JPH06186233A - New measurement method of antistreptolysin o - Google Patents

Abstract

PURPOSE:To easily, rapidly, and accurately measure antistreptolysin O by using liposome which is prepared by a method for eliminating a surface-active agent. CONSTITUTION:When using liposome which is prepared by the method for eliminating a surface-active agent for the method for measuring antistreptolysin O value using liposome including a labeled substance instead of a red cell, the fluctuation in the measurement sensitivity for each manufacturing lot of liposome does not change much. Although one type or more of phosphor lipid such as natural lecithin and dipalmitoylphosphachizircolin or their mixtures, etc., with cholesterol are listed as the main film-constitution constituent of the liposome, phosphor lipid containing unsaturated fat acid such as the natural lecithin is more desirable. Joint use of a compound with a negative charge such as phosphachijin acid improves reactivity with streptolysin O of the obtained liposome and hence increases measurement sensitivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring anti-streptolidine O (ASO) value using liposomes and a reagent kit for the measurement.

[0002]

BACKGROUND OF THE INVENTION The determination of ASO titer is a widely used test for demonstrating a history of hemolytic streptococcal infection.
Streptolidine O (SLO) is a type of lytic toxin produced by hemolytic streptococci and has antigenicity. Therefore, when infection of hemolytic streptococcus occurs, antibody titer against it (ASO value) , It is possible to prove the history of hemolytic Streptococcus infection by finding it.

The methods conventionally used as ASO titer determination methods include the Lands-Rendal method using a rabbit, sheep or human O-type red blood cell and the microtiter method. All of these utilize the so-called neutralization reaction that inactivates toxin activities such as hemolytic activity and cytocidal activity by reacting SLO, which is an antigen, with ASO in the serum of a sample.
This is a method of measuring the SO value. However, these methods inevitably require fresh erythrocytes of animals and humans (which are extremely unstable), so that variations between measured values are likely to occur and the operation method is complicated. Moreover, the reaction time is relatively long, and automation is extremely difficult.

On the other hand, in order to solve such problems, a measuring method by an immunoturbidimetric method utilizing a kind of precipitation reaction has been developed (Japanese Patent Laid-Open No. 61-25062, etc.). However, the degree of turbidity due to the antigen-antibody reaction of SLO-ASO is much smaller than that of other antigen-antibody reactions. Therefore, a so-called aggregation promoter or the like must be added in a large amount so as to non-specifically precipitate other proteins. As a result, the system becomes complicated and lacks accuracy, and the prozone phenomenon occurs. However, there are many problems in automating this method.

As a method for solving the above problems,
Some of the inventors of the present invention have been able to automate A by using a liposome encapsulating a labeling substance instead of red blood cells.
A new method for measuring SO value has been developed (Japanese Patent Laid-Open No. 63-184063). This method does not require the use of unstable substances such as erythrocytes, is a method that enables simple, rapid, and accurate measurement, and is a method that can be automated, but the production of liposomes to be used There is a problem that the measurement sensitivity varies from lot to lot, and further improvements have been desired.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and it is possible to perform a simple, quick and accurate measurement, can be automated, and there is almost no fluctuation in measurement sensitivity between liposome production lots. It is an object of the present invention to provide a novel method for measuring an ASO value and a reagent kit for measuring an ASO value used in the method.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention uses an ASO-containing sample, a liposome encapsulating a labeling substance and SLO, and measures the amount of the labeling substance which is released when the liposome membrane is destroyed by streptolysin O. In the method for measuring ASO value, the invention is a method for measuring an ASO value, characterized in that the liposome is prepared by a method for removing a surfactant, and a reagent kit for measuring an ASO value used in the method.

That is, the inventors of the present invention should solve the problem that the measurement sensitivity varies depending on the production lot of liposomes in the method of measuring the ASO value using liposomes containing a labeling substance instead of red blood cells. Focusing on the fact that the properties of liposomes change variously depending on the type of preparation method during the research, we thought that the above problems could be solved by selecting the preparation method. Using liposomes prepared by the agent removal method
When the SO value was measured, it was found that there was almost no change in measurement sensitivity between liposome production lots, and the present invention was completed.

The main membrane constituents of the liposome according to the present invention include natural lecithin (eg, egg yolk lecithin, soybean lecithin, etc.) and dipalmitoylphosphatidyl which are used as membrane constituents in the preparation of ordinary liposomes. Choline (DPPC), Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine
(DSPC), Dioleoylphosphatidylcholine (DOPC),
Dimyristoylphosphatidylethanolamine (DMP
E), egg yolk phosphatidylglycerol, dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), palmitoyloleoylphosphatidylcholine (POPC), etc. One or more of the phospholipids of
Alternatively, a mixed system of these and cholesterols, or a system in which these are further combined with lipopolysaccharide (LPS) and the like can be mentioned. As the phospholipid, any of those mentioned above or others may be used, but natural lecithin (eg, egg yolk lecithin, soybean lecithin, etc.), palmitoyl oleoylphosphatidylcholine (POPC), dioleoylphosphatidyl is used. More preferred are phospholipids containing unsaturated fatty acids such as choline (DOPC). As one of the membrane components of the liposome, for example, a compound having a negative charge such as LPS, phosphatidylglycerol such as egg yolk phosphatidylglycerol or DPPG, or phosphatidic acid such as DMPA or DPPA is used. This is more preferable because the reactivity of the obtained liposome with SLO is increased, and as a result, the measurement sensitivity can be increased.

