JP2020076692A - Manufacturing method of latex particle for measuring anti-streptolysin o - Google Patents

Abstract

To provide an anti-streptolysin O measurement reagent allowing a high sensitivity by using a small amount of streptolysin O.SOLUTION: A manufacturing method of a latex particle for measuring an anti-streptolysin O includes: using a latex particle having a carboxyl group on its surface; covalent-binding a streptolysin O to the latex particle; performing a blocking processing; and then performing cleaning process with a buffer solution including a surfactant, for example Tween20.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing latex particles used as an anti-streptolidine O measuring reagent (hereinafter sometimes referred to as “ASO reagent”). More specifically, the present invention relates to a method for producing latex particles for measuring anti-streptolysin O using a latex aggregation turbidimetric method using streptolidine O as a measurement principle.

  In order to produce a measurement reagent based on the latex aggregation turbidimetric method, it is necessary to sensitize latex particles with an antibody or an antigen that binds to a substance to be measured. For example, when the latex particles are sensitized with streptolysin O (hereinafter also referred to as “SLO”) produced outside the cells by Streptococcus pyogenes, the antibody titer against SLO (ASO titer) can be obtained. ) Can be measured, and SLO is widely used as a raw material of an ASO measuring reagent for diagnosing the presence or absence of infection with hemolytic streptococcus.

Conventionally, a physical adsorption method based on hydrophobic interaction between latex particles and SLO has been widely adopted, and the obtained complex of latex particles and SLO is a buffer solution in which an immunologically inactive protein such as BSA is dissolved. It was generally stored in the form of a reagent suspended in (Patent Document 1). However, in the reagent thus prepared, the reactivity of the reagent decreases due to self-aggregation of the carriers with each other or desorption of the sensitized SLO depending on the conditions for preparing the reagent. In some cases, the measurement performance could not be exhibited. Therefore, a method has been proposed in which the latex particles and SLO are covalently bonded to prevent the SLO from being desorbed and improve the stability of the reagent (Patent Documents 2 and 3).
However, since the boric acid compound contained in the borate buffer solution used in Patent Document 2 or Patent Document 3 is a substance of high concern listed in the list of substances subject to authorization under the European REACH regulation, the boric acid compound It was desired to develop a manufacturing method that does not use

On the other hand, unlike the physical adsorption method, in the preparation of a latex reagent by covalent bonding, the type of covalent bond, the type of latex particles, the conditions for sensitizing antibodies or antigens, the blocking conditions, the washing conditions, the final storage of latex particles There are many factors that affect the reagent performance such as the type of buffer solution, and it was very difficult to systematically evaluate those conditions and find the optimum conditions.

JP-A-5-203642 JP, 2003-344410, A Japanese Patent No. 4704662

  The present invention has been made in view of such a situation, and an object thereof is to provide an ASO reagent that can obtain high sensitivity with a small amount of SLO.

  The present inventors have conducted extensive studies to improve the stability of the measurement reagent, and as a result, the stability of the reagent can be improved by adding a surfactant, particularly preferably Tween 20 (polyoxyethylene sorbitan monolaurate), to the washing buffer after blocking. The inventors have found that it is improved and have completed the present invention.

That is, the present invention is exemplified by those listed below.
[Item 1]
Latex for measuring anti-streptolysin O including a step of washing with a buffer solution containing a surfactant after using latex particles having a carboxyl group on the surface thereof, covalently binding streptolysin O to the latex particles, and performing a blocking treatment. Method for producing particles.
[Item 2]
Item 2. The method for producing latex particles for measuring anti-streptolysin O according to Item 1, which uses at least one surfactant selected from the group consisting of Tween 20, Triton X-100, CHAPS, SDS, and deoxycholic acid.
[Item 3]
Item 3. The method for producing latex particles for measuring anti-streptolysin O according to Item 2, wherein the surfactant is Tween20.
[Item 4]
Item 4. The anti-streptolidine O measurement reagent according to any one of Items 1 to 3, wherein the concentration of the surfactant in the buffer is 0.01% to 0.1%.

