JPH08254532A - Determination of steptolysin o - Google Patents

Abstract

PURPOSE: To determine streptolysin O in a liquid to be determined stably by using a carrier fixed with a steroid compound having a specified structure to be bonded with the streptolysin O in a liquid to be determined. CONSTITUTION: A steroid compound represented by a specified formula is fixed physically or chemically to a carrier, e.g. a latex particle or a bead. In the formula, R1 represents a substituted/unsubstituted, saturated/unsaturated cholesterol side chain group or coal acid side chain group, R2 represents hydrogen atom or hydroxy group, and the breaking line position represents a saturated or double bond position. The group R1 represents a saturated/unsaturated cholesterol side chain group or coal acid side chain group having one or two double bond substituted/unsubstituted by hydroxy group, lower alkyl group or lower acyl group. When the carrier is caused to react on a solution to be determined containing streptolysin O derived from group A hemolytic streptococci, only the streptolysin O is bonded specifically onto the carrier.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for immunologically determining streptolysin O in a test solution, wherein the following general formula (1)

[0002]

Embedded image

(Wherein R1 is a substituted or unsubstituted, unsaturated or saturated, cholesterol side chain group or cholic acid side chain group, R2 is a hydrogen atom or a hydroxyl group, and the broken line site is saturated or divalent. Indicates the heavy binding site.)
The present invention relates to a method for determining streptolysin O, which comprises using a carrier having one or more steroid compounds represented by the formula (1) immobilized thereon.

[0004]

2. Description of the Related Art Streptolysin O
ysin O) is, for example, Lansfield (Lan
serologic classification (Lancefield)
d, R.D. C. J. Exp. Med. , Volume 57, 57
1-595, 1933), group A hemolytic streptococcus.
Tococci) -produced toxin, which is generally in a reducing state, for example, in the presence of 2-mercaptoethanol, has a molecular weight of 50,000-
It is known as a protein contained in the range of 70,000, has a high affinity for cholesterol, and has an activity of binding to the cell membrane to lyse cells (Streptococcal infection [above] -its basic and clinical). -, Edited by Yuichi Shiokawa, Morimasa Yoshioka and Shigeyuki Hamada, pp. 88-97, Hirokawa Shoten Co., Ltd., June 5, 1992).

Further, it is known that due to infection with group A hemolytic streptococcus, a reaction-specific antibody against streptolysin O (anti-streptolysin O antibody) is present in patient serum (Kenji Tomoeyama et al., Sanitary. Inspection, Volume 23, 8
03-807, 1974). Streptolidine O
As a method for determining the erythrocyte, a method using the hemolytic activity, which is a biological activity of streptolysin O, as an index has been widely used. However, since this method necessarily requires fresh erythrocyte, the erythrocyte used However, there is a problem in that variations in measured values are likely to occur due to the stability of, the difference between lots, and the like.

Further, as a general method for specifically determining an antigenic substance, a carrier having adsorbed an antibody against the antigenic substance to be determined is brought into contact with the antigenic substance, and as a result, the antibody is mediated. A method for confirming or quantifying an antigenic substance bound to a carrier by an immunological method, for example, "enzyme-linked immunosorbent assay, protein nucleic acid enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 13 P. To p. 26, Kyoritsu Shuppan Co., Ltd., issued September 10, 1987 ”and the like.

[0007] However, in order to specifically bind an antigenic substance to a carrier, an antibody that has the ability to react specifically with the antigenic substance and is highly purified is required (immunity). New application examples of assay method and application to diagnostic reagent / therapeutic drug development, immunoassay method development study group, pages 3-6, management education publication, published June 20, 1985). Obtaining such an antibody requires a great deal of labor and cost, resulting in a high measurement cost. Further, it has been pointed out that the carrier on which the antibody is adsorbed does not necessarily have good storage stability, and that the antibody is easily released from the carrier by contact with a buffer solution or an acidic solution having increased ionic strength. A (Takasaka Akira, clinical examination, 32nd
Vol. 8, pp. 936-938, 1988).

[0008]

In order to solve the problems of these methods for determining streptolysin O, to provide a stable and inexpensive carrier to which streptolysin O can be specifically bound, and to use the same. To provide a method for reaction-specifically determining streptolysin O in a test solution.

[0009]

As a result of earnest research, the present inventors have found that the following general formula (1)

[0010]

Embedded image

(Wherein R 1 is a substituted or unsubstituted, unsaturated or saturated, cholesterol side chain group or cholic acid side chain group, R 2 is a hydrogen atom or a hydroxyl group, and the broken line site is saturated or divalent. Indicates the heavy binding site.)
In a solution containing streptolidine O, which is a test liquid, by reacting a carrier, which is physically or chemically immobilized with a steroid compound represented by, with a solution containing streptolidine O derived from group A hemolytic streptococcus. Of Streptolidine O can be specifically bound to a carrier, and the Streptolidine O thus obtained can be obtained.
It was found that the binding carrier retains the reactivity with the anti-streptolidine O antibody, and that the amount of streptolysin O in the test solution can be determined by utilizing this property, and the present invention is completed. Came to.

The present invention has been completed based on the above findings, and in the immunological determination method of streptolysin O in a test liquid, the following general formula (1)

[0013]

[Chemical 4]

(Wherein R1 is a substituted or unsubstituted, unsaturated or saturated, cholesterol side chain group or cholic acid side chain group, R2 is a hydrogen atom or a hydroxyl group, and the broken line site is saturated or divalent. Indicates the heavy binding site.)
The method for determining streptolysin O is characterized by using a carrier on which one or more steroid compounds represented by are immobilized.

The steroid compound according to the present invention is represented by the above general formula (1), in which R is
1 represents a substituted or unsubstituted, unsaturated or saturated cholesterol side chain group or cholic acid side chain group, R2 represents a hydrogen atom or a hydroxyl group, and a broken line site represents a saturated or double bond site. is there. Further, specific examples of the group R1 include, for example, an unsaturated or saturated cholesterol side chain having 1 or 2 double bonds, which is substituted or unsubstituted with a hydroxyl group, a lower alkyl group or a lower acyl group. Group or cholic acid side chain group. The cholesterol side chain group has the following general formula (2)

[0016]

Embedded image

(In the formula, R3 is a hydrogen atom, a hydroxyl group,
A lower alkyl group or a lower acyl group, R4 represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower acyl group, R5 represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower acyl group; The heavy binding site is shown. ) Or the following general formula (3)

[0018]

[Chemical 6]

(In the formula, the broken line site represents a saturated or double bond site), and a side chain group of cholesterol can be mentioned, preferably R in the general formula (2).
3 and R4 are hydrogen atoms, and R5 is a hydrogen atom, a hydroxyl group, a methyl group or an ethyl group, and a cholesterol side chain group is mentioned. Specific examples thereof include a cholesterol side chain group and β- Examples thereof include a sitosterol side chain group, a stigmasterol side chain group, a campesterol side chain group, an ergosterol side chain group, a cerebrosterol side chain group, and a desmosterol side chain group.