The method for preparing the liposome according to the present invention will be described in detail below. That is, first, the phospholipids and cholesterols as described above are dissolved in an appropriate organic solvent (eg, chloroform, ethers, alcohols, etc.), concentrated to dryness under reduced pressure using, for example, a rotary evaporator, and then in a desiccator. Sufficiently dry under reduced pressure. Then, an aqueous surfactant solution (usually 50 mM to 3
00 mM, preferably 100-200 mM) is added and dispersed evenly. Examples of the surfactant used here include cholic acid, tritons (trade name of Rohm and Haas Co.), octyl glucoside, octyl thioglucoside, etc. which have been often used in this field. Desirably, a surfactant having a high critical micelle concentration (CMC) such as octyl glucoside or octyl thioglucoside is preferable. Next, if necessary, the lipopolysaccharides are added as a powder or as a solution, and a solution containing a desired labeling substance is further added and thoroughly mixed with stirring. Immediately after this, it is most desirable to remove the surfactant immediately, and examples of the method include dialysis method, gel filtration method, resin adsorption method and the like known per se. The treatment conditions are 1 minute to 1 day and 1 night, preferably 30 minutes to 3 hours,
The temperature varies slightly depending on the components of the liposome membrane,
It may be appropriately selected in the range of 0 to 70 ° C. Among these treatment methods, the dialysis method is most preferable. The liposomes thus obtained are used or stored after being concentrated to a predetermined concentration by ultrafiltration or the like.

When the liposome film according to the present invention is prepared as described above, when a surfactant solution or a solution containing a labeling substance is added to the obtained lipid film, for example, serum albumin (eg bovine serum) is added. Origin, human serum origin, etc.), water-soluble gelatin, polyethylene glycol (PE
G), polyvinyl alcohol (PVA), and other water-soluble polymer compounds are added, and as a result, the liposomes are more stably stored during storage, which is preferable. . The water-soluble polymer compound may be added as a powder together with the surfactant solution or the solution containing the labeling substance, or may be dissolved in the solution containing the surfactant or the labeling substance in advance and then added. Good. The amount added is not particularly limited as long as it does not hinder the preparation of liposomes, but when it is added by being dissolved in a solution containing a labeling substance, the concentration in the solution is usually 1 to 10
It is added by dissolving so as to be in the range of 0 mg / ml, preferably 10 to 30 mg / ml.

The labeling substance retained in the liposome according to the present invention may be any detectable labeling substance without particular limitation, and examples thereof include alkaline phosphatase and glucose-6. -Phosphate dehydrogenase, β-galactosidase and other enzymes, eg nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and other coenzymes, eg Luminol, luciferin, bis (2,4,6-trichlorophenyl) oxalate, N-methylacridinium ester and other luminescent compounds, such as carboxyfluorescein and other fluorescent compounds, such as arsenazo III, 4- (2-pyridylazo) ) Resorcinol, 2- (5-bromo-2-pyridylazo) -5- (N-propyl-N-sulfopropylamino) fluoro Enol sodium salts and other pigments, for example sugars such as glucose, ionic compounds such as potassium dichromate, sodium dichromate and sodium chloride, radical compounds such as nitroxide compounds, such as 2,2,6,6 Typical examples include spin-labeling agents represented by -tetramethylpiperidine-1-oxyl (TEMPO) and the like.

The method for measuring the amount of the labeling substance differs depending on the type of the labeling substance used. For example, when the labeling substance is an enzyme, for example, "enzyme immunoassay, protein / nucleic acid / enzyme separate volume No.31, Kitagawa Tsuneki" is used. Hiro, Minamihara Toshio, Tsuji Akio, Eiji Ishikawa, 51-63, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”, etc. When the labeling substance is a coenzyme, the labeling substance may be measured according to the method described in, for example, U.S. Pat.No. 4,704,355, and when the labeling substance is a fluorescent substance, for example, "illustration" Fluorescent antibody, Akira Kawao, 1st edition,
The labeled substance may be measured according to the method described in "Soft Science Co., Ltd., 1983" and the like. When the labeling substance is a luminescent substance, for example, "enzyme immunoassay,
Protein Nucleic Acid Enzyme Separate Volume No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 252-263, Kyoritsu Shuppan Co., Ltd., 19
It is sufficient to measure the labeling substance according to the method described in "Issue September 10, 1987", etc. When the labeling substance is a dye,
For example, Andrews, Janoff et al., Clin. Chem., Vol. 29, p. 1587,
The labeled substance may be measured according to the method described in 1983, and when the labeled substance is a saccharide, for example, T.Kat
The labeled substance may be measured according to the method described in aoka et al., Eur. J. Biochem., Volume 21, p. 80, 1971. Furthermore, when the labeling substance is an ionic compound, for example, YU
mezawa et al., Talanta, Vol. 31, p. 375, 1984 etc. may be measured according to the method described in the labeling substance, when the labeling substance is a radical compound, for example, Wu R, Alving
Et al., J. Immunal Methods, Volume 9, 165, 1975, etc., the labeled substance may be measured according to the method described above, and the labeled substance has a property as a spin labeling agent. In the case of, for example, “enzyme-linked immunosorbent assay, protein / nucleic acid / enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 264-271, Kyoritsu Shuppan Co., Ltd., September 1987.
The labeled substance may be measured in accordance with the method described in “Issue 10 days”.

