 was only an alkali metal hydroxide. The system was implemented. In any of the comparative examples, the average particle size was as small as less than 0.5 μm, and the monodispersity of the particle size distribution was poor. In Comparative Example 2 and Comparative Example 5, aggregates were generated, and dispersed latex particles could not be obtained. In Comparative Example 3, although the Group 2 metal sulfate was used, the Group 2 metal could not be detected in the latex particles.

(Examples 5 and 6)
A polymerization reaction was carried out in the same manner as in Example 2 except that the type of salts used in Example 2 was changed, and Examples 5 and 6 were obtained. Table 1 shows the type, concentration, average particle diameter value, CV value of the particle diameter value, and Group 2 metal content used. Example 5 was carried out by changing the alkali metal phosphate and alkali metal hydroxide from Example 2, but latex particles having good monodispersity of the particle size distribution were obtained. Further, Example 6 was carried out by changing the group 2 metal sulfate from Example 2, but latex particles having good monodispersity of the particle size distribution were obtained.

(Comparative Examples 8-11)
A polymerization reaction was carried out in the same manner as in Example 2 except that the Group 2 metal sulfate used in Example 2 was changed to a salt other than the salts used in the present invention to obtain Comparative Examples 8 to 11. Table 1 shows the type, concentration, average particle diameter value, CV value of the particle diameter value, and Group 2 metal content used. In Comparative Examples 8 to 10, the average particle size was as small as less than 0.5 μm, and the monodispersity of the particle size distribution was poor. Further, in Comparative Example 11, latex particles in which aggregates were generated and the particles were dispersed were not obtained. In Comparative Example 9 and Comparative Example 10, a Group 2 metal could not be detected in the latex particles even though a Group 2 metal salt was used.

(Comparative Examples 12-13)
A polymerization reaction was carried out in the same manner as in Example 2 except that the same salt as in Example 2 was used and the concentration was changed, and Comparative Example 12 and Comparative Example 13 were obtained. Table 1 shows the type, concentration, average particle diameter value, CV value of the particle diameter value, and Group 2 metal content used. In Comparative Example 12 and Comparative Example 13, the content of magnesium in the latex particles was outside the specified range of the present invention, and the monodispersity of the particle size distribution was poor.

(Comparative Examples 14-15)
Comparative Example 14 and Comparative Example 15 were carried out by carrying out the polymerization reaction in the same manner as in Example 2 except that magnesium hydroxide and magnesium oxide were used as the salts and the concentration was changed. Table 1 shows the type, concentration, average particle diameter value, CV value of the particle diameter value, and Group 2 metal content used. In Comparative Example 14 and Comparative Example 15, the average particle size value was 0.72 to 0.95 μm, but magnesium could not be detected in the latex particles, and the monodispersity of the particle size distribution was poor.

  From the above results, in order to obtain latex particles having an average particle size value of 0.5 μm to 1.0 μm and good monodispersity of the particle size distribution, the particles of Group 2 within the specified range of the present invention are contained in the particles. It has been confirmed that it contains a metal and that it is necessary to use three types of salts defined in the present invention.

(Evaluation 1)
In this evaluation, production reproducibility when repeated polymerization was performed under the same conditions was confirmed by comparing variations in the average particle size of the obtained latex particles. Polymerization was performed 10 times each under the conditions of Examples and Comparative Examples except Comparative Examples 2, 5, and 11 where latex particles were not obtained, and the average particle size was measured by the method of Example 1. Table 2 shows the average value of the average particle diameter values (n = 10) and the CV value of the average particle diameter values in 10 polymerization reactions. In the examples, the CV value representing the degree of variation in the average particle size value was as good as 5% or less, whereas in the comparative examples, the CV value was large and the polymerization reproducibility was poor.

