JPS6329249A - Latex reagent - Google Patents

Abstract

PURPOSE:To obtain a reagent having excellent preservable stability by forming a latex reagent constituted by sensitizing an immune material to latex particles in such a manner said reagent has the average particle size of a prescribed range and that the ratio between the average particle size thereof and the average particle size of the latex particles attain the value of a specified range. CONSTITUTION:The average particle size of the latex particles to be used is not particularly limited and the latex particles having the average particle size ranging 0.05-0.38, more preferably 0.07-0.3mu are particularly adequate; further, the reproducibility is better with the particles having the smaller dispersion value of the particle sizes. The latex reagent is obtd. by sensitizing the immune active material with the latex particles. This reagent is required to have the average particle size D ranging; for example, 0.1-0.5mum. The ratio D/d of the average D of the particle of the reagent to the average particle size (d) of the latex particles is required to be in; for example, 1.3-3.0 range. The reagent has the high preservable stability but hardly detects the antigen of a low concn. if D/d is <1.3. Large agglutinated particles are generated at the time of sensitization and the agglutination arises non-specifically at the time of preservation when D/d exceeds 3.0.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a latex reagent with excellent storage stability.

[Problems to be solved by the prior art and the invention] In immunological tests that utilize antigen-antibody reactions, agglutination reactions are used together with precipitation reactions and complement fixation reactions, or because they are much simpler and more sensitive than these reactions. It is used as a reaction.

The agglutination reaction, along with the reaction to detect antigens localized on the surface of free cells and bacterial membranes, has enabled highly specific antiserum to be obtained due to advances in antigen/antibody purification technology.
Highly specific antibodies to blood cell particles and hentonite particles.

Scope of application in clinical testing, such as testing antigen temperature by immobilizing on a particle carrier such as kaolin particles or latex particles, reacting with the corresponding antigen to cause agglutination, and measuring the agglutination directly or indirectly. is expanding significantly.

Moreover, in recent years, with the advancement of antigen purification technology, the development of highly specific antibodies, and furthermore, the development of quantitative analysis technology, it has become highly sensitive, does not cause non-specific agglutination reactions, and has excellent storage stability. There is a demand for the development of diagnostic reagents with properties such as:

However, the demand for improving the stability of diagnostic reagents during storage while retaining the rapidity and sensitivity of immunoagglutination reactions is a desire to satisfy both mutually contradictory properties. is extremely difficult. That is,
Diagnostic reagents with excessive colloidal chemical stability have excellent storage stability, but many of them do not have sufficient rapidity and sensitivity for immunological agglutination reactions (and, conversely, If an attempt is made to improve the rapidity and sensitivity of the agglutination reaction, it will tend to spontaneously aggregate non-specifically and have poor storage stability, making it unusable as a diagnostic reagent.For this reason, currently known diagnostic reagents cannot be used. It can be said that one of the above properties is sacrificed.

[Means for solving problems]

The present inventors have conducted extensive and systematic research in order to develop a reagent that has excellent storage stability and is rapid and sensitive for immunological agglutination reactions. In addition to these results, we have also discovered that these contradictory properties can be achieved by controlling the particle size of the latex particles and the particle size of the latex reagent that sensitizes the latex particles with an immunoactive substance, and have completed the present invention. And I have come to propose it here.

That is, the present invention provides a latex reagent having an average particle diameter (D) of 0.1 to 0.5 μm, which is obtained by sensitizing latex particles with an immunologically active substance, and the latex reagent has an average particle diameter (D) of 0.1 to 0.5 μm. The latex reagent is characterized in that the ratio (D/d) of the average particle diameter (D) of the latex reagent is in the range 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 (1): , an acyloxy group, an alkoxy group, or a cyano group.

Here, substituents for the phenyl group are not particularly limited, but include halogen atoms, haloalkyl groups, alkyl groups, and the like. Among such hydrophobic vinyl monomer units, hydrophobic vinyl monomer units in which R2 is a substituted or unsubstituted phenyl group, chlorine atom, or alkoxycarbonyl group are preferred. Examples of monomers providing such monomer units include styrene, vinyltoluene, chloromethylstyrene, chlorostyrene, vinyl chloride, vinyl bromide, methyl methacrylate, ethyl methacrylate, propyl methacrylate, and 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 employed. In addition, in the present invention, latex particles made of a high molecular weight polymer have a monomer unit represented by the following formula (II) (wherein, R is a hydrogen atom or an alkyl group) on the surface. Suitably used. Examples of the monomer providing the monomer unit represented by formula (n) include glycidyl acrylate, glycidyl methacrylate, and the like.