  The method for producing latex particles used for the ASO reagent of the present invention is useful in that it enables highly sensitive measurement even when a small amount of SLO is used.

(SLO)
The SLO used in the present invention is not particularly limited, and examples thereof include gene-recombinant SLO produced by using SLO or recombinant E. coli produced by Streptococcus pyogenes extracellularly, A modified SLO in which a part of the amino acid sequence is deleted, or a fused SLO in which MBP or the like is fused may be used. For example, the SLO disclosed in WO2017 / 204325 is specifically exemplified.

(Latex particles)
The latex particles used in the present invention are not particularly limited, but for example, latex particles for a covalent bonding method in which a carboxyl group is introduced into polystyrene particles obtained by copolymerizing styrenesulfonic acid, methacrylic acid, acrylic acid, acrylic acid ester and the like. Is mentioned. Also, latex particles containing polyacrylamide can be used in order to suppress physical adsorption, that is, in order to increase hydrophilicity. Preferred are polystyrene particles copolymerized with acrylic acid, and more preferred are polystyrene particles copolymerized with acrylic acid whose hydrophilicity is increased by polyacrylamide.

The introduction efficiency of the carboxyl group is appropriately selected, but preferably 50 μeq / g to 300 μeq / g, more preferably 70 μeq / g to 250 μeq / g, and further preferably 90 μeq / g to 200 μeq / g. Latex particles.

The average particle size of the latex particles used is appropriately selected according to the purpose and application, but in the present invention, if it is for an automatic analyzer, it is preferably 50 nm to 250 nm, more preferably 60 nm to 200 nm, further preferably Is preferably latex particles having a particle size of 70 nm to 150 nm. Further, for a reagent for visual judgment, latex particles having a particle size of preferably 100 nm to 500 nm, more preferably 200 nm to 400 nm are preferable.

(Method of sensitizing SLO to latex particles)
The method for binding the amino group of SLO to the carboxyl group on the surface of the latex particles is not particularly limited, but for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Water soluble carbodiimides such as N-hydroxysuccinimide may be used in combination to convert the labile acylisourea intermediate to a more stable succinimidyl ester that is more reactive with amines.

(blocking)
In addition, the immunoassay reagent of the present invention is not particularly limited in order to suppress non-specific reactions and to enhance the stability of the immunoassay reagent itself, but examples thereof include proteins such as albumin, casein, and gelatin. One or more of these protein degradation products, amino acids, and surfactants may be further supported on the insoluble carrier. Although the concentration of the blocking agent is appropriately determined, it is preferably 0.1% to 5%, more preferably 1% to 3%. The treatment temperature is also appropriately determined, but is preferably 4 ° C to 30 ° C, more preferably 4 ° C to 20 ° C. The treatment time is also appropriately determined, but it is preferably 0.5 hour to 24 hours, more preferably 1 hour to 16 hours.

(Washing)
In the washing step after the blocking reaction, it is desirable to carry out washing with a buffer solution containing various surfactants. As the surfactant, Tween 20, Triton X-100 (polyethylene glycol mono-p-isooctyl phenyl ether), CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate), SDS ( Sodium dodecyl sulfate), deoxycholic acid and the like. These surfactants may be used alone or in combination of two or more. The concentration to be used can be appropriately determined, but for example, phosphate buffered saline (pH 7.4) containing 0.1% Tween 20 is suitable.

(Measurement reagent)
In the present invention, the ASO reagent is composed of two reagents, and the buffer solution is the first reagent and the latex reagent solution containing latex particles sensitized with streptolidine O is the second reagent. Then, it is preferable that the buffer solution of the first reagent contains polyvinylpyrrolidone.

