JPH0212484B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field) The present invention relates to a latex useful as an immunoserological diagnostic reagent. (Prior art) With the progress of immunoserology, the technology in clinical testing has improved tremendously. Immune serology tests are performed in test tubes, and volumetric pipettes are used for serum dilution. The complexity of such work using test tubes and pipettes and the processing of a large amount of specimens accompanying an increase in the number of tests are also the causes of low accuracy in immunoserological tests. However, with the introduction of automation in clinical testing, a micro-volume test method (microtiter method) has been implemented that reduces the amount of blood collected from patients and obtains accurate test data with a small amount of test material in the field of immunoserological tests. It has come to be. This microtiter method was devised by Hungarian Takatsy in 1955, and further developed in the United States in 1962.
Improved by Sever. Since the microtiter kit went on the market in the United States in 1963, it has been adopted for immunoserological testing and is used all over the world. 1967, American CDC
(Center for Disease Control) has adopted it as the standard test for complement fixation. Public
The microtiter method is used in the Health Service's training manual. In Japan, it was introduced relatively quickly by virology researchers, and imported products are used for virus serological tests (hemagglutination reaction, hemagglutination inhibition reaction, complement fixation reaction, etc.) and cell/tissue testing. Cultivation is being carried out. The characteristics of these microtiter methods are:
It is possible to perform multiple tests with a small amount of serum; it is easy to operate and can quickly dilute a large number of samples in a short period of time; it is economical because it requires a smaller amount of antigens, antisera, reagents, etc. than the current method; 1
The overall reaction can be seen on a single plate, and the agglutination image and hemolysis are clear and easy to judge; the sensitivity and accuracy of the reaction is unchanged from the current method, and the reproducibility is also good. The main blood cells used in these microtiter methods are sheep red blood cells and chicken blood cells. When these animal blood cells are used, the blood cells undergo severe decomposition and degeneration, resulting in a lack of long-term storage, and due to large individual differences, test values vary widely, making it difficult to obtain accurate data. There is a problem. Moreover, since the blood cells themselves contain antigens, these react with various antibodies in the serum being tested, which tends to cause misleading reactions. In recent years, synthetic latex has been used as a substitute for these blood cells. For example, JP-A-51-9716 discloses a synthetic latex reagent. This synthetic latex is manufactured using a surfactant (emulsifier).
Other synthetic latex reagents using surfactants are also known. The manufacturing method involves, for example, mixing anionic, nonionic, or cationic emulsifiers in water, coexisting styrene monomer, water-soluble radical initiators, etc., and heating the mixture to an appropriate temperature, preferably in an atmosphere excluding oxygen. It's about holding it for an appropriate amount of time. Generally, in these synthesized latexes, a part of the emulsifier used during polymerization is adsorbed or chemically bonded to the surface of polystyrene latex particles, and the rest exists in the latex in a free state. Between these states, an equilibrium of adsorption and desorption of the emulsifier to the surface of the polystyrene latex particles is established. In polystyrene latex manufactured by conventional methods, emulsifiers are essential for the formation of stable latexes, but on the other hand, free emulsifiers are unfavorable for the agglutination reaction of latexes caused by antigens or antibodies. influence To produce diagnostic reagents,
First, polystyrene latex is sensitized with an antigen or antibody as described above. However, if a latex containing free emulsifier is used, it will already coagulate at this stage. Next, when detecting an antibody or antigen that reacts with the antigen or antibody using latex sensitized with the antigen or antibody by a latex agglutination reaction, the serum containing the antibody or antigen to be detected is The sensitized latex will agglutinate on contact, and it is essential that the sensitized latex does not agglutinate on contact with serum that does not contain such antibodies or antigens. However, in the case of sensitized latexes containing these free emulsifiers, they often aggregate even when they come into contact with negative serum, resulting in a so-called non-specific agglutination reaction. Although it is possible to remove these emulsifiers from the latex using ion exchange or dialysis techniques, if these free emulsifiers are removed from the latex, as mentioned above, the free emulsifiers and the latex particles will be adsorbed onto the surface of the latex particles. The stability of the latex due to the establishment of adsorption/desorption equilibrium with the emulsifier is disrupted, and the stability of the latex becomes extremely poor, making it practically unusable. JP-A-57-14610 discloses a latex obtained by polymerizing styrene in water using a persulfate as a polymerization initiator in the absence of an emulsifier and then heating the polymer under alkaline conditions. When the latex thus obtained is made into a reagent, no self-aggregation reaction or non-specific agglutination reaction occurs because no emulsifier is used. It also has excellent latex stability. However, the specific gravity of such latex is 1.03