Specific examples of the steroid compound represented by the above general formula (1) include, for example, cholesterol (cholesterol), 7-dehydrocholesterol (7-dehydrocholesterol),
Cholestanol (cholestanol), coprostanol (coprostanol), Δ ⁷ - cholesteryl Nord ^{(Δ 7 -cholestenol), Δ 7} - Kopurosu tenor (Δ ⁷ -coprostenol), β-
Sitosterol (β-sitosterol), 7-dehydro-β-sitosterol (7-dehydro-β
-Sitosterol), stigmasterol (st
igmasterol), 7-dehydrostigmasterol (7-dehydrostigmasterol)
l), campesterol,
7-dehydrocampesterol (7-dehydroc
ampsterol), stigmastanol (sti
gmastanol), Δ ^7- stigmastenol (Δ
^7- stigmastanol), 11α-hydroxycholesterol (11α-hydroxycholes
terol), Δ ²² -stigmastenol (Δ ²² -st
igmastenol), α-spinasterol (α-
spinasterol), campestanol (cam)
pestanol), Δ ⁷ - campesterol scan tenor (Δ ⁷ -
camstenol), 20α-hydroxycholesterol (20α-hydroxycholester
ol), brush casterol (brassicaste)
rol), ergosterol (ergostero)
l), cerebrosterol
l), 7-dehydro cerebrosterol (7-dehy
drocerebrosterol), celebrity Ross ethanol (cerebrostanol), Δ ⁷ - Celebrity loss tenor (Δ ⁷ -cerebrostenol), desmosterol (desmosterol), 7- dehydrocholesterol desmosterol (7-dehydrodesmo
sterol), 3β-hydroxycholanic acid (3β-h
ydroxycholonic acid) or 3β
-Hydroxy-Δ ⁵ -cholenic acid (3β-hydroxy
-Δ ⁵ -cholenic acid) and the like.

Preferably, cholesterol, 7-dehydrocholesterol, cholestanol, coprostanol, Δ ⁷ -cholestenol, Δ ⁷ -coprostenol,
β-sitosterol, 7-dehydro-β-sitosterol, stigmasterol, 7-dehydrostigmasterol, campesterol, 7-dehydrocampesterol and the like can be mentioned.

[0022] More preferably, cholesterol, 7-dehydrocholesterol, cholestanol, coprostanol, delta ⁷ - cholesteryl Nord or delta ⁷ - Kopurosu tenor and the like. Among these steroid compounds, those containing one kind or two or more kinds can be mentioned.

The steroid compound represented by the general formula (1) in the present invention (hereinafter, simply referred to as the present steroid compound) can be immobilized on a carrier, preferably by a method well known to those skilled in the art. Is preferably by physically adsorbing the steroid compound on the surface of a carrier, or chemically reacting a reactive derivative of the steroid compound to a surface having a functional group capable of reacting with the steroid compound or an activated surface. It is done by combining.

In the practice of the present invention, as an advantageous embodiment of the carrier on which the steroid compound is immobilized, the steroid compound is usually used in an amount of 25 per unit area (cm ² ) of the carrier.
0 nmol-0.025 nmol, preferably 25 nm
A carrier bonded in the range of ol to 0.25 nmol can be mentioned. The carrier according to the present invention is not limited in its material, shape, size and the like as long as it can carry out the present invention, and examples thereof include polystyrene, polyethylene, polypropylene, acrylonitrile butadiene styrene copolymer, and butadiene. Styrene copolymer, polycarbonate, polyvinyl chloride, polyvinylidene chloride, polyamide, polymethylmethacrylate, polymethylpentene, polyacetal, polyvinyl acetate, polyvinyl alcohol, polyvinyl butyral, polyisobutylene, vinyl chloride vinyl acetate copolymer, fluororesin (Polytetrafluoroethylene, tetrafluoroethylene ethylene copolymer, perfluoroalkoxy copolymer, etc.), polyacrylonitrile, polystyrene acrylate, polyethylene terephthalate , Polybutylene terephthalate, polyurethane, urea resin,
Epoxy resin, melamine resin, phenol resin, unsaturated polyester resin, silicone, nitrocellulose, cellulose acetate, cellulose acetate butyrate,
A molded polymer such as cellulose acetate propionate, cellulose propionate, ethyl cellulose, carboxymethyl cellulose or polyvinylidene difluoride, or a functional group such as carboxyl group, nitrile group, primary amino group or secondary amino group on the polymer. A shaped polymer obtained by further copolymerizing a substance having these functional groups at the time of synthesizing the above-mentioned polymer for the purpose of holding it, or a molding obtained by introducing these functional groups by surface treatment after forming the above-mentioned polymer. Polymers, molded glass or metal, etc. can be used.

In the present invention, the material, shape and size, etc., which are commonly used in immunological determination methods well known to those skilled in the art, such as microtiter plate, latex particles, beads, balls, test tube are used. Alternatively, it is particularly advantageous to use a membrane or the like as a carrier. As an example of a specific method for immobilizing the steroid compound on the carrier by physically adsorbing the steroid compound on the carrier, for example, in the case of using a microtiter plate as the carrier, Usually 1 μg / m in a hydrophilic or lipophilic solvent, optimally an ethanol or methanol solution, which can dissolve, disperse or suspend the steroid compound without damaging the structure of the titer plate.
1 to 10 mg / ml, preferably 10 μg / ml to 1 m
Dissolve, disperse or suspend in a concentration range of g / ml, and normally add 2 to each well of a microtiter plate.
5 μl to 200 μl, preferably 50 μl to 100 μl
Addition, usually in the temperature range of 4 ° C to 60 ° C, preferably 4 ° C to 30 ° C, usually 10 minutes to 24 hours, preferably 30
An example is a method of immobilizing the steroid compound of the present invention by allowing the well to stand for a reaction in the time range of from 6 minutes to 6 hours and then washing the well with water or an appropriate buffer solution.

At this time, the reaction solution may contain water, and does not impair the structure of the microtiter plate,
And it may contain chloroform, acetone, hexane, dichloroethane, propanol, isopropanol, xylene, toluene, benzene, ether, petroleum ether, methyl acetate, ethyl acetate or other solvent in a concentration range that does not greatly affect the reaction. .

Alternatively, the steroid compound is usually added to 10
0 ng / ml to 1 mg / ml, preferably 1 μg / ml
In a concentration range of up to 100 μg / ml, a hydrophilic or lipophilic solvent, optimally an ethanol or methanol solution, which can dissolve, disperse or suspend the steroid compound without damaging the structure of the microtiter plate, etc. Dissolve, disperse or suspend the liquid in wells of a microtiter plate, usually 25 μl per well
Add 200 μl, preferably 50 μl to 100 μl,
Usually, the steroid compound is immobilized by standing still in the temperature range of 20 ° C. to 90 ° C., preferably 40 ° C. to 70 ° C. to evaporate the solvent, and washing the wells with water or an appropriate buffer solution. The method of doing is also convenient.