Latex particles composed of only one monomer unit represented by the above formula (1) or (II),
Alternatively, latex particles composed of both the monomer units represented by the above formulas (1) and (II) are particularly preferably used in the present invention.

When the above latex particles are used as a raw material for an adsorption type latex reagent, the content of the monomer unit represented by the above formula (II) in the latex particles is 0 to 20 mol%, preferably 0.05 to 20 mol%. It is suitably 15 mol%. In addition, when the latex particles are used as a raw material for a covalently bonded latex reagent, the content of the monomer unit represented by the formula (II) in the latex particles is 20 to 20.
It is suitable to select from the range of 100 mol%, preferably from 30 to 99 mol%.

In addition, when latex particles are used as a raw material for a diagnostic latex reagent, the hydrophilic vinyl monomer unit may be added to the latex particles in an amount of 20 mol% or less within a range that does not adversely affect the properties of the latex reagent. It may be included inside. Examples of monomers providing hydrophilic vinyl monomer units include methacrylic acid, acrylic acid, styrene sulfonic acid, sodium styrene sulfonate, 2-hydroxyethyl (meth)acrylate, vinylpyrrolidone, and polyethylene glycol (meth)acrylate. Examples include acrylic esters. Furthermore, crosslinkable monomers such as divinylhenzene, ethylene glycol dimethacrylate, diethylene glycol methacrylate, and bisphenol A diglycidyl ether can also be suitably used if necessary.

The polymerization method for obtaining latex particles made of a high molecular weight polymer used in the present invention is not particularly limited, and known methods are preferably employed. 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, and a method of emulsion polymerization using a water-soluble radical initiator in an aqueous medium without using a surfactant. A method of heterogeneous polymerization using a chemical agent, a method of suspension polymerization in the presence of a protective colloid such as partially hydrogenated polyhinyl alcohol or polyvinylpyrrolidone, and an organic solvent that dissolves the vinyl monomer but not the polymer. A method such as precipitation polymerization in a medium is adopted.

The average particle diameter of the latex particles used in the present invention is not particularly limited, but those in the range of 0.05 to 0.38 microns, preferably 0.07 to 0.3 microns are generally suitably used. Furthermore, it is desirable for the latex particles to have a smaller particle diameter dispersion value because of better reproducibility. for example,
The particle size dispersion value is preferably 10% or less, more preferably 5% or less. The above-mentioned dispersion value is a value obtained by dividing the standard deviation by the average particle diameter and multiplying by 100, and is expressed in %.

The latex particles thus obtained are preferably used as a diagnostic reagent for immunological reactions, especially after being sensitized with an immunologically active substance to obtain a latex reagent. The immunologically active substance is not particularly limited, and may be any of various antigens, antibodies, etc. that can be used in, for example, agglutination reactions, agglutination inhibition reactions, and the like. To give a typical example,
For example, denatured gamma globulin, rheumatoid factor, antinuclear factor, human albumin, anti-human albumin antibody, immunoglobulin G (IgG), immunoglobulin A (Ig
A), immunoglobulin M (IgM), streptolysin O1 anti-streptolysin 0 antibody, C-reactive protein, anti-C-reactive protein antibody, alpha-fetoprotein (AFP), anti-AFP antibody, carcinoembryonic antigen (CEA) )
, anti-CEA antibody, human placental lufftogen (HPL), anti-H
PL antibody, human chorionic gonadotropin (HCG), anti-C
E antibody, anti-estrogen antibody, anti-insulin antibody, B
Hepatitis surface antigen (HBS), anti-HBS antibody, Tryponema pallidum antigen, rubella antigen, complement component C1Q, anti-complement component C
Known immunologically active substances such as No. 14 antibodies can be mentioned.

The latex reagent of the present invention can be obtained by sensitizing the latex particles with an immunoactive substance.

The latex particles generally have good dispersibility and non-aggregation, but when sensitized with an immunoactive substance, they become agglomerated.
The latex reagent exists as aggregated particles. The latex reagent provided by the present invention has a particle diameter (D) of 0.1 to
It needs to be in the range of 0.5 μm. Latex reagents with a particle size smaller than the above range are extremely stable and are insufficient in terms of rapidity and sensitivity required in immunological agglutination reactions. On the other hand, if the particle size of the latex reagent is larger than the above range, it is not preferable because it may have strong aggregation properties and may not be a stable reagent.

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 particles to the particle diameter (d) of the latex particles must be in the range of 1.3 to 3.0. There is. When the above (D/d) is less than 1.3, it has high storage stability, but it becomes difficult to detect antigens or antibodies at low concentrations.