(Evaluation 2)
In this evaluation, evaluation was performed when the latex particles obtained in Example 1, Example 2, Comparative Example 1, Comparative Example 4, Comparative Example 12, and Comparative Example 13 were used as a diagnostic reagent carrier.
(1) Preparation of latex particles sensitized with HBs antigen Each latex particle (solid) in Examples and Comparative Examples was added to 7.5 mL of 36 mmol / L phosphate buffer (pH 6.6) in which 2.0 mg / mL of HBs antigen was dissolved. 1 mL each of 10% (W / V) was added and stirred at 30 ° C. for 60 minutes. To this buffer solution, 8.5 mL of 100 mmol / L phosphate buffer solution (pH 8.0) containing 1.0% by weight of bovine serum albumin (hereinafter “BSA”) was added and stirred at 30 ° C. for 60 minutes. After stirring, the mixture was centrifuged at 18000 rpm for 20 minutes at 4 ° C. To the resulting precipitate, 20 mL of a 100 mmol / L phosphate buffer (pH 8.0) containing 0.5% by weight of BSA was added. The obtained mixture was incubated at 30 ° C. for 7 days to prepare HBs antigen-sensitized latex particles. This was used as the first reagent.

(2) Preparation of Sample Dilution Solution A 50 mmol / L phosphate buffer solution (pH 7.0) containing 1.0% by weight of BSA and polyethylene glycol having an average molecular weight of 500,000 to a concentration of 1.0% by weight. It dissolved so that it might become. This was used as the second reagent.

(3) Preparation of calibration curve Human serum containing HBs antibody at each concentration of 0, 150, 300, 600, 1200 mIU / mL was used as a standard solution for preparing a calibration curve. 20 μL of each standard solution and 120 μL of the second reagent were mixed and heated at 37 ° C., and then 120 μL of the first reagent was added and stirred. Absorbance at 750 nm was measured 1 minute and 5 minutes after the addition of the first reagent, and the absorbance difference (ΔAbs) between the two was measured with an automatic analyzer (automatic analyzer 7170 manufactured by Hitachi, Ltd.). The obtained calibration curve is shown in FIG.

(4) Reproducibility at the time of sample measurement In place of 20 μL of the standard solution in (3) above, except that 20 μL of sample was used, the same operation as in (3) above was performed, and the obtained ΔAbs was extrapolated to the calibration curve. The amount of HBs antibody in the sample was calculated. Table 3 shows the reproducibility (CV value) of measured values when 10 types of HBs antibody-positive sera (positive specimens) were measured 10 times each. When the latex particles of Examples 1 and 2 were used, the CV value was 5% or less, indicating good reproducibility. When the latex particles of the comparative example were used, the CV value was 6% or more and the reproducibility was poor. When the latex particles of the comparative example were used, it was possible to measure the standard solution and the specimen, but it was confirmed that the CV value in the reproducibility test was significantly low. In addition, when the latex particles of other Examples 3 to 6 were used, the CV value was similarly 5% or less, showing good reproducibility.

  According to the present invention, it is possible to provide monodisperse latex particles for a diagnostic reagent having an average particle size of 0.5 to 1.0 μm. In addition, according to the method of the present invention, soap-free polymerization of an aromatic vinyl monomer is performed in a dispersion medium in which specific salts are dissolved, so that simple operations can be performed without impairing monodispersity of the particle size distribution. In addition, latex particles for diagnostic reagents of the present invention can be produced with good reproducibility.

Claims (5)

  Diagnostic reagent latex particles comprising an aromatic vinyl polymer containing a Group 2 metal, wherein the content of the Group 2 metal in the latex particles is 0.005 to 0.05% by weight. Latex particles for diagnostic reagents.   The latex particle for diagnostic reagent according to claim 1, wherein the Group 2 metal is magnesium.   The latex particle for diagnostic reagent according to claim 1 or 2, wherein the average particle size of the latex particle for diagnostic reagent is 0.5 to 1.0 µm.   4. The soap-free polymerization of an aromatic vinyl monomer in the presence of a Group 2 metal sulfate, an alkali metal phosphate, and an alkali metal hydroxide. A method for producing latex particles for diagnostic reagents according to claim 1. Step 1 of dissolving Group 2 metal sulfate, alkali metal phosphate and alkali metal hydroxide in dispersion medium, Step 2 of adding aromatic vinyl monomer to dispersion medium, polymerization initiator Step 3 of performing soap-free polymerization in the presence of
The manufacturing method of the latex particle for diagnostic reagents in any one of Claims 1-3 containing these.

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