The amount of the liposome, the labeling substance, SLO, etc. used in the assay method of the present invention is the final reaction solution (in the assay method using the reaction stop solution, the reaction before the reaction stop solution is added). It is as follows when it is shown as the concentration in the liquid). The amount of liposomes is usually 1 to 500 nm as the amount of phospholipid.
ol / ml, preferably 5-100 nm ol / ml. The amount of the labeling substance encapsulated in the liposome varies depending on the type of the labeling substance and is not uniform. For example, in the case of alkaline phosphatase (hereinafter abbreviated as AP), it is usually 0.01 to 0.6u.
nit / ml, preferably 0.02-0.2 unit / ml. The amount of SLO varies depending on the difference in the constituent components of the liposome membrane, and is not particularly limited, but is usually 5 to 5000 HU / m 2.
It is used in the range of l, preferably in the range of 50 to 1000 HU / ml.

The HU value of SLO is measured by the following method. That is, a mixture of 2 volumes of 1% rabbit erythrocyte solution and 1 volume of SLO solution diluted to a predetermined dilution ratio was incubated at 37 ° C. for 30 minutes and then centrifuged (30
00 rpm × 5 min.) To confirm the dilution factor of the SLO solution that causes 100% hemolysis, and use it as the HU value of the SLO stock solution.

Further, in the assay method of the present invention, the group mass used when an enzyme is used as a labeling substance varies depending on the type of enzyme or substrate used, and is not uniform, but when AP is used, for example. Is usually 0.5 to 10 mM, preferably 2 to 5 mM as the concentration of the substrate p-nitrophenyl phosphate in the substrate solution.

Examples of the buffer used in the measuring method of the present invention include phosphoric acid and its salts, boric acid and its salts, tris (hydroxymethyl) aminomethane (Tris),
Good, N-2-hydroxyethylpiperazine-N'-2-
Examples thereof include buffer agents such as ethanesulfonic acid (HEPES) and veronal, but are not particularly limited as long as they are commonly used buffer agents.

The measuring reagent used in the measuring method of the present invention includes proteins such as bovine serum albumin (BSA) and gelatin,
It is optional to appropriately add reagents, such as sugars, chelating agents, reducing agents, and preservatives, which are usually used in a measuring reagent solution in this field, if necessary. The concentration range and the like may be appropriately selected from, for example, the concentration range and the like normally used in immunoassay reagents using the antigen-antibody reaction known per se. However, since SLO is relatively unstable in an aqueous solution, it is desirable to add proteins such as BSA and gelatin to the buffer solution used for the SLO solution in order to stabilize SLO. The amount of addition is not particularly limited, but usually about 0.05 to 2.0 W / V% is added to the buffer solution.
The pH of the reagent solution for measurement used in the assay method of the present invention is usually selected in the range of 5 to 9 and preferably in the range of 6 to 8 by the buffering agent as described above.

Regarding the assay method of the present invention, for example, a liposome containing AP as a labeling substance is used and the substrate is used as a substrate.
An example of using p-nitrophenylphosphoric acid is as follows. That is, first, the sample and SLO are mixed and pre-incubated for about 1 to 10 minutes.
After adding liposomes encapsulating the same, or after mixing the sample with SLO and the liposomes, incubating for 5 to 30 minutes, and then adding the substrate p-nitrophenyl phosphate to the mixture for further 5 to 5 minutes. Incubate for 30 minutes. Then, a reaction stop solution (eg, NaOH solution, KOH solution, etc.) is added to this to stop the reaction, and the absorbance at 410 nm is measured. In the above procedure, the SLO and liposome may be added in any order, and all reagents (ie, SLO, liposome and substrate) are allowed to act on the sample at the same time, and after incubation for 5 to 30 minutes, The same operation can be carried out by adding a reaction stop solution to stop the reaction and measuring the absorbance at 410 nm. Although any of the above methods can be performed, the method described at the end, that is, the method in which all the reagents are simultaneously acted on the sample to perform the measurement is superior in that the operation is simple and the measurement can be performed in a short time. .

When an enzyme is used as the labeling substance, the final reaction solution is preheated for a predetermined time and then the change in the absorbance per unit time is measured, or a mixture of the sample, liposome and SLO is used. The ASO value can also be measured by pre-heating for a predetermined time, adding a substrate thereto, and then measuring the amount of change in absorbance per unit time.