It is as small as ~1.05, which is comparable to polystyrene. Therefore, it takes a long time to convert latex into a reagent and examine its agglutination with a specimen. In order to obtain a latex with a high specific gravity, polymerization or copolymerization of vinyl toluene, chlorostyrene, methyl methacrylate, vinyl chloride, vinylidene chloride, etc., has also been carried out. but,
For example, chlorostyrene is highly reactive, making it difficult to control the polymerization reaction. The resulting latex also has an unpleasant odor due to the presence of residual monomers. (Object of the Invention) An object of the present invention is to provide a latex for diagnostic reagents that is free from lot-to-lot variation and can be stably maintained for a long period of time. Another object of the present invention is to provide a soap-free latex that is low in self-aggregation and non-specific aggregation and can be easily evaluated by anyone in a short period of time, and is suitable for the microtiter method. Still another object of the present invention is to provide a latex from which test values can be obtained with high precision. Yet another object of the present invention is to provide a latex of desired particle size and specific gravity. (Structure of the Invention) The latex for diagnostic reagents of the present invention is produced by polymerizing styrene under weakly alkaline conditions using persulfate as an initiator in the absence of an emulsifier to obtain a suspension of polystyrene particles. Polystyrene particles containing crosslinked polystyrene are obtained by heat treatment under neutral to acidic conditions, and polystyrene particles containing crosslinked polystyrene are then obtained by chlorination treatment, thereby achieving the above object. Examples of persulfates used as initiators in the present invention include ammonium persulfate, potassium persulfate, and sodium persulfate. The ratio of these persulfates to styrene is 8% by weight.
The content is preferably 0.09 to 6% by weight, more preferably 0.1 to 5% by weight. In the present invention, styrene and an initiator are added to a reactor filled with water and heated under weakly alkaline conditions while stirring to carry out a polymerization reaction to obtain a polystyrene suspension. The temperature of the polymerization reaction is 50-100°C, preferably 60-85°C. The reaction time varies depending on the concentration of styrene, type and concentration of initiator. The reaction time is usually 5 to 35 hours, and the reaction time is 7.1 to 7.1.
7.8 is preferred. In order to set the pH during the reaction within this range, alkaline earth metal hydroxides, oxides, carbonates, bicarbonates, etc. are added to the reaction solution. For example, sodium hydroxide, potassium hydroxide, sodium carbonate, etc. are used. The polystyrene suspension thus obtained is then heated under neutral or acidic conditions. The reaction conditions vary depending on the desired degree of crosslinking of polystyrene, but the reaction temperature is usually 50 to 100°C, preferably
60-85℃; reaction time is 5-30 hours; and pH is
2.4-7.0, preferably PH3.0-6.8. When heated under neutral or acidic conditions, a partial crosslinking reaction occurs between polystyrenes. The resulting polystyrene containing crosslinked polystyrene is then subjected to chlorination treatment. The temperature at that time varies depending on the amount of suspension charged and processing conditions, but is usually 5 to 65℃.
The temperature is preferably 10 to 60°C. The final amount of chlorination varies depending on the degree of crosslinking and particle size of the latex, but is usually 5 to 40%, preferably 10 to 30%. The chlorination treatment time is 10 to 500 minutes, preferably 30 to 400 minutes, and more preferably 60 to 360 minutes. The specific gravity of the latex can be determined in the range of 1.06 to 1.50, preferably 1.10 to 1.50, by appropriately selecting the chlorination conditions.
is adjusted to the range of If excessive chlorination occurs outside the above treatment conditions,
The surface of the resulting latex is severely damaged and causes self-agglomeration. Even if the treatment is successful, the specific gravity of the latex itself is too high, and when evaluated using the microtiter method, it settles too quickly, resulting in ±~
All aggregation near + is reversed, making it impossible to obtain accurate data. In the present invention, polystyrene having a partially crosslinked structure as described above is subjected to chlorination treatment.