Further, for example, when a test tube or the like is used as the carrier, the micro volume is determined in consideration of the internal volume of the test tube or the like and other factors under the condition that the structure of the carrier such as the test tube is not impaired. It may be performed according to the titer plate method. Furthermore, for example, when latex particles are used as a carrier, the present steroid compound and the latex particles are hydrophilic or hydrophilic so that the present steroid compound can be dissolved, dispersed or suspended without impairing the structure of the latex particles. Contact in an oily solvent, optimally ethanol or methanol solution, usually in the temperature range of 4 ° C to 60 ° C, preferably 4 ° C to 30 ° C, usually 10 minutes to 24 hours, preferably 30 minutes to 10 hours. Examples of the method include immobilizing the steroid compound by stirring, shaking, or leaving still for reaction within a time range of 1, and then washing the latex particles with water or an appropriate buffer solution as needed.

At this time, the solid content concentration expressed as the concentration of latex particles in the reaction solution is usually 0.01% to 10%.
%, Preferably 0.05% to 5%, and the concentration of the steroid compound is usually 0.1 μg / ml.
-10 mg / ml, preferably 1 μg / ml-1 mg /
A concentration range of ml is desirable. The reaction solution may contain water, and does not impair the structure of the latex particles, and chloroform, acetone, hexane, dichloroethane, propanol, isopropanol, xylene, toluene in a concentration range that does not greatly affect the reaction,
It may contain benzene, ether, petroleum ether, methyl acetate, ethyl acetate or other solvent.

Further, for example, when beads or balls are used as the carrier, the surface area, volume, number and other factors of the beads or balls, etc. are determined under the condition that the structure of the material such as the beads or balls serving as the carrier is not impaired. It may be carried out according to the method of latex particles in consideration of factors. Furthermore, for example, when a membrane or the like is used as a carrier, a hydrophilic or lipophilic solvent that can dissolve, disperse or suspend the present steroid compound without impairing the structure of the membrane or the like, which is a carrier, is most suitable. The steroid compound dissolved, dispersed or suspended in an ethanol or methanol solution is usually used in an amount range of 250 nmol to 0.025 nmol, preferably 25 nmol to 0.25 nmol per unit area (cm ² ) of the membrane, preferably A method well known to those skilled in the art, for example, a method of contacting with a membrane or the like by a dot spot or the like to immobilize the present steroid compound can be mentioned. At this time,
The size of the spot, etc. is arbitrary, and the solution in which the steroid compound is dissolved, dispersed or suspended may contain water, does not impair the structure of the membrane, etc. and has a great influence on the reaction. No concentration range of chloroform,
Acetone, hexane, dichloroethane, propanol,
It may contain isopropanol, xylene, toluene, benzene, ether, petroleum ether, methyl acetate, ethyl acetate or other solvent.

The method of chemically bonding the steroid compound to the carrier in the present invention is carried out by adding functional groups such as carboxyl group, hydroxyl group, thiol group, 1 to the steroid compound.
Having a reactive derivative of the present steroid compound having a secondary amino group, a secondary amino group, an amide group, a nitro group or an aldehyde group introduced thereinto, and a functional group capable of reacting with the functional group introduced into the steroid compound The carrier or the activated carrier can be prepared by a method known per se, for example, “Experimental and Applied Affinity Chromatography, Ichiro Chibata.
Tetsuya Tosa and Yushi Matsuo, pp. 30-109, Kodansha Ltd., published September 10, 1976 "or" enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 3rd edition.
Ed., Pp. 75-151, Medical Shoin Co., Ltd., May 1987
It can be carried out by chemically bonding in accordance with the method described in "Issued on March 15," and the like.

Examples of the method of chemically binding the steroid compound to the carrier in the present invention include, for example, the following general formula (4)

[0033]

[Chemical 7]

(In the formula, R6 is a substituted or unsubstituted saturated cholesterol side chain group or cholic acid side chain group, R7 is a hydrogen atom or a hydroxyl group, and the broken line site is a saturated or double bond site. In the steroid compound represented by the formula (1), R6 is a substituted or unsubstituted saturated cholesterol side chain group or a cholic acid side chain group, R7 is a hydrogen atom or a hydroxyl group, and any of the broken line sites is present. A steroid compound having a double bond at one site was used as a reaction starting material, and "Hosoda Hiro et al., Ch.
em. Pharm. Bull. , Vol. 28, p. 1294 to p. 1299, 1980 ", etc.
5,6-epoxy body, 7,8-epoxy body or 1
A carboxyl group, a primary amino group, a secondary amino group, an amide group, a nitro group or an aldehyde group which is crosslinked at the 6-, 7- or 15-position by a thioether bond through a ring-opening reaction of a 4,15-epoxy compound And the like, a reactive derivative of the present steroid compound is formed, and the compound is bonded to the surface of a carrier having a functional group capable of reacting with the compound.

More specifically [0035] Among the steroid compound represented by the general formula (4), for example, delta ⁷ - cholesteryl Nord, delta ⁷ - Kopurosu tenor, delta ⁷ - stigmastanol mass tenor, delta ⁷ - campesterol By using a steroid compound having a double bond at the 7-8 position such as stenol or Δ ⁷ -cerebrostenol as a reaction starting material, the reactivity of the steroid compound can be improved by introducing a carboxyl group at the 7 position of this reaction starting material. To show using an example of forming a derivative, first, in a hydrophilic organic solvent (preferably methanol or ethanol etc.) capable of dissolving, dispersing or suspending the above steroid compound as a reaction starting material. , Contact with peracid (preferably hydrogen peroxide etc.) and alkaline reagents (preferably sodium hydroxide or potassium hydroxide etc.) Touch, usually in the temperature range of 0 ° C to 40 ° C, preferably 0 ° C to 10 ° C, usually for 30 minutes to 1
Stirring for 2 hours, preferably 1 to 6 hours,
After shaking or standing, the reaction solution is neutralized to form an epoxy derivative as a reaction starting material.

At this time, the concentration of the steroid compound as a reaction starting material in the reaction solution is usually 0.01 mmol /
ml to 1 mmol / ml, preferably 0.05 mmol
The concentration of peracid is usually 0.1% to 10%, preferably 0.5%.
The concentration of the alkaline reagent is usually 0.5% to 10%, preferably 1% to 5%, and the water content in the reaction solution is preferably 20%. %
The following range is desirable. The method for separating the formed epoxy derivative is described in, for example, “Biochemical Research Method I, No. 7”.
Edition, edited by K. Ando, Hiroshi Terayama, Kazutoshi Nishizawa, Tamio Yamakawa, 49
Pp.-96, Asakura Shoten Co., Ltd., March 20, 1973 ”and the like.