The measuring method of the present invention is not limited to the manual method, but can be sufficiently applied to the measuring method using an automatic analyzer, and the measurement can be carried out easily and quickly. There are no particular restrictions on the combination of reagents when performing measurements using an automatic analyzer, and the combination of reagents that seems to be the best can be selected appropriately according to the model or taking other factors into consideration. And use it.

The method of the present invention is a reagent kit comprising, for example, a combination of (A) a labeling substance and a water-soluble polymer compound prepared by a surfactant removal method, and (B) an SLO solution. Can be carried out.

It is desirable that the liposome in the reagent kit used in the present invention contains at least one phospholipid containing an unsaturated fatty acid as a membrane constituent and at least one lipid having a negative charge. Further, in the reagent kit used in the present invention, for example, when the labeling substance is an enzyme,
As a matter of course, in addition to the above (A) and (B), a substrate solution of the enzyme or the like is included.

The present invention is characterized by the use of liposomes prepared by the method of removing a surfactant in the ASO value measurement method using liposomes, which causes the problems of the conventional measurement methods. It has a remarkable effect in solving the problem that the measurement sensitivity varies depending on the production lot of liposomes, which is a vortexing method or a reverse phase evaporation method (a method generally used as a liposome preparation method). Even if a liposome obtained by the REV method) or the like is used, the effect of the present invention cannot be obtained. The reason is not clear, but it can be considered as follows, for example.

That is, the method for measuring the ASO value of the present invention is SL
It utilizes the liposome dissolution reaction of O, and the liposome dissolution rate of SLO in this dissolution reaction is considered to change depending on the liposome membrane structure (monolayer, bilayer, or multilayer) and its particle size. To be Therefore, according to the surfactant removal method, liposomes having a uniform membrane structure and almost constant particle size can always be obtained. Therefore, the dissolution rate of liposomes obtained by this method by SLO hardly changes from production lot to production lot. As a result, it is considered that the measurement sensitivity of ASO value hardly changes for each liposome production lot. on the other hand,
In the vortexing method and the reverse phase evaporation method (REV method),
Since liposomes with different membrane structures are obtained in unspecified proportions for each production lot and the particle size of the liposomes varies widely, the dissolution rate of liposomes obtained by these methods by SLO differs depending on the production lot, resulting in an ASO value. It is considered that the measurement sensitivity of is different for each liposome production lot. Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Experimental Examples and Examples, but the present invention is not limited thereto.

[0026]

EXAMPLES Reference Example 1. Preparation of liposomes by vortexing method 2 ml of a 40 mM chloroform solution of egg yolk lecithin and 2 ml of a 40 mM chloroform solution of cholesterol were mixed in a round bottom flask, the solvent was distilled off by a rotary evaporator, and then vacuum dried in a desiccator for about 2 hours. Then, a thin film of lipid was prepared on the wall surface of the flask. AP solution [2000U / ml, 0.01M HEPES buffer (pH 7.4)]
250 μl was added, and the mixture was stirred with a vortex mixer while heating to about 30 ° C. until uniform. Then, this was subjected to ultracentrifugation (4 ° C., × 100,000 G, 60 min.), The supernatant was removed, and the resulting pellet was mixed with 0.01 M HEPES buffer (pH 7.
4) It was suspended in 5 ml to obtain a liposome solution. This operation was repeated 4 times to obtain 4 lots of liposome solution.

Reference Example 2. Preparation of liposomes by REV method 0.325 ml of a 20 mM chloroform solution of egg yolk lecithin and 0.325 ml of a 20 mM chloroform solution of cholesterol were mixed in a round bottom flask, and the solvent was distilled off by a rotary evaporator, followed by vacuum in a desiccator for about 2 hours. Drying was performed to form a thin film of lipid on the wall surface of the flask. 0.5 ml each of chloroform and diethyl ether
Dissolve by adding and then AP aqueous solution [2000U / ml, 0.01M HE
Dissolved in PES buffer (pH 7.4)] 80 μl was added and vigorously stirred with a vortex mixer to emulsify.
Then, this was subjected to rotary evaporation in a water bath at 43 to 48 ° C. to remove the solvent, and the resulting gelled product was adjusted to 0.
1 ml of 01M HEPES buffer (pH 7.4) was added, and the mixture was stirred with a vortex mixer until uniform. Then, this was subjected to ultracentrifugation (4 ° C, × 100000G, 40min. × 5), the supernatant was removed, and the resulting pellet was mixed with 0.01M HEPES buffer (pH 7.4).
Suspension in 2 ml gave a liposome solution. This operation was repeated 3 times to obtain 3 lots of liposome solution.

Reference Example 3. Preparation of liposomes by surfactant removal method-1 2 ml of a 40 mM chloroform solution of egg yolk lecithin and 2 ml of a 40 mM chloroform solution of cholesterol were placed in a round bottom flask and mixed, and then the solvent was distilled off by a rotary evaporator, followed by a desiccator for about 2 hours. Vacuum drying was carried out in order to form a thin film of lipid on the wall surface of the flask. to this
1.2 ml of 200 mM n-octyl glucoside aqueous solution and 0.01 M HEP
1.8 ml of ES buffer (pH 7.4) was added and stirred with a vortex mixer until uniform. Next, AP aqueous solution [2000
U / ml, dissolved in 0.01M HEPES buffer (pH 7.4)] 250 μl
Was added and mixed with stirring for a while. This was placed in a dialysis tube and dialyzed in 0.01 M HEPES buffer (pH 7.4) at 50 ° C for 1 hour, and then the liposome suspension was subjected to ultracentrifugation (4 ° C, × 10000).
0G, 60 min.), Remove the supernatant, and remove the resulting pellet.
A liposome solution was obtained by suspending in 5 ml of 0.01 M HEPES buffer (pH 7.4).