Since such polystyrene has a high charge density on the particle surface, the reaction can be easily controlled when performing the chlorination reaction. Therefore, a latex with a desired specific gravity can be easily obtained. There is no possibility of excessive chlorination resulting in damage to the latex surface. Since the obtained latex has a partially crosslinked structure, the surface of the latex will not be damaged or destroyed during freezing or redispersion even if it is freeze-dried. Therefore, self-aggregation and non-specific agglutination reactions do not occur. For example, latex reagents prepared using streptococcus, which are unstable and whose titer tends to drop easily, can be stored for a long period of time by freeze-drying. According to the present invention, by controlling the reaction conditions of polymerization and chlorination, it is possible to obtain a latex with uniform particle size and desired specific gravity. The latex thus obtained is particularly suitable as a latex for use in the microtiter method since it does not cause non-specific agglutination reactions. (Example) The present invention will be described below with reference to Examples. Example 1 (A) Preparation of latex: 90 g of styrene monomer,
Potassium persulfate 0.48g and ion exchange water 470g
g was charged into a reaction vessel. Then, homopolymerization was carried out for 24 hours while purging the container with nitrogen gas and controlling the reaction temperature to fall within the range of 70°C to 72°C. The pH at this time was 7.5. After the polymerization was completed, the pH of the polystyrene suspension was adjusted to 6.3. This was then reacted at 70°C for 24 hours while maintaining the pH at 6.3. The reactor was stopped, and the polystyrene latex containing crosslinked polystyrene was taken out from the container and purified by filtration using Toyo Roshi No. 2 (diameter 12.5 cm). Then 70℃
When this latex was dried using a dryer and the purified solid content obtained was weighed, it was found to be 12.6 (W/
W)%. Next, add 2000 water to 3 reaction vessels.
After adding 500 g of purified latex diluted to 7.0% with distilled water, the reaction temperature was maintained at 13 to 15 °C under natural light.
Chlorination treatment was carried out by blowing chlorine gas for 270 minutes.
After replacing the inside of the container with nitrogen gas for 180 minutes, take out the obtained chlorinated latex and use Toyo Roshi No.
2 was used for filtration and purification. This latex was treated with an ultrasonic generator for 30 seconds and dispersed in water. Furthermore, the latex was purified with distilled water using a dialysis membrane for 24 hours. The amount of chlorine in the latex after chlorination treatment was 14.7%. As a result of observing the latex after chlorination using an electron microscope, the average particle size was 0.48 μm, and the variation in particle size was 0.005 expressed as a coefficient of variation (standard deviation of particle size/average particle size). The specific gravity of this latex was 1.31. A portion of the polystyrene latex containing crosslinked polystyrene before chlorination treatment was taken out into a beaker, weighed, and dried at 70°C for 5 hours. This was placed in a test tube, methyl ethyl ketone (MEK) was added, and the test tube was sealed. This was heated to 90°C in a silicon bath and extracted for 48 hours. The MEK-insoluble portion was taken out and dried at 70°C for 8 hours, and its weight was measured. The gel fraction is
It was 8.1%. (B) Latex evaluation by R-PHA method agglutination reaction: The polystyrene latex obtained in section (A) was
1 volume of 1% solids dispersed in phosphate buffer with pH 7.4 and HBs produced by guinea pigs.
Monospecific antibody (Sepharose 4B
HBs antigen purified by affinity chromatography, which was passed twice through a column of immobilized HBs antigen) was dissolved in phosphate buffer at a concentration of 40 μg/ml. The antibody was allowed to bind to the latex by incubating for a minute. Next, apply this sensitized latex to
Centrifugation was performed at 15,000 rpm for 15 minutes to remove unadsorbed antibodies. The antibody titer in this supernatant was measured by PHA (passive hemagglutination) method, and it was found that at least 99.5% of the antibody was adsorbed to the latex. This settled latex
Centrifuge at 18,000 rpm for 8 minutes, discard the supernatant, and collect the precipitated sensitized latex at pH 7.0.