That is, the electrostatic repulsion between latex particles sensitized with the corresponding antigen or antibody is too strong to allow them to aggregate in the presence of a low concentration of antigen or antibody. In addition, the above (D
/d) exceeding 3.0 indicates that large aggregates are formed during sensitization, which causes nonspecific aggregation during storage. Furthermore, since the final aggregate size in the presence of antigen or antibody is fixed, it becomes difficult to detect high concentrations of antigen or antibody.

Although the method for producing the latex reagent of the present invention is not particularly limited, the properties can generally be selected depending on the particle size of the latex used as a raw material and the conditions for sensitizing the 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 average particle diameter (D) of the latex reagent is D/d=1.3 to 3.
0, preferably in the range of 1.5 to 2.5, the following method is suitable, for example. For example, by inserting a motorized stirring rod into an aqueous suspension of latex particles and stirring the suspension, the immunoactive substance-containing solution is quickly added.
Continue stirring. 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, but is preferably 100 to 500 revolutions/minute, preferably 300 to 400 revolutions/minute. In addition, in the case of the above-mentioned shaking, 25 to 250 times/min, preferably 50 to 150 times/min is suitably used. The sensitization treatment is generally preferably carried out at a pH of about 6.0-8.6 and a temperature of about 4-37°C. Furthermore, it is generally advisable to use the immunoactive substance in an amount slightly in excess of the saturated adsorption amount of the latex particles, and remove the excess amount by centrifugation or the like after sensitization. In general, the immunologically active substance is preferably selected such that the ratio of antigen or antibody (■)/latex particle surface area (rd) is in the range of 0.05 to 10 ■/tolerance.

The latex reagent obtained as described above is washed with an aqueous solvent if necessary, and then generally subjected to an immunological agglutination reaction while suspended in an aqueous solvent. Generally, the concentration of latex reagent when suspended in an aqueous solvent is 0.01 to 0.
．． It is preferable to select from the range of 1% by weight.

In addition to the properties described above, the latex reagent of the present invention has a zeta potential of 120 mV to 10 mV, preferably 110 mV to On+ when suspended in the aqueous solvent.
V range, and the absorbance is 0.5 in a cell with an optical path length of 10 m.
It is most preferable to prepare, for example, by the method described above, so that the molecular weight is in the range of 1.5 to 1.5. These properties are particularly suitable for obtaining a latex reagent with good sensitivity.

[For production]

The latex reagent of the present invention having the above-mentioned characteristics generally has extremely high storage stability as it has an average of 2 to 3 latex particles agglomerated, but it also has high sensitivity and rapidity due to moderate aggregation. are doing.

〔effect〕

The latex reagent of the present invention is extremely stable in an aqueous medium, and has extremely high value as it provides a latex reagent that not only has excellent storage stability but also has a wide measurement range.

The following examples demonstrate 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 diameter of the latex reagent in the examples was measured using a laser rotation grading automatic surface charge spectrum analyzer LASERZEE"' Sy
It was determined using stem 3000 (manufactured by PEN KEM) (hereinafter abbreviated as particle analyzer).

Example 1 (1) Preparation of rheumatoid factor side “regular reagent” Polystyrene latex particles with an average particle diameter of 0.178 μm were mixed with 0.1M NaCl at pH 8.2 containing 120.05M.
A suspension having a latex concentration of 1% by weight is prepared by diluting it with a borate buffer solution (hereinafter abbreviated as BBS).

Then, the IgG heated at 60°C for 10 minutes was
Dilute with S and adjust to 1 μ/ml. 1 volume of heat-denatured human 1 gG was added to 1 volume of the above latex suspension while stirring at 350 rpm, and then left at room temperature for 2 hours while stirring at the same speed. After centrifugation and removal of the supernatant, the precipitate was redispersed in BBS containing bovine serum albumin at a concentration of 0.05% by weight to give a latex concentration of 0.03%.
% by weight to obtain a rheumatoid factor measuring reagent. As a result of analysis using a particle analyzer, the average particle diameter of the latex reagent was 0.240 μm. Also LAS [!
The result of measuring the zeta potential with RZEE Model 501 (manufactured by PEN KEM) was -2.5 ± 0.2 mV (
BBS). The absorbance of latex reagent is 1°2
5 (optical path length 101 m, 580 nm).

(2) Measurement method Absorbance was measured using a Hitachi υ-3200 self-recording spectrophotometer equipped with a temperature controller and a magnetic stirrer attached to the photometry section. A cylindrical stirrer was placed in a glass optical cell with an optical path length of 10 mm, and then 1980μ of the rheumatoid factor measurement reagent obtained in (1) was added. was dispensed, inserted into the photometric section, and kept at 37°C.