Reference Example 4. Preparation of Liposomes by Detergent Removal Method-2 2 ml of a 40 mM chloroform solution of egg yolk lecithin and 2 ml of a 40 mM chloroform solution of cholesterol were placed in a round bottom flask and mixed, and then the solvent was distilled off by a rotary evaporator, followed by a desiccator for about 2 hours. Vacuum drying was carried out in order to form a thin film of lipid on the wall surface of the flask. to this
1.2 ml of 200 mM n-octyl glucoside aqueous solution (containing 6.6 mg / ml of LPS) and 1.8 ml of 0.01 M HEPES buffer (pH 7.4) were added, and the mixture was stirred with a vortex mixer until uniform. Next, AP aqueous solution [2000U / ml, 0.01M HEPES buffer (pH
Dissolved in 7.4)] 250 μl was added and mixed with stirring for a while.
Place this in a dialysis tube and add 0.01M HEPES buffer (pH 7.
After dialysis in 50 ° C for 1 hour in 4), the liposome suspension was subjected to ultracentrifugation (4 ° C, × 100000G, 60 min.), The supernatant was removed, and the resulting pellet was mixed with 0.01M HEPES buffer. (pH7.4) 5m
It was suspended in l to obtain a liposome solution. This operation was repeated 4 times to obtain 4 lots of liposome solution.

Reference Example 5. Preparation of Liposomes by Surfactant Removal Method-3 2 ml of a 40 mM chloroform solution of egg yolk lecithin, 2 ml of a 40 mM chloroform solution of cholesterol and 0.47 ml of a 17 mM chloroform solution of egg yolk phosphatidylglycerol were placed in a round bottom flask and mixed, and then rotary. The solvent was distilled off by an evaporator, and then vacuum drying was performed for about 2 hours in a desiccator to form a lipid thin film on the wall surface of the flask. 200m n-octyl glucoside aqueous solution 1.2m
1.8 ml of 0.01 M HEPES buffer (pH 7.4) was added and stirred with a vortex mixer until uniform. Then A
250 μl of a P aqueous solution [dissolved in 2000 U / ml, 0.01 M HEPES buffer (pH 7.4)] was added and mixed with stirring for a while. Put this in a dialysis tube, and add 50M in 0.01M HEPES buffer (pH 7.4).
After dialysis at ℃ for 1 hour, the liposome suspension is ultracentrifuged.
(4 ° C., × 100,000 G, 60 min.), The supernatant was removed, and the obtained pellet was suspended in 5 ml of 0.01 M HEPES buffer (pH 7.4) to obtain a liposome solution.

Reference Example 6. Preparation of Liposomes by Detergent Removal Method-4 2 ml of a 40 mM chloroform solution of egg yolk lecithin and 2 ml of a 40 mM chloroform solution of cholesterol were placed in a round bottom flask and mixed, and then the solvent was distilled off by a rotary evaporator, followed by a desiccator for about 2 hours. Vacuum drying was carried out in order to form a thin film of lipid on the wall surface of the flask. to this
1.2 ml of 200 mM n-octyl glucoside aqueous solution (containing 6.6 mg / ml of LPS) and 1.8 ml of 0.01 M HEPES buffer (pH 7.4) were added, and the mixture was stirred with a vortex mixer until uniform. Next, an aqueous AP solution [2000 U / ml, 0.0 mg containing 20 mg / ml of a predetermined water-soluble polymer compound (BSA, gelatin, PEG 20000, PVA, water-soluble polymer compound shown in Table 2).
Dissolved in 1M HEPES buffer (pH7.4)] 250 μl was added and mixed with stirring for a while. Place this in a dialysis tube and add 0.01
After dialyzing in M HEPES buffer (pH 7.4) at 50 ° C for 1 hour,
Ultracentrifugation of liposome suspension (4 ℃, × 100000G, 60mi
n.), remove the supernatant, and add the resulting pellet to 0.01M HE.
By suspending in 5 ml of PES buffer solution (pH 7.4), a liposome solution containing various water-soluble polymer compounds was obtained.