Then, the obtained epoxy derivative is dissolved in a hydrophilic organic solvent capable of dissolving, dispersing or suspending it, optimally methanol or ethanol, and mercaptomonocarboxylic acid (preferably 2-mercaptoacetic acid). Or 3-mercaptopropionic acid etc.) and an alkaline reagent (preferably sodium hydroxide or potassium hydroxide etc.), and usually in the temperature range of 0 ° C. to 60 ° C., preferably 10 ° C. to 40 ° C., usually for 30 minutes. After stirring, shaking or standing for ˜12 hours, preferably 1 to 6 hours, the pH of the reaction solution is changed to acidic to give 7% of steroid compound as a reaction starting material.
A carboxyl group crosslinked by a thioether bond can be introduced at the position.

At this time, the concentration of the epoxy derivative in the reaction solution is usually 0.01 mmol / ml to 1 mmol / ml,
Preferably 0.05 mmol / ml to 0.5 mmol /
The concentration of mercaptomonocarboxylic acid is usually 0.05 mmol / ml to 5 mmol / ml,
The concentration is preferably 0.1 mmol / ml to 1 mmol / ml, and the concentration of the alkaline reagent is usually 0.5% to
It is desirable that the concentration is in the range of 10%, preferably 1% to 5%, and the water content in the reaction solution is preferably in the range of 20% or less. Further, the reaction liquid may contain dioxane or the like. The method for separating the formed derivative may be similar to the above-mentioned method for separating the epoxy derivative.

[0039] By the above method, among the steroid compound represented by the general formula (4), for example, delta ⁷ - cholesteryl Nord, delta ⁷ - Kopurosu tenor, delta ⁷ - stigmastanol mass tenor, delta ⁷ - Kanpesu Using a steroid compound having a double bond at the 7-8 position such as tenol or Δ ^7- cerebrostenol as a reaction starting material, thioether at the 7-position such as cholestanol, coprostanol, stigmasteranol, campestanol or cerebrostanol, respectively. It is possible to form a reactive derivative in which a carboxyl group cross-linked by a bond is introduced.

Further, according to the above method, for example, 5-6
Cholesterol, which has a double bond at the position, β-sitosterol, campesterol, cerebrosterol, etc. as reaction starting materials, respectively, carboxyls crosslinked to the 6-position by thioether bond such as cholestanol, stigmasteranol, campestanol or cerebrostanol. A reactive derivative having a group introduced can be formed.

The reactive derivative of the present steroid compound having the carboxyl group introduced therein can be prepared by a method known per se, for example, the acid anhydride method, the carbodiimide method or the N method.
Immobilization can be carried out by reacting with the surface amino group of the carrier having an amino group on the surface by a hydroxysuccinimide method or the like. In the present invention, a method for immunologically determining streptolysin O in a test solution using a carrier to which the steroid compound of the present invention is immobilized is a method for determining an immunoreaction between streptolysin O and an anti-streptolidine O antibody in the test solution. Conditions that do not interfere with the antigen-antibody reaction and that do not inactivate the properties of the reagents used (for example, the detectable properties of the labeling agent when a labeling agent is used), etc. Below, the carrier to which the steroid compound according to the present invention is immobilized and the test solution and the anti-streptolidine O antibody solution are reacted sequentially or simultaneously, and as a result, the antigen-antibody complex bound on the carrier is confirmed or quantified. Is characterized by:

The test liquid according to the present invention includes all liquids containing streptolysin O, which can be determined in the present invention, for example, A.
Group, C group or G group, produced by hemolytic streptococcus, or produced by E. coli, yeast, or other host by using gene recombination technology, streptolysin O, its derivative or mutant, or the like. Streptolysin O-like compound that retains the ability to bind to a steroid compound in the present invention and that reacts with an anti-streptolidine O antibody, for example, a synthetic protein that is a part or all of the hemolytic active site deleted Or serum, plasma containing synthetic peptides, etc.
Examples thereof include biological fluids such as saliva and urine, tissue extracts, tissue culture solutions, microbial cell extracts, microbial cell cultures and the like. These test liquids are the test liquid stock solutions or the test solution stock solutions are appropriately buffered in consideration of the principle of the method for determining streptolysin O used in the present invention, the detection sensitivity, the measurement range and other factors. It is used in the present invention after being appropriately diluted with a liquid or the like.

As additives for the purpose of stabilizing streptolysin O in the test solution or preventing non-specific adsorption of proteins and the like to the carrier as described above, for example, bovine serum albumin (BSA), gelatin It is optional to appropriately add proteins such as skim milk or milk-derived proteins, sugars, glycerol, ethylene glycol, chelating agents, reducing agents and the like to water or a buffer solution.
The concentration range and the like may be appropriately selected from, for example, the concentration range and the like usually used in the immunological determination method of anti-streptolidine O antibody utilizing the antigen-antibody reaction known per se. However, since streptolysin O is relatively unstable in an aqueous solution, for the purpose of stabilization, proteins such as bovine serum albumin (BSA), gelatin, skim milk, or proteins derived from milk are usually added to 0.1%. 001% to 10%, preferably 0.01% to 1
It is desirable to add it in water or a buffer solution in a concentration range of%.

For the purpose of preventing non-specific adsorption of protein or the like to the carrier, a carrier on which the steroid compound of the present invention has been immobilized is used, for example, protein such as bovine serum albumin (BSA), gelatin, skim milk or milk-derived protein. It is optional to pretreat the above with a solution in which water or a buffer solution is dissolved. The concentration range and the like may be appropriately selected, for example, from the concentration range ordinarily used in the immunological determination method of an antibody utilizing an antigen-antibody reaction known per se, but usually 0.05% to 10%, preferably It suffices if it is selected from the concentration range of 0.5% to 5%.

The anti-streptolidine O antibody used in the present invention is not particularly limited as long as it binds to streptolysin O. For example, as one of the sources of the anti-streptolidine O antibody used in the present invention, , Sera of patients infected with group A hemolytic streptococci, which may be used after being appropriately diluted or purified. Also, Japanese Patent Publication No. 6-503002 and Japanese Patent Laid-Open No. 1-290.
In accordance with the method disclosed in Japanese Patent No. 698, etc., experimental animals,
For example, a polyclonal antibody or a monoclonal antibody obtained by immunizing a rabbit or mouse with an immunogen containing streptolysin O can be used. Furthermore, for convenience, a domestic standard anti-streptolidine O antibody that can be obtained from the National Institute of Preventive Health may be used.

Further, naturally, in consideration of the principle of the method for determining streptolysin O used in the present invention, these antibodies can be obtained by reacting a proteolytic enzyme such as papain or pepsin. Possible enzymatic degradation fragments, eg F (ab ')
_{The use of 2} , F (ab) ₂ , Fab ′, Fab and the like is optional.

The buffer solution used in the present invention is not particularly limited as long as it is a commonly used buffer solution.
It is desirable to use a buffer having a pH range of H4 to 10, preferably pH 6 to 8, for example, phosphate buffer, borate buffer, carbonate buffer, Tris buffer, veronal buffer, Good's. A buffer (eg, HEPES buffer, PIPES buffer,
A scalpel (MES) buffer solution or the like can be used.