The preparation of the latex reagent was completed by redispersing it in phosphate buffer. The R-PHA (reverse passive hemagglutination) test method was attempted using the latex thus prepared and replacing the sensitized red blood cells of the currently commercially available River Cell (manufactured by Yamanouchi Pharmaceutical) with the latex. Using a microdropper in a microtiter, 50μ of the buffer solution was dispensed into each tube. And 1μ
A sample of 50 μg containing HBs antigen was taken out and immediately diluted several times with a diluter. Then, the latex obtained by the above method was dispensed into each 50μ tube, and then shaken with a mixer for 30 seconds.
This was left standing for 270 minutes and 420 minutes, and then the agglutination image was determined. For comparison, instead of latex, R-PHA cells, in which anti-HBs antibodies were adsorbed to sheep red blood cells, were used at the same time and evaluated in comparison with latex agglutination. The results are shown in Tables 1 and 2. Tables 1 and 2 show the results of agglutination images after standing for 270 minutes and 420 minutes, respectively. The meanings of the symbols in the table below are as follows. -: No aggregation is observed ±: Slow aggregation is observed +: Clear aggregation is observed: Complete aggregation is significantly observed Example 2 (A) Preparation of latex: 75 g of styrene monomer,
Potassium persulfate 0.40g and ion exchange water 450g
g was charged into a reaction vessel. Then, homopolymerization was carried out for 30 hours while purging the container with nitrogen gas and controlling the reaction temperature to fall within the range of 70°C to 72°C. The pH at this time was 7.3. After the polymerization was completed, the pH of the latex suspension was adjusted to 6.0. This was then reacted at 70°C for 28 hours while maintaining the pH at 6.0. The reactor was stopped, and the polystyrene latex containing crosslinked polystyrene was taken out from the container and purified by filtration using Toyo Roshi No. 2. Next, this latex was dried using a 70°C dryer, and the purified solid content obtained was weighed and found to be 12.3 (W/W)%.
Next, add 1800 g of water to 3 reaction containers and add latex diluted purified latex to 10.0% with distilled water.
After adding 400 g of the reactor, chlorine gas was blown into the reactor for 160 minutes under a 100 W mercury lamp while maintaining the reaction temperature at 25 to 27°C. After purging the inside of the container with nitrogen gas for 140 minutes, the obtained chlorinated latex was taken out and filtered and purified using Toyo Roshi No. 2. This latex was treated with an ultrasonic generator for 1 minute and dispersed in water. Furthermore, the latex was purified with distilled water using a dialysis membrane for 24 hours. The amount of chlorine in the latex after chlorination treatment was 25.5% as a result of HCl analysis in the reaction solution. As a result of observing the latex after chlorination using an electron microscope, the average particle size was 0.50 μm, and the variation in particle size was 0.008 expressed as a coefficient of variation (standard deviation of particle size/average particle size). The specific gravity of this latex was 1.43. A portion of the polystyrene latex containing crosslinked polystyrene before chlorination treatment was taken out into a beaker, weighed, and dried at 70°C for 5 hours. This was placed in a test tube, MEK was added, and the test tube was sealed. This was heated to 90°C in a silicon bath and extracted for 48 hours. Take out the MEK insoluble part and heat it at 70℃.
It was dried for an hour and its weight was measured. The gel fraction is
It was 7.02%. (B) Evaluation of latex by R-PHA method agglutination reaction: Using the polystyrene latex obtained in Section (A) of this Example, the latex was evaluated according to the method in Section (B) of Example 1. The results are shown in Tables 1 and 2.