Reference Example 7. Preparation of Liposomes by Detergent Removal Method-5 5 ml of a 40 mM chloroform solution of egg yolk lecithin, 2 ml of a 40 mM chloroform solution of cholesterol and 0.47 ml of a 17 mM chloroform solution of egg yolk phosphatidylglycerol were placed in a round bottom flask and mixed, followed by rotary. The solvent was distilled off by an evaporator, and then vacuum drying was performed for about 2 hours in a desiccator to form a lipid thin film on the wall surface of the flask. To this, 200 mM n-octyl glucoside aqueous solution (LPS
Containing 6.6 mg / ml) and 0.01 M HEPES buffer (pH 7.
4) 1.8 ml was added and stirred with a vortex mixer until uniform. Next, AP aqueous solution [2000 U / ml, BSA 20
Dissolved in 0.01 M HEPES buffer (pH 7.4) containing mg / ml]
250 μl was added and mixed with stirring for a while. Place this in a dialysis tube and place it in 0.01M HEPES buffer (pH 7.4) at 50 ℃ for 1 hour.
After dialysis for an hour, the liposome suspension was ultracentrifuged (4
The resulting pellet was suspended in 5 ml of 0.01 M HEPES buffer (pH 7.4) to obtain a liposome solution encapsulating BSA.

Experimental Example 1. Liposomes and S produced by different manufacturing methods
Examination of reactivity with LO (1) 50 μl of 10 μl of the liposome solution obtained in Reference Example 2 (REV method) or Reference Example 4 (method of the present invention: surfactant removal method)
2 mM was added to 200 μl of mM Tris buffer (pH 7.8).
p-Nitrophenylphosphoric acid solution (containing 220 HU / ml SLO)
Was added and incubated at 37 ° C for 30 minutes, then 0.1N
The reaction was stopped by adding 1.5 ml of NaOH solution, and the absorbance at 410 nm of the obtained reaction solution was measured. The results are shown in Table 1.

[0034]

[Table 1]

From the results in Table 1, the method of Reference Example 2 (REV
The reactivity of the liposomes obtained by the method) with SLO varies greatly depending on the production lot, and the reactivity of the liposomes obtained by the method of Reference Example 4 (surfactant removal method) with SLO is It can be seen that each manufacturing lot hardly changes. Experimental example 2. Examination of reactivity between liposomes produced by different manufacturing methods and SLO (2) 50 mM of the liposome solution obtained in Reference Example 1 or Reference Example 4
To 250 μl of a solution diluted 11 times with Tris buffer (pH 7.8), 5 μl of ASO solution having a predetermined concentration was added, and the mixture was incubated at 37 ° C. for 5 minutes, and then SLO solution (200 HU / m)
l) 150 μl and 2 mM p-nitrophenylphosphoric acid 150 μl were added, and after incubation at 37 ° C. for 5 minutes, the rate of change in absorbance of the obtained solution at 410 nm per unit time was measured. The calibration curve obtained using the liposome solution obtained in Reference Example 4 is shown in FIG. 1, and the calibration curve obtained using the liposome solution obtained in Reference Example 1 is shown in FIG. still,
1 and 2, the horizontal axis represents the ASO value (Todd unit) in the ASO solution, and the vertical axis represents the relative value of the absorbance change rate (ASO
The rate of change in absorbance obtained when the value is 0 Todd unit is 100. ) Are shown respectively. From the results of FIG. 1 and FIG. 2, the reactivity of the liposomes obtained by the method of Reference Example 4 (surfactant removal method) with SLO hardly changed between production lots, and the method of Reference Example 1 (Voltexing method)
It can be seen that the reactivity of the liposomes obtained with SLO with SLO varies greatly depending on the production lot.

Experimental Example 3. The average particle size of the liposomes in the liposome solutions obtained in Reference Examples 4 and 6 was measured by a laser dynamic light scattering method [measurement device: LPA 3000/3100 (manufactured by Otsuka Electronics Co., Ltd.)]. The results are shown in Table 2.

[0037]

[Table 2]

From the results shown in Table 2, it is understood that liposomes having a more uniform particle size can be obtained by adding a water-soluble polymer compound to prepare liposomes.

Experimental Example 4. Examination of stability of liposomes during storage Reference Examples 4 and 6 (BS as a water-soluble polymer compound)
The liposome solution obtained in (A) was stored refrigerated for a predetermined number of days, and 10 μl of the liposome solution was added with 50 mM Tris buffer (pH 7.8) for 20 days.
To the solution diluted to 0 μl, 400 μl of 2 mM p-nitrophenylphosphate solution (containing 88 HU / ml of SLO) was added, and the mixture was incubated at 37 ° C for 30 minutes.
After incubation for 1 minute, the reaction was stopped by adding 1.5 ml of 0.1N NaOH solution, and the absorbance at 410 nm of the obtained reaction solution was measured. Results are shown in FIG. In FIG. 3, □ shows the results obtained with the liposome solution of Reference Example 4, and ◯ shows the results obtained with the liposome solution of Reference Example 6, respectively. Further, in FIG. 3, the horizontal axis represents the number of days of refrigerated storage, and the vertical axis represents the absorbance at 410 nm. From the results shown in FIG. 3, it is understood that by preparing a liposome by adding a water-soluble polymer compound, a liposome whose reactivity with SLO does not change for a long period of time can be obtained.