  The immunoassay method of the present invention is not particularly limited as long as it uses the ASO reagent of the present invention, and can be performed by a usual method. For example, the immunoassay reagent of the present invention was added to a medium containing the substance to be measured, and the substance to be measured and the antigen immobilized on the latex particles contained in the immunoassay reagent of the present invention were bound to each other by an antigen-antibody reaction to generate. Aggregation is observed optically or visually. The preferable lower limit of the pH of the antigen-antibody reaction is 5, the upper limit is 10, and the more preferable lower limit is 6 and the upper limit is 9. The preferable lower limit of the reaction temperature is 4 ° C and the upper limit thereof is 50 ° C, and the more preferable lower limit thereof is 20 ° C and the upper limit thereof is 40 ° C. Although the reaction time is appropriately determined, it is desirable that the reaction is performed for about 1 to 10 minutes after mixing with the second reagent. As a method for visually observing and determining, for example, a method in which a sample and the immunoassay reagent of the present invention are mixed on a determination plate at room temperature and shaken for 1 to 5 minutes, and then the presence or absence of aggregation is determined, etc. Can be mentioned

  As the medium, various buffer solutions suitable for the type of the substance to be measured are used. The buffer solution may be one that does not inactivate the substance to be measured, and has an ion concentration or pH that does not inhibit the antigen-antibody reaction, for example, a phosphate buffer solution, a glycine buffer solution, Buffer solutions such as Tris buffer and various Good's buffers are preferred. These buffer solutions may be used alone or in combination of two or more kinds.

  The medium contains a water-soluble additive such as polyethylene glycol, carboxymethyl cellulose, methyl cellulose, dextran, polyvinylpyrrolidone, polyglycosylethyl methacrylate, pullulan, dextran, or erucinan in order to enhance the sensitivity of the reaction. You may. For the purpose of improving specificity and reagent stability, proteins such as casein and gelatin, or degradation products thereof, denatured products; quaternary ammonium salts such as choline chloride, EDTA, polyanions, chaotropic ions (Cl- , I-, SCN-, etc.), amino acids, surfactants and the like.

As an example of a particularly preferred embodiment, it is particularly desirable to use polyvinylpyrrolidone in the first reagent. The concentration of polyvinylpyrrolidone added to the first reagent is 0.05% to 0.4%, more preferably 0.07% to 0.36%, still more preferably 0. 09% to 0.33%, particularly preferably 0.1% to 0.3% is desirable.

(ASO reagent evaluation method)
The performance of the ASO reagent includes items such as detection sensitivity, measurement range, measurement accuracy, reproducibility, and stability. According to the present invention, the reagent becomes stable for a long period of time when Tween 20 is added to the washing buffer when the latex particles are blocked. The stability can be evaluated by an acceleration test. Generally, the latex reagent is usually stored at 2 to 10 ° C. In order to evaluate the long-term storage stability of such a reagent, an accelerated test is carried out at 20 to 40 ° C. for performance evaluation after short-term storage. The temperature used is preferably an accelerated test conducted at 37 ° C. The unit (IU / ml) of the ASO reagent used for the measurement is based on the international unit of anti-streptolidine O antibody determined by the World Health Organization (WHO).

  The present invention will be specifically described below with reference to examples. The present invention is not limited to the embodiments.