In the present invention, the method for determining streptolysin O naturally varies depending on the type of the carrier on which the steroid compound is immobilized or the principle of the method for confirming or quantifying the antigen-antibody complex. The reaction conditions may be selected in accordance with the examples shown below, and the reaction conditions may be selected from the known reaction conditions usually used for each determination method, which are considered to be optimal.

For example, in the case of using a microtiter plate, beads, balls, test tubes or the like on which the present steroid compound is immobilized, first, a stock solution of the test solution or a diluted test solution and the carrier are prepared. Contact, usually 4
C. to 40.degree. C., preferably in the temperature range of 4.degree. C. to 30.degree. C., usually for 5 minutes to 24 hours, preferably for 10 minutes to 12 hours, after standing, stirring or shaking to react, The carrier is washed with water or the aforementioned buffer solution to remove components not bound to the carrier.

Next, for example, an enzyme immunoassay (enzyme immunoass) well known to those skilled in the art.
Say; EIA) method and the like, and the carrier and the anti-streptolysin O antibody, and a second antibody that can react with the anti-streptolysin O antibody and is labeled with a labeling agent (hereinafter, labeled second antibody). , Or) and then the activity of the labeling agent bound to the carrier via the antigen-antibody complex is measured, and streptolysin O is determined from the calibration curve obtained with any streptolidine O standard solution. Method or biotin, which is one of the B vitamins,
Utilizing the property of egg white to selectively bind to the basic glycoprotein avidin, in the above measurement method, a biotin-bound antibody is used instead of the labeled second antibody, and then avidin labeled with a labeling agent is also used. Reaction with streptavidin, and then the activity of the labeling agent bound to the carrier through the antigen-antibody complex is measured.
Streptolysin O in the test solution can be determined by a method of determining streptolysin O from the calibration curve obtained from the standard solution.

For example, when a microtiter plate, beads, balls, test tubes or the like on which the present steroid compound is immobilized are used, the undiluted test solution or diluted test solution is contacted with the carrier. And usually 4 ℃
~ 40 ° C, preferably 4 ° C to 30 ° C, usually 5 minutes to 24 hours, preferably 10 minutes to 12 hours, after allowing to stand, stirring or shaking to react,
The carrier is washed with water or the above-mentioned buffer to remove non-binding components from the carrier, and then, according to a well-known enzyme immunoassay method, an anti-streptolidine O antibody labeled with a labeling agent is used. (Hereinafter, labeled anti-streptolysin O
Antibody), and then the activity of the labeling agent bound to the carrier through the antigen-antibody complex is measured, and streptolysin O can be determined from the calibration curve obtained using any streptolidine O standard solution. . The labeled anti-streptolysin O antibody used at this time is, 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 37-45, Kyoritsu. It is sufficient to use a labeled anti-streptolidine O antibody that can be used in the present invention, according to the method described in "Publishing Co., Ltd., published September 10, 1987".

Furthermore, for example, when a membrane or the like on which the present steroid compound is immobilized is used, for example, immunostaining, which is one of the enzyme immunoassay methods well known to those skilled in the art, is used.
g) In accordance with the method etc., first, the test solution stock solution or diluted test solution is brought into contact with the membrane in a suitable reaction container, or the test solution stock solution or diluted test solution is treated with the steroid compound. Normally, 4
C. to 40.degree. C., preferably in the temperature range of 4.degree. C. to 30.degree. C., usually for 5 minutes to 24 hours, preferably for 10 minutes to 12 hours, after standing, stirring or shaking to react, Wash the membrane with water or the buffer described above.
Then, according to the method described above, the anti-streptolidine O antibody and the labeled second antibody are reacted sequentially or simultaneously, or the labeled anti-streptolidine O antibody is reacted. Furthermore, the activity of the labeling agent bound to the carrier via the antigen-antibody complex can be detected, and the activity can be visually confirmed or quantified with a densitometer to determine the streptolysin O in the test solution. it can.

In the above-mentioned method, the labeling agent includes, for example, all the enzymes usually used in the enzyme immunoassay method, preferably peroxidase, alkaline phosphatase, β-galactosidase or glucose oxidase can be appropriately selected. For the detection of the enzyme activity, "Enzyme-linked immunosorbent assay, protein nucleic acid enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 51-63, Kyoritsu Shuppan Co., Ltd., 1987.
Issued September 10, 2014 "and the like.

Further, it may be a radioactive substance ordinarily used in the radioimmunoassay method or a fluorescent substance ordinarily used in the immunofluorescence method or the like well known to those skilled in the art. The second antibody or biotin-conjugated antibody to be labeled is not particularly limited as long as it binds to the anti-streptolysin O antibody used in the reaction, and it may be a polyclonal antibody, a monoclonal antibody, or an enzymatic degradation fragment of these antibodies, for example, , F (ab ') ₂ , F
It can be used regardless of (ab) ₂ , Fab ′, Fab and the like. Further, the antibody protein may be derived from any animal species, or may be produced by Escherichia coli, yeast or other hosts by gene recombination technology. Usually, for example, when the anti-streptolidine O antibody is derived from human, conveniently, an antibody obtained by immunizing a non-human animal with human immunoglobulin as an antigen may be used. An anti-human immunoglobulin antibody may be used. Also,
For example, when the anti-streptolidine O antibody is derived from a mouse, an antibody obtained by immunizing an animal other than a mouse with mouse immunoglobulin as an antigen may be conveniently used. More conveniently, a commercially available anti-mouse may be used. An immunoglobulin antibody may be used.

The method for labeling the antibody with the labeling agent is, for example,
"Enzyme-linked immunosorbent assay, protein nucleic acid enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 37-45
Page, Kyoritsu Shuppan Co., Ltd., published September 10, 1987 ”and the like. In addition, as a matter of course, it may be appropriately selected from the commercially available labeled secondary antibodies usually used in general immunological assay methods and used in the present invention.

Furthermore, in the present invention, as an example of the method for determining streptolidine O, for example, when latex particles having the present steroid compound immobilized thereon are used, first, a test solution stock solution or a diluted test solution is used. The liquid and latex particles having the present steroid compound immobilized thereon are contacted with each other in water or the above-mentioned buffer solution, and the temperature is usually 0 ° C to 50 ° C, preferably 4 ° C to 40 ° C, and usually 5 minutes to 24 hours. , Preferably standing in a time range of 10 minutes to 12 hours,
After the reaction is carried out by stirring or shaking, the latex particles are washed with water or the above-mentioned buffer solution or the like, if necessary.