Example 1. ASLO value was measured using Hitachi 7150 type automatic analyzer. First, 5 μl of a sample and 50 μl of the first reagent solution R1 [liposome solution obtained in Reference Example 4
Diluted 20-fold with mM HEPES buffer (pH 7.4). ] 250
Perform blank correction by mixing with μl, and after 5 minutes, add 2 ml of the second reagent solution [SLO150HU / ml and p-nitrophenyl phosphate].
50 mM HEPES buffer (pH 7.4) containing M] 150 μl was added,
After a further 5 minutes, the change in absorbance per minute at 415 nm / 700 nm was measured. The calibration curve of ASO value in this method is shown in FIG. In FIG. 4, the horizontal axis represents the ASO value (Todd unit), and the vertical axis represents the change in absorbance per minute at 415 nm / 700 nm. From the results of FIG. 4, it can be seen that a good calibration curve can be obtained by the method of the present invention. Further, the correlation between this method and the conventional method (Lands-Landal method) is shown in FIG. From the results in FIG. 5, it can be seen that the measured values obtained by the method of the present invention show a good correlation with those obtained by the conventional method.

Example 2. Automatic analyzer Cobasmira
s Mira, trade name of F. Hoffmann-La Roche) was used to measure the ASO value. First, sample 2μl, 50mM
80 µl of HEPES buffer (pH 7.4) and first reagent R1 [SLO20
50m containing 0 HU / ml and 2mM p-nitrophenyl phosphate
M HEPES buffer (pH 7.4)] 160 μl was mixed to perform blank correction, and after 5 minutes, the second reagent solution [liposome solution obtained in Reference Example 4 was 101 times with 50 mM HEPES buffer (pH 7.4)]. Diluted with. ] 80 μl was added, and 15 minutes later, the absorbance at 415 nm was measured. A calibration curve of ASO value in this method is shown in FIG. In FIG. 6, the horizontal axis shows the ASO value (Todd unit), and the vertical axis shows the absorbance at 415 nm. From the results of FIG. 6, it can be seen that a good calibration curve can be obtained by the method of the present invention.
Further, the correlation between this method and the conventional method (Lands-Landal method) is shown in FIG. From the results in FIG. 7, it can be seen that the measured values obtained by the method of the present invention show a good correlation with those obtained by the conventional method.

Example 3. ASO value was measured using Hitachi 7150 type automatic analyzer. First, 5 μl of the sample and the first reagent solution R1 [50 mM of the liposome solution obtained in Reference Example 5
Diluted 20 times with HEPES buffer (pH 7.4)] 250 μl was mixed to perform blank correction, and after 5 minutes, the second reagent solution [S
LO 150 HU / ml and 50 mM HEPES buffer (pH 7.4) containing 2 mM of p-nitrophenyl phosphate] 150 μl was added, and further 5
After that, the amount of change in absorbance per minute at 415 nm / 700 nm was measured. A calibration curve of ASO value in this method is shown in FIG. Figure 8
, The horizontal axis is ASO value (Todd unit) and the vertical axis is 415n
The amount of change in absorbance per minute at m / 700 nm is shown. From the results shown in FIG. 8, it can be seen that a good calibration curve can be obtained by the method of the present invention. Further, the correlation between this method and the conventional method (Lands-Landal method) is shown in FIG. From the results of FIG. 9, it can be seen that the measured values obtained by the method of the present invention show a good correlation with those obtained by the conventional method.

Example 4. ASLO value was measured using Hitachi 7150 type automatic analyzer. First, 5 μl of the sample and the first reagent solution R1 [the liposome solution obtained in Reference Example 6 (B
SA encapsulation) was diluted 20-fold with 50 mM HEPES buffer (pH 7.4)] and mixed with 250 μl to perform blank correction.
After 2 minutes, the second reagent solution [SLO 150 HU / ml and 50 mM HEPES buffer solution (pH 7.4) containing 2 mM of p-nitrophenyl phosphate] 15
After addition of 0 μl, the amount of change in absorbance per minute at 415 nm / 700 nm was measured after 5 minutes. The calibration curve of ASO value in this method is shown in FIG. In FIG. 10, the horizontal axis represents the ASO value (To
dd unit), and the vertical axis shows the amount of change in absorbance per minute at 415 nm / 700 nm. From the results shown in FIG. 10, it can be seen that a good calibration curve can be obtained by the method of the present invention. FIG. 11 shows the correlation between this method and the conventional method (Lands-Landal method). From the results of FIG. 11, it can be seen that the measured values obtained by the method of the present invention show a good correlation with those obtained by the conventional method.

Example 5. ASO value was measured using Hitachi 7150 type automatic analyzer. First, 5 μl of the sample and the first reagent solution R1 [50 mM of the liposome solution obtained in Reference Example 7
Diluted 20 times with HEPES buffer (pH 7.4)] 250 μl was mixed to perform blank correction, and after 5 minutes, the second reagent solution [S
LO 150 HU / ml and 50 mM HEPES buffer (pH 7.4) containing 2 mM of p-nitrophenyl phosphate] 150 μl was added, and further 5
After that, the amount of change in absorbance per minute at 415 nm / 700 nm was measured. The calibration curve of ASO value in this method is shown in FIG. In FIG. 12, the horizontal axis is the ASO value (Todd unit) and the vertical axis is
The amount of change in absorbance per minute at 415 nm / 700 nm is shown. From the results shown in FIG. 12, it can be seen that a good calibration curve can be obtained by the method of the present invention. 13 shows the correlation between this method and the conventional method (Rands-Landal method). From the results in FIG. 13, it can be seen that the measured values obtained by the method of the present invention show a good correlation with those obtained by the conventional method.