【table】

【table】

[Table] From the above test results, the latex according to the present invention has the following properties:
It can be seen that because the composition has a partially crosslinked structure, there is little self-aggregation and non-specific aggregation, and it has excellent storage stability and no variation between lots. Because the particles are well-aligned and have a relatively large specific gravity, anyone can easily evaluate aggregation reactions in a short time. Furthermore, it is clear that the latex has little effect on test values and is suitable for microtiter methods and the like. Comparative Example (A) Preparation of latex: 90 g of styrene monomer,
Charge 2 g of nonionic emulsifier (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., Emuljit 49), 0.6 g of potassium persulfate, and 450 g of ion-exchanged water into a reaction container, and replace the container with nitrogen gas to keep the reaction temperature within the range of 70 to 72 °C. Polymerization was carried out for 24 hours under controlled conditions. Observation of the obtained latex with an electron microscope revealed that the average particle size was 0.75 μm and the variation in particle size was 0.127 expressed as a coefficient of variation. The specific gravity of this latex was 1.04. (B) Latex evaluation by R-PHA method agglutination reaction: The polystyrene latex obtained in section (A) was
1 volume of 1% solids dispersed in phosphate buffer with pH 7.4 and HBs produced by guinea pigs.
Monospecific antibody (Sepharose 4B
HBs antigen purified by affinity chromatography, which was passed twice through a column of immobilized HBs antigen) was dissolved in phosphate buffer at a concentration of 40 μg/ml. The antibody was allowed to bind to the latex by incubating for a minute. Next, apply this sensitized latex to
Centrifugation was performed at 15,000 rpm for 15 minutes to remove unadsorbed antibodies. This settled latex
Centrifuge at 15,000 rpm for 15 minutes, discard the supernatant, and collect the precipitated sensitized latex at pH 7.0.
The preparation of the latex reagent was completed by redispersing it in phosphate buffer. The latex prepared in this way was used to detect HBs antigen (HBs antigen detection).
R- using EIA Kit (manufactured by Yamanouchi Pharmaceutical)
We tried the PHA test method. The test method is the same as in Example 1. Judgments after 270 minutes and 420 minutes showed no change in the agglutination image compared to before standing. A latex reagent was prepared according to Example 1 using the latex obtained in Comparative Example (A), and the agglutination strength of human serum containing various concentrations of HBs antigen was measured using this reagent. The latex reagent used was 25μ, and the human serum sample containing HBs antigen was 100μ. The results are shown in Table 3.

[Table] Next, using Reverseaia (HBs antigen detection EIA kit manufactured by Yamanouchi Pharmaceutical Co., Ltd.), the HBs antigen in serum was found to be 0.4 ng/ml or less158
A similar test was performed on normal human serum. Of the 158 samples, 20 were positive and 27 were false positive. From the above test results, it is clear that the latex prepared as a reagent using the latex of the comparative example causes a non-specific agglutination reaction. (Effects of the Invention) As described above, according to the present invention, a latex is produced which is stable at all times, is sensitive to antigen-antibody reactions, and exhibits high aggregation properties, even though it does not contain any emulsifier. Moreover, this latex has a well-uniformed particle size. Since this latex does not contain any emulsifier, when used as an immunoserological diagnostic reagent, there is no variation in test values due to non-specific agglutination reactions. By controlling the reaction conditions during the preparation of the latex, a latex with a desired particle size and specific gravity can be obtained. Since a portion of latex has a crosslinked structure, the surface of latex is not damaged or destroyed even when stored by freeze-drying. Therefore, even unstable latex reagents can be stored for long periods of time by freeze-drying. The latex according to the present invention can be used effectively in microtiter methods and the like.

Claims (1)

[Claims] 1. Polymerize styrene under weakly alkaline conditions using persulfate as an initiator in the absence of an emulsifier to obtain a suspension of polystyrene particles, and then polymerize the suspension under neutral to acidic conditions. A latex for diagnostic reagents obtained by heat-treating polystyrene particles containing cross-linked polystyrene, and then chlorinating the polystyrene particles containing cross-linked polystyrene. 2 The polymerization reaction under the weakly alkaline conditions
7.1 to 7.8, latex according to claim 1, which is carried out at 50 to 100°C for 5 to 35 hours. 3. The latex according to claim 1, wherein the heat treatment is performed at a pH of 2.4 to 7.0 and a temperature of 50 to 100°C for 5 to 30 hours. 4. The latex according to claim 1, wherein the chlorination treatment is performed at 10 to 65°C for 0.3 to 50.0 hours. 5 Claim 1 whose specific gravity is 1.10 to 1.50
Latex as described in Section. 6. The latex according to claim 1, having a particle size of 0.1 to 10.0 μm.