At this time, the concentration of the latex particles in the reaction solution is usually 0.01% to 10%, preferably 0.05% to 5% as the solid concentration.
Next, for example, latex slide agglutination method (Satomi Shibata, et al., Equipment / Reagents, Vol. 11, 338-342, 19
1988), a method of mixing the latex particles and an anti-streptolidine O antibody solution on a determination plate, and visually confirming the presence or absence of aggregation of the latex particles due to an antigen-antibody reaction, an immunoratio measurement (nephelomericim).
Munioassay; NIA) method (Yamagishi Yasuko, Laboratory, Volume 23, Extra edition, pages 1286 to 1289, 1
979), the antigen-antibody complex generated by mixing the latex particles and the anti-streptolidine O antibody solution is irradiated with light (laser light), and the amount of change in the intensity of scattered light is measured. , A method for determining streptolysin O from a calibration curve obtained using an arbitrary standard solution of streptolysin O, latex agglutination immunoassay (latex photo
metric immunoassay; LPIA) method (Ikunosuke Sakurabayashi et al., Japan Clinic, 48th volume, special issue (second volume), pp. 1356-1361, 1990), according to the latex particles and the anti-streptolidine O antibody solution. And a method of determining streptolysin O from a calibration curve obtained by arbitrarily measuring streptolysin O standard solution by optically measuring turbidity resulting from the antigen-antibody reaction, or a counting immunoassay (counting immunoassay).
unoassay; CIA) method (Yoshikazu Hashimoto et al., Inspection and Technology, Volume 22, No. 5, Special Issue, pp. 67-68, 19
1994), mixing the latex particles with an anti-streptolidine O antibody solution, measuring the size and number of aggregates resulting from the antigen-antibody reaction, and calibrating with any streptolidine O standard solution. Streptolidine O in the test solution can be determined by a method of determining streptolysin O from the line.

The method for confirming or quantifying the product of the antigen-antibody reaction in the present invention is not limited to the manual method, and may be a method using an automatic analyzer. In the present invention, for example, in the case of determining streptolysin O in a test solution using the present steroid compound-immobilized microtiter plate, the present steroid compound-immobilized test tube or the like by an enzyme immunoassay method, for example, BECKM is an automated system for liquid dispensing, reagent dispensing, washing operation, absorbance measurement, data processing, etc.
AN Biomek 1000 Automated
Laboratory Workstation (manufactured by Beckman), CELL ASSAY-2000ROB
OTIC ASSAY SYSTEM (manufactured by Moritex Co., Ltd.) and the like can be used. Specifically, a test solution, an anti-streptolidine O antibody solution, and an enzyme-labeled second solution are applied to the steroid compound-immobilized microtiter plate or the steroid compound-immobilized test tube. The antibody solution and the substrate solution are sequentially reacted by inserting a washing operation, and after reacting with an enzyme reaction stopping solution as needed, the color development in the enzymatic reaction due to the relationship between the labeled enzyme and the substrate used is determined under known measurement conditions. Streptolysin O in the test solution can be determined by measuring at an arbitrary wavelength selected and comparing the result with a calibration curve obtained with an arbitrary streptolidine O standard solution.

Further, for example, in the case where streptolysin O in a test solution is determined using beads, balls or the like having the present steroid compound immobilized by an enzyme immunoassay method, for example, the test solution is dispensed. , ALOCA AEC-2000PO, which is an automated system for reagent dispensing, washing operation, absorbance measurement, data processing, etc.
SEIDONII (manufactured by Aloka Co., Ltd.) can be used. Specifically, the test solution, anti-streptolidine O antibody solution, enzyme-labeled second antibody solution and substrate solution are washed on beads or balls on which the steroid is immobilized. Then, after reacting with the enzyme reaction stop solution as needed, the color development in the enzyme reaction due to the relationship between the labeled enzyme and the substrate used is measured at any wavelength selected from known measurement conditions. By comparing this result with a calibration curve obtained using an arbitrary streptolidine O standard solution, streptolysin O in the test solution can be determined.

The method of the present invention is easier to operate if the reagents necessary for carrying out the present invention are provided in a kit.
It is also advantageous when the present invention is carried out using an automatic analyzer. The reagent kit used in the present invention is
It is particularly advantageous that the steroid compound-immobilized carrier used in the present invention is one of the kit-constituting reagents. The reagent kit used in the present invention naturally includes other reagents necessary for carrying out the present invention (for example, buffers, standard substances, labeled antibodies, substrates, substrate lysing agents, reaction terminators, etc. ) May be appropriately contained as a component reagent of the kit.

[0061]

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited thereto.

[0062]

Example 1 Investigation of Reactivity between Immobilized Steroid Compound and Streptolidine O (1) Preparation of Streptolidine O Todd-Hewitt B
roth: Difco) A with 2.5 liters of medium
Streptococcus py
genes) type 3 strain D58X strain (ATCC1238
After culturing 3) at 37 ° C. for 16 hours, the sterilized culture solution was recovered by centrifugation and filtration. Saturated ammonium sulfate was added to this sterilized culture solution to 40% saturation, and the resulting precipitate was 100 ml.
Dissolved in purified water. This solution was dialyzed against purified water and PBS to remove ammonium sulfate, and then subjected to gel filtration with Sephadex G-100 (gel amount: 1 liter) equilibrated with PBS, and the hemolytic activity was used as an index for streptolysis. The lysine O fraction was collected. Upon concentration, the absorbance measured at 280 nm was 0.825, and the hemolytic activity was 8,19.
12 ml of a sample containing streptolysin O showing 2 hemolytic units (HU) / ml was obtained.

To measure the hemolytic activity, fresh rabbit defibrinated erythrocytes (manufactured by Japan Biotest Institute Co., Ltd.) were centrifugally washed 5 times with PBS, and then PB was adjusted to a 1% erythrocyte suspension.
2 ml of the solution prepared in S and 1 ml of a solution in which a sample containing streptolysin O was diluted in PBS at an equal ratio were mixed in a glass test tube, incubated at 37 ° C. for 30 minutes, and then streptolysin O showing 100% hemolysis. Check the highest dilution factor of the contained sample and check it
It was defined as the HU value per 1 ml of the contained sample. (2) Preparation of cholesterol-immobilized carrier Microtiter plate (Sumitomo Bakelite Co., Ltd.)
100 μl / well of ethanol solution of cholesterol (manufactured by Hanai Chemical Co., Ltd.) with a concentration of 1 mg / ml.
A microtiter plate with immobilized cholesterol was prepared by adding the solution, allowing it to stand at 60 ° C. for 2 hours, and distilling off the solvent. (3-1) Reaction of cholesterol-immobilized carrier with streptolidine O-containing solution 1 The streptolidine O-containing sample prepared by (1) is diluted with PBS to 20 times,
50 μl per well was added to the wells of the cholesterol-immobilized microtiter plate and cholesterol-unimmobilized microtiter plate prepared in (2), and the mixture was reacted at room temperature for 2 hours. The reaction solution was recovered from the well, and the recovered solution was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and hemolytic activity measurement (the method described in Example 1 (1)), and compared with that before the reaction.