Next, while stirring the reagent for measuring rheumatoid factor using the stirring device, 20 µm of the test liquid was added. was added.

Measurement of absorbance was started at the same time as the addition. Absorbance measurements were performed using a light beam with a wavelength of 580 hm. Note that stirring was stopped 3 seconds after the addition of the test liquid.

(3) Measurement of serum with known concentration The pooled serum of rheumatoid patients with a rheumatoid factor concentration of 16001 U/- was diluted with physiological saline to obtain a rheumatoid factor concentration of 5.10.20.40.80.100.200゜50.
Test liquids of 0 and 1000 mm/ml were obtained.

Under the measurement conditions of (2), the absorbance was measured five times for each of the nine test solutions and BBS.

Among the obtained absorbances, the difference in absorbance for a fixed interval of time was obtained from the absorbances 1 minute and 5 minutes after addition of the test liquid.

Further, the reagent obtained in (1) was stored at 4° C. for 12 months, and then the difference in absorbance was determined in the same manner. The results are shown in Table 1.

From the results in Table 1, 7 days after reagent preparation and 4°C.

The differences in absorbance after 12 months of storage are within the coefficient of variation for all measured concentrations.

The average particle size of the reagent after storage at 4°C for 12 months is 0.2
40 μm, the zeta potential was −2.3±0.2 mV, and the absorbance was 1.26.

(Leaving space below next summer) Table 1 (Notes 1 and 2) From the results of each five measurements, the average value, standard deviation, and coefficient of variation expressed as a percentage were calculated by dividing the standard deviation by the average value.

0 Main 1) Measurement utilization on the 7th day after reagent preparation, 0 Main 2) Measurement results after 12 months storage at 4°C after reagent preparation.

Comparative Example 1 Heat-denatured human IgG was added to 1 volume of the same latex suspension as used in Example 1. , 1 volume of the latex suspension was added while the latex suspension was still standing, and then left standing for 2 hours.

Thereafter, the same treatment as in Example 1 was carried out. As a result of analysis using a particle analyzer, the average particle size of the latex reagent was 0.
．． It was 720 μm and the absorbance was 2.35. The ratio of the average particle diameter (d) of the latex particles to the average particle diameter CD of the latex reagent was D/d = 4.0. Table 2 shows the results of measuring the pooled serum of rheumatoid arthritis patients with known concentrations in the same manner as in Example 1.

As is clear from Table 2, on the 7th day after reagent preparation and at 4℃, 1
The difference in absorbance after 2 months storage is different.

The average particle diameter after 12 months at 4℃ after reagent preparation was 0.931.
The absorbance in μm was 2.65, and nonspecific aggregation occurred during storage.

(Left space below the wooden page) Table 2 (Notes 1 and 2) From the results of each five measurements, the average value, standard deviation, and coefficient of variation expressed as a percentage were determined by dividing the standard deviation by the average value.

0 Main 1) Measurement result 0 main on 7th day after reagent preparation 2) Measurement result 0 main after storage at 4℃ for 12 days after reagent preparation 3)
BBS measurement results Comparative Example 2 1/20 volume of a nonionic surfactant (HLB = 15) was added to 1 volume of the same latex suspension as used in Example 1, and the mixture was allowed to stand at room temperature for 1 hour. . Then human IgG solution 1
quickly add the volume while stirring at 400 rpm,
Stirring was continued at 400 revolutions/minute. The following treatment was carried out in the same manner as in Example 1. As a result of analysis using a particle analyzer, the average particle diameter of the latex reagent was 0.196 μm, the zeta potential was 35 mV, and the absorbance was 0.47. The ratio of the average particle diameter (D) of the latex particles to the average particle diameter (D) of the latex reagent is D/d = 1.
It was 10.

Table 3 shows the results of measuring the pooled serum of patients with rheumatoid arthritis having known concentrations in the same manner as in Example 1.

From the results in Table 3, the storage stability is excellent, but there is a drawback that no aggregation occurs at 5 mm1/ml, which is required for clinical measurement. The average particle diameter after 12 months at 4°C after reagent preparation was 0.198 μm, and the surface charge density was -37 μm.
The absorbance was 0.48 at V.

(Margin below next summer) Table 3 (Note 1.2) From the results of each five measurements, the average value, standard deviation, and coefficient of variation expressed as a percentage were calculated by dividing the standard deviation by the average value.

0 main 1) Measurement results on the 7th day after reagent preparation 0 main 2) Measurement results after 12 months storage at 4°C after reagent preparation.