[Example 1] Preparation of ASO latex reagent (second reagent) 0.14 mL of carboxyl group-introduced latex particles (product number: PSI90-21) manufactured by Merck & Co., Inc. was centrifuged, and the supernatant was extracted. Next, 1 mL of 50 mM MES (pH 4.5) was added and sufficiently suspended. Then, centrifugation was performed again and the supernatant was extracted. Furthermore, 0.7 mL of 50 mM MES (pH 4.5) was added. To this, 0.35 mL of 4% carbodiimide (EDC) and 0.35 mL of 2.4% N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at 25 ° C. for 15 minutes under a state where the latex particles had a concentration of 1%. After activating the group, centrifugation was performed and the supernatant was removed. To this, 0.7 mL of 50 mM potassium phosphate buffer (hereinafter referred to as "KPB") (pH 6.5) was added, the mixture was centrifuged again, and the supernatant was removed. 0.7 mL of 50 mM KPB (pH 6.5) was added thereto and dispersed by ultrasonic waves to obtain activated latex particles. Streptolysin O (SLO) prepared by the method described in Example 2 of WO / 2017/204325 was dissolved in 50 mM KPB (pH 6.5) to a concentration of 3.0 (mg / mL). Next, 0.7 mL of the activated latex particles and 0.7 mL of the SLO solution were mixed and stirred and mixed at 25 ° C. for 2 hours to covalently bond the SLO to the latex particles.

Next, centrifugation was performed to remove the supernatant, and then the cells were resuspended in 2 mL with a blocking solution (1 × PBS (pH 7.4) containing 2% BSA). The supernatant was removed again by centrifugation, 1.4 mL of the blocking solution was added, and the mixture was ultrasonically dispersed, and then stirred overnight at 4 ° C. Then, centrifugation was performed, the supernatant was removed, and the cells were resuspended in 1.4 mL of a final buffer solution (1 × PBS (pH 7.4) containing 0.5% BSA and 0.05% sodium azide). Here, in order to confirm the effect of Tween 20 in the final buffer solution, the level at which Tween 20 was added to a final concentration of 0.01% and the level at which Tween 20 was added to a final concentration of 0.1%, a total of 3 The reagents were prepared at two levels. Then, after dispersing with ultrasonic waves, the mixture was stirred at 4 ° C. for 1 hour. Again, the supernatant was removed by centrifugation, 2 mL of the final buffer solution was added, and the mixture was sufficiently dispersed by ultrasonic waves, and then 2.67 mL of the final buffer solution was added to obtain the second reagent.

[Example 2] Stability evaluation of ASO latex reagent (second reagent) As the first reagent in the ASO latex reagent of the present invention, 1 x PBS (pH 7.4) containing 0.5% BSA was prepared. Next, using an automatic analyzer Hitachi 7180 (manufactured by Hitachi High Technologies), the sensitivity of each second reagent prepared in Example 1 immediately after the reagent preparation was measured. First, to 120 μl of the first reagent, 1.5 μl of ASO standard solution of each concentration (0, 125, 250 IU / ml) was added and stirred, and the mixture was kept at 37 ° C. for an appropriate time, and then 30 μl of the second reagent was added and stirred, and then The change in absorbance (ΔmAbs) at a wavelength of 546 nm from 300 seconds to 600 seconds was evaluated. Subsequently, the second reagent prepared in Example 1 was placed in a bottle, sealed, and placed in a 37 ° C. incubator for an accelerated test. Seven days after storage, the sample was taken out from the incubator, the sensitivity was measured again, and the change in the measured value was evaluated. As a result, as shown in Table 1, it was found that the stability of the reagent was improved depending on the concentration of Tween 20 contained in the washing buffer.

  The present invention provides a technique useful as a diagnostic agent having excellent measurement sensitivity, particularly in the medical field.

Claims (4)

Latex for measuring anti-streptolysin O including a step of washing with a buffer solution containing a surfactant after using latex particles having a carboxyl group on the surface, covalently binding streptolysin O to the latex particles, and performing a blocking treatment. Method for producing particles. The method for producing latex particles for measuring anti-streptolidine O according to claim 1, wherein at least one kind of surfactant selected from the group consisting of Tween 20, Triton X-100, CHAPS, SDS and deoxycholic acid is used. The method for producing latex particles for measuring anti-streptolysin O according to claim 2, wherein the surfactant is Tween 20. The anti-streptolidine O measuring reagent according to claim 1, wherein the concentration of the surfactant in the buffer is 0.01% to 0.1%.