SDS-polyacrylamide gel electrophoresis was carried out using a 12.5% polyacrylamide gel, using Laemmli, UK, Nature, 2nd.
27, p. 680-685, 1970). In the same gel, SDS-PAGE molecular weight standard low range (phosphorylase B: 9
2,500, bovine serum albumin: 66,200, ovalbumin: 45,000, carbonic anhydrase: 31,000, soybean trypsin inhibitor: 21,500, lysozyme: 14,400; manufactured by Bio-Rad) , Was used as a standard for confirming the molecular weight of each protein band of the recovered solution and the pre-reaction solution separated by SDS-polyacrylamide gel electrophoresis (lane 1 in FIG. 1). The gel after electrophoresis was subjected to silver staining with Silver Staining Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
(3-2) Reaction of cholesterol-immobilized carrier with streptolidine O-containing solution 2 The streptolidine O-containing sample prepared by 2 (1) is diluted with PBS to 20 times,
100 μl per well was added to the wells of the cholesterol-immobilized microtiter plate prepared in (2), and the mixture was reacted at room temperature for 4 hours. After removing the reaction solution from the well and washing the well three times with PBS, 100 μl of PBS containing 0.1% sodium dodecyl sulfate (SDS) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and the mixture was allowed to stand at room temperature for 1 hour.
The adsorbed material was extracted by shaking the plate for an hour.

This extract was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis,
It was compared with that before the reaction. For SDS-polyacrylamide gel electrophoresis, 12.5% polyacrylamide gel was used.
Laemmli, UK, Nature
e, Vol. 227, pp. 680-685, 1970).

In the same gel, SDS-PAGE molecular weight standard low range (phosphorylase B: 9
2,500, bovine serum albumin: 66,200, ovalbumin: 45,000, carbonic anhydrase: 31,000, soybean trypsin inhibitor: 21,500, lysozyme: 14,400; manufactured by Bio-Rad) , Was used as a standard for confirming the molecular weight of each protein band in the extract and the pre-reaction solution separated by SDS-polyacrylamide gel electrophoresis (lane 3 in FIG. 2). The gel after electrophoresis was subjected to silver staining with Silver Staining Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.). (4) Results As a result of (3-1), the SDS-PAGE pattern shown in FIG. 1 was obtained. In FIG. 1, lane 2 is a solution (pre-reaction solution) before reacting with a cholesterol-immobilized microtiter plate or a cholesterol-unimmobilized microtiter plate, that is, a sample containing streptolysin O prepared in (1) with PBS. It means the separation pattern of the proteins contained in the 20-fold diluted solution, and FIG.
In the figure, lane 3 means the separation pattern of the proteins contained in the recovered solution recovered from the cholesterol-immobilized microtiter plate, and in FIG. 1, lane 4 represents the recovered solution recovered from the cholesterol-immobilized microtiter plate. It means the separation pattern of the protein contained in, and from this result, the molecular weight of the result of (3-1) is 66,000 to 69,000 and 55,000 to 5.
It was confirmed that the band of streptolysin O showing 8,000 was selectively bound and disappeared by reacting with the cholesterol-immobilized carrier. Further, the hemolytic activity of the recovered liquid residue from the cholesterol-fixed microtiter plate was only 0.8% of the activity before the reaction was recovered.

On the other hand, as a result of (3-2), SD shown in FIG.
An S-PAGE pattern was obtained. In FIG. 2, lane 1 is from a cholesterol-fixed microtiter plate (3-
2) means the separation pattern of the protein contained in the extract extracted by the method of 2), and also shows lane 2 in FIG.
Means a separation pattern of proteins contained in a solution before reacting with the cholesterol-immobilized microtiter plate (pre-reaction solution), that is, a solution obtained by diluting the streptolysin O-containing sample prepared in (1) with PBS 20 times. From this result, from the result of (3-1), the molecular weight 66 disappeared from the solution containing streptolysin O,
000 to 69,000 and 55,000 to 58,000
It was confirmed that streptolysin O showing 0 was actually bound to the cholesterol-immobilized carrier.

From the above results, it was revealed that streptolidine O having biological activity in the streptolidine O-containing solution specifically binds to the cholesterol-immobilized carrier.

[0069]

Example 2 Measurement of Streptolidine O (1) Preparation of Cholesterol-Immobilized Carrier A solution prepared by suspending cholesterol (manufactured by Hanai Chemical Co., Ltd.) in a 10% ethanol aqueous solution to a concentration of 100 μg / ml was prepared. 50 μl per well was added to the wells of a microtiter plate (manufactured by Sumitomo Bakelite Co., Ltd.) and allowed to stand at room temperature for 2 hours, and then the wells were washed twice with purified water. Then, 20 mM Tris-HCl buffer (pH
A solution (hereinafter referred to as a blocking solution) in which skim milk (manufactured by Snow Brand Milk Products Co., Ltd.) was dissolved in 7.6, containing 0.14 M sodium chloride to a concentration of 5% (hereinafter referred to as blocking solution) was added to 10 wells.
After adding 0 μl and reacting at room temperature for 2 hours, the wells were washed twice with 20 mM Tris-hydrochloric acid buffer (pH 7.6, containing 0.14 M sodium chloride) (hereinafter referred to as a washing solution). (2) Preparation of Streptolidine O Standard Solution Commercially available streptolysin O (S-5265, Lot34
H4124, Sigma) was dissolved in 20 mM Tris-hydrochloric acid buffer (pH 7.6, containing 0.14 M sodium chloride), and this streptolidine O solution was dissolved in Example 1
The hemolytic activity was determined according to the method described in (1). Based on the hemolytic activity of this streptolidine O lysate, with 20 mM Tris-HCl buffer (pH 7.6, containing 0.14 M sodium chloride) containing 0.5% skim milk (hereinafter referred to as the diluent) The streptolidine O solution was diluted to prepare a solution having a hemolytic activity of 160 hemolytic units (HU) / ml, and this solution was used as a streptolysin O standard solution. (3) Preparation of calibration curve BECKMAN Biomek 1000 Autom
arated Laboratory Workstati
was used to measure streptolysin O.
To the cholesterol-immobilized microtiter plate prepared in (1), 50 μl per well of a solution obtained by diluting the streptolidine O standard solution prepared in (2) with a diluent was added and reacted at room temperature for 2 hours. Let
After washing the wells 7 times with the washing solution, a solution of anti-streptolidine O antibody domestic standard (obtained from the National Institute of Preventive Health) was prepared with a diluting solution to 1 international unit (IU) / ml per well. After adding 50 μl and reacting at room temperature for 1 hour, the wells were washed 7 times with a washing solution.

Then, 50 μl per well of a 1,000-fold dilution of alkaline phosphatase-labeled goat F (ab ′) ₂ anti-human IgG (manufactured by Tago) was added to each well, and the mixture was reacted at room temperature for 1 hour. The wells were washed 7 times with the washing solution. Then, p-nitrophenyl phosphoric acid (manufactured by Wako Pure Chemical Industries, Ltd.) as a substrate solution was adjusted to a concentration of 0.5 mg / ml in diethanolamine buffer (pH 9.5).
100 μl of the solution dissolved in 1 was added to each well and reacted at room temperature for 15 minutes. Further, 100 μl of 0.5N sodium hydroxide aqueous solution was added to each well to stop the enzyme reaction, and ImmunoReader NJ-2001 was added.
The absorbance (ΔE) at 405 nm was measured by (manufactured by Intermed). (4) Results The calibration curve of this method is shown in FIG. From the results of FIG. 3, it was found that a good calibration curve can be obtained by the method of the present invention.