0 Main 3) Measurement results at BBS.

Example 2 Polystyrene latex particles with an average particle diameter of 0.134 μm were mixed with pi (0.1 of 8,2) containing 0.05 M of NaCe.
- dilute with glycine buffer (hereinafter abbreviated as CBS) to prepare a suspension having a latex concentration of 1% by weight. One volume of the heat-denatured IgG melt was added to one volume of the latex suspension in the same manner as in Example 1, and then left under stirring for 2 hours. Thereafter, the same treatment as in Example 1 was carried out. As a result of analysis using a particle analyzer, the average particle diameter of the latex reagent was 0.2.
old μm, and the zeta potential is -3,4 ± 0.2 mV (C
(in BS), and the absorbance was 1.03. In the same manner as in Example 1, pooled sera of rheumatoid arthritis patient sera with known concentrations were measured. The results are shown in Table 4.

From the results in Table 4, 7 days after reagent preparation and 4°C.

The differences in absorbance after 12 months of storage are within the coefficient of variation for all measured concentrations.

The average particle diameter of the reagent after storage at 4°C for 12 months was 0.
At 205 pm, the zeta potential was -3.3±0.1 mV and the absorbance was 1.06.

Table 4 (Notes 1 and 2) From the results of each of the five measurements, the average value, standard deviation, and coefficient of variation expressed as a percentage were determined by dividing the standard deviation by the average value.

0 Main 1) Measurement results on the 7th day after making the reagent Q Main 2) Measurement results after storing the reagent at 4°C for 12 months.

0 main 3) Measurement results with BBS, Example 3 +mm Preparation average diameter of C-reactive protein measurement reagent 0
．． A suspended S solution having a latex concentration of 1% by weight is prepared by diluting 123 μm polystyrene latex particles with an aluminum chloride-ammonia buffer (pH=8.0). Next, anti-CR was fractionated by salting out from anti-CRP serum obtained by immunizing a goat with C-reactive protein (hereinafter abbreviated as CRP).
The goat IgG fraction is diluted with ammonium chloride-ammonia buffer (pH-8,0) to prepare a solution with a protein concentration of 2/ml. Add 1 volume of anti-CRP goat IgG fraction solution to 1 volume of the above latex suspension in the same manner as in Example 1.
The reaction was carried out at 7°C for 2 hours. After centrifugation and removal of the supernatant, the precipitate was added to an ammonium chloride-ammonia buffer containing bovine serum albumin at a concentration of 0.05% by weight.
Redisperse at pH = 8.0 > to reduce latex concentration to 0.
．． 05% by weight to obtain a CRP measurement reagent.

As a result of analysis using a particle analyzer, the average particle diameter of the latex reagent was 0.213 μm. The zeta potential was -2.0±0.2+mmV (in ammonium chloride-ammonia buffer) and the absorbance was 1.30.

(2) Measurement method Measurement was carried out 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 with known concentration A purified CRP solution with a CRP concentration of 240 mg/dl was
C that has undergone absorption treatment to become substantially free of CRP.
Diluted with RP-free serum to give a CRP concentration of 0.10.0
．． 25.0.50.1.0.2.5.5.0.10゜1
5.20.30.40.60 mg7dl of test liquid was obtained.

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

Among the obtained absorbances, the difference in absorbance for a fixed interval of time was obtained from the absorbances 1 minute and 5 minutes after addition of the test liquid. Further, the reagent obtained in (1) was stored at 4° C. for 12 months, and then the difference in absorbance was determined in the same manner.

The results are shown in Table 5.

Table 5 From the results in Table 5, 7 days after reagent preparation and 4°C.

The differences in absorbance after 12 months of storage are within the coefficient of variation for all measured concentrations.

The average particle diameter of the reagent after storage at 4°C for 12 months was 0.
At 214 pm, the zeta potential is -2.1 ± 0.3 mV,
The absorbance was 1.31.

Claims (1)

[Scope of Claims] 1. A latex reagent having an average particle diameter (D) of 0.1 to 0.5 μm, which is obtained by sensitizing latex particles with an immunoactive substance; A latex reagent characterized in that the ratio (D/d) of the average particle diameter (D) of the latex reagent to d) is in the range of 1.3 to 3.0. 2. The zeta potential of the latex reagent is -20mV to 10m
The latex reagent according to claim 1, which has a range of V. 3. The absorbance of the latex reagent is 0 in a cell with an optical path length of 10 mm.
．． The latex reagent according to claim 1, which has a molecular weight in the range of 5 to 1.5.