[0045]

INDUSTRIAL APPLICABILITY As described above, the present invention uses the liposomes prepared by the surfactant removal method in the method for measuring the ASO value using liposomes, and thus the conventional vortexing method or REV method is used. Solved the problem that the measurement sensitivity greatly varies depending on the production lot of liposomes when the measurement is performed using the prepared liposomes, and one of phospholipids containing unsaturated fatty acid as a membrane constituent component of the liposomes. By using the above and one or more kinds of lipids having a negative charge in combination, it was possible to enhance the measurement sensitivity in ASO value measurement, and further, to encapsulate a water-soluble polymer compound in a liposome together with a labeling substance. It is possible to obtain liposomes having a uniform particle size, and the remarkable effect is that the stability of the liposomes during storage is improved, contributing to the industry. Rutokoro is a large made invention.

[0046]

[Brief description of drawings]

FIG. 1 shows the results obtained in Experimental Example 2, in which anti-streptolidine O (ASO) obtained by using 4 lots of liposomes prepared by a surfactant removal method.
It shows the calibration curve of the value.

FIG. 2 shows the results obtained in Experimental Example 2, in which liposome 4 prepared by the vortexing method was used.
It is what shows the calibration curve of ASO value obtained using the lot.

[Fig. 3] Fig. 3 shows liposomes and streptolysin O (SL) obtained in Experimental Example 4 after refrigerated storage for a predetermined number of days.
It shows the results of examining the reactivity with O).

FIG. 4 shows a calibration curve of ASO value obtained in Example 1.

FIG. 5 shows the correlation between the measuring method of the present invention obtained in Example 1 and the conventional method (Lands-Rendal method).

FIG. 6 shows a calibration curve of ASO value obtained in Example 2.

FIG. 7 shows the correlation between the measurement method of the present invention obtained in Example 2 and the conventional method (Lands-Rendal method).

FIG. 8 shows a calibration curve of ASO value obtained in Example 3.

FIG. 9 shows the correlation between the measuring method of the present invention obtained in Example 3 and the conventional method (Lands-Rendal method).

FIG. 10 is an ASO obtained in Example 4.
It shows the calibration curve of the value.

FIG. 11 shows the correlation between the measuring method of the present invention obtained in Example 4 and the conventional method (Lands-Rendal method).

FIG. 12 is an ASO obtained in Example 5.
It shows the calibration curve of the value.

FIG. 13 shows the correlation between the measurement method of the present invention obtained in Example 5 and the conventional method (Lands-Rendal method).

[Explanation of symbols]

In FIG. 1, ◯, □, +, and Δ indicate differences in the production lots of the four types of liposome solutions obtained in Reference Example 4. Figure 2
In the table, ◯, □, +, and Δ indicate differences in production lots of the four types of liposome solutions obtained in Reference Example 1. In FIG. 3, □ shows the results obtained using the liposome solution obtained in Reference Example 4, and ◯ shows the results obtained using the liposome solution obtained in Reference Example 6, respectively.

Front page continued (72) Inventor Shin Takano 6-1 Takada-cho, Amagasaki-shi, Hyogo Wako Pure Chemical Industries, Ltd. Osaka Research Institute

Claims (8)

[Claims] 1. Using a sample containing anti-streptolysin O, a liposome encapsulating a labeling substance and streptolysin O, the amount of the labeling substance released by breaking the liposome membrane by streptolysin O is measured, and In the method for measuring the O value of streptolidine, the liposome is prepared by a method for removing a surfactant, and the method for measuring the O value of anti-streptolidine. 2. The measuring method according to claim 1, wherein the liposome comprises at least one phospholipid containing an unsaturated fatty acid as a membrane constituent and at least one lipid having a negative charge. 3. The measuring method according to claim 1, wherein the liposome contains a water-soluble polymer compound together with a labeling substance. 4. The labeling substance is an enzyme, a coenzyme, a luminescent compound, a fluorescent compound, a dye, a saccharide, an ionic compound, a radical compound or a spin labeling agent.
The measuring method according to any one of 3 above. 5. A reagent kit for measuring the anti-streptolidine O value in a sample, which comprises the following constitution. (A) a labeling substance and a water-soluble polymer compound, which are prepared by a method of removing a surfactant, or a posome solution, and (B) a streptolidine O solution 6. The reagent kit according to claim 5, wherein the liposome comprises one or more phospholipids containing an unsaturated fatty acid as a membrane constituent and one or more lipids having a negative charge. 7. The reagent according to claim 5, wherein the labeling substance is an enzyme, a coenzyme, a luminescent compound, a fluorescent compound, a dye, a saccharide, an ionic compound, a radical compound or a spin labeling agent. kit. 8. The reagent kit according to claim 5, wherein the labeling substance is an enzyme, and a substrate solution of the enzyme is contained in addition to (A) and (B).