[0071]

Example 3 Test for Examining Stability of Streptolysin O Bound to Cholesterol-Immobilized Carrier (1) Preparation of Cholesterol-Immobilized Carrier 100 μg of cholesterol (manufactured by Hanai Chemical Co., Ltd.) in 10% ethanol aqueous solution 50 μl per well was added to the well of a microtiter plate (Sumitomo Bakelite Co., Ltd.), and the suspension was allowed to stand at room temperature for 2 hours, and then the well was washed twice with purified water. Washed. Then, 20 mM Tris-HCl buffer (pH
A solution (hereinafter referred to as a blocking solution) in which skim milk (manufactured by Snow Brand Milk Products Co., Ltd.) was dissolved in 7.6, containing 0.14 M sodium chloride to a concentration of 5% (hereinafter referred to as blocking solution) was added to 10 wells.
0 μl was added, and the mixture was reacted at room temperature for 2 hours. The wells were washed twice with 20 mM Tris-hydrochloric acid buffer (pH 7.6, containing 0.14 M sodium chloride) (hereinafter referred to as a washing solution). (2) Measurement of streptolysin O BECKMAN Biomek 1000 Autom
arated Laboratory Workstati
was used to measure streptolysin O.
For the cholesterol-immobilized carrier prepared in (1), commercially available streptolysin O (S-5265, Lot
34H4124, Sigma) in 20 mM Tris-HCl buffer (pH 7.6, containing 0.5% skim milk).
Well containing a solution prepared so that the hemolytic activity determined by the method described in Example 1 (1) was 20 hemolytic units (HU) / ml (containing 0.14 M sodium chloride) (hereinafter referred to as a diluent). 50 μl was added to each well, and the mixture was reacted at room temperature for 2 hours.

After washing the wells 7 times with the washing solution, the following various media (medium having high ionic strength, medium in acidic region to weak alkaline region, medium containing chelating agent, medium containing cholesterol-soluble solvent, etc.), 1M NaCl PBS containing, 1
MKCl-containing PBS, 0.1 M citric acid aqueous solution,
0.1M sodium bicarbonate aqueous solution, 0.2% ED
TA-containing PBS, 20% ethanol aqueous solution, or
PBS as a control solution, 50μ per well
1 was added and treated at room temperature for 15 minutes. After washing the wells 3 times with the washing solution, the anti-streptolidine O antibody domestic standard product (obtained from the National Institute of Preventive Health) was prepared with a diluting solution to 1 international unit (IU) / ml, and the solution was added per well. 50 μl was added and reacted at room temperature for 1 hour.

After washing the wells 7 times with the washing solution, alkaline phosphatase-labeled goat F was used as the enzyme-labeled second antibody.
(Ab ′) ₂ anti-human IgG (manufactured by Tago) was diluted 1,000 times with a diluent, and 50 μl of the solution was added to each well, and the mixture was reacted at room temperature for 1 hour. The wells were washed 7 times with the washing solution, and then p-nitrophenyl phosphoric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the diethanolamine buffer solution (pH 9.5) to a concentration of 0.5 mg / ml as a substrate solution. Add 100 μl of the dissolved solution to each well and
The reaction was performed for 5 minutes. Further, 100 μl of 0.5 N sodium hydroxide aqueous solution was added to each well to stop the enzyme reaction, and ImmunoReader NJ-2001 was added.
The absorbance (ΔE) at 405 nm was measured by (manufactured by Intermed). (3) Results The results of measuring streptolidine O by this method are shown in FIG.
From the result of FIG. 4, 1 MN was compared with the control solution (PBS).
aCl-containing PBS, 1M KCl-containing PBS, 0.1M citric acid aqueous solution, 0.1M sodium bicarbonate aqueous solution,
No change in the measured value of streptolysin O due to contact with 0.2% EDTA-containing PBS or 20% ethanol aqueous solution was observed, and medium with high ionic strength, medium in acidic range to weakly alkaline range, medium containing chelating agent, and cholesterol It was found that the binding between the steroid-immobilized carrier and streptolysin O in the present invention is stable and can be quantified with extremely reproducibility even when various media containing a solvent are used.

[0074]

Example 4 Test for Examining Storage Stability of Cholesterol-Immobilized Carrier (1) Preparation of Cholesterol-Immobilized Carrier Cholesterol (manufactured by Hanai Chemical Co., Ltd.) at a concentration of 100 μg / ml was added to 10% ethanol aqueous solution. The thus-suspended solution was added to the wells of a microtiter plate (Sumitomo Bakelite Co., Ltd.) in an amount of 50 μl per well, allowed to stand at room temperature for 2 hours, and then the wells were washed twice with purified water. After washing, 250 μl of purified water containing 0.02% sodium azide was added to the wells. Eight cholesterol-fixing microtiter plates formed by this operation were prepared and stored at 4 ° C until the day of use. (2) Measurement of Streptolidine O Using the cholesterol-immobilized microtiter plate prepared in (1), the microtiter plate preparation date and 1, 2, 5, 10,
BECKMAN Biomek on days 20, 30 and 35
1000 Automated Laborator
Streptolidine O was measured using y Workstation. First, 20 mM Tris-hydrochloric acid buffer solution (pH 7.6, containing 0.14 M sodium chloride) was added to the cholesterol-immobilized carrier prepared in (1).
Skim milk (Snow Brand Milk Products Co., Ltd.)
100 μl was added per well to a solution (hereinafter referred to as blocking solution) in which the product was dissolved, and the mixture was reacted at room temperature for 2 hours.

Then, 20 mM Tris-HCl buffer (p
The wells were washed twice with H7.6 containing 0.14 M sodium chloride (hereinafter referred to as a washing solution). Commercially available streptolysin O (S-5265, Lot34H4124,
Sigma) 20m containing 0.5% skim milk
Example 1 with M Tris-hydrochloric acid buffer solution (pH 7.6, containing 0.14 M sodium chloride) (hereinafter referred to as a diluent)
The hemolytic activity determined by the method described in (1) is 20.
A solution prepared so as to have a hemolytic unit (HU) / ml was added at 50 μl per well and reacted at room temperature for 2 hours. After washing the wells 7 times with the washing solution, a solution of anti-streptolidine O antibody domestic standard (obtained from the National Institute of Preventive Health) was prepared with a diluting solution to 1 international unit (IU) / ml per well. 50 μl was added and reacted at room temperature for 1 hour.

After washing the wells 7 times with the washing solution, alkaline phosphatase-labeled goat F was used as the enzyme-labeled secondary antibody.
(Ab ′) ₂ anti-human IgG (manufactured by Tago) was diluted 1,000 times with a diluent, and 50 μl of the solution was added to each well, and the mixture was reacted at room temperature for 1 hour. The wells were washed 7 times with the washing solution, and then p-nitrophenyl phosphoric acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the diethanolamine buffer solution (pH 9.5) to a concentration of 0.5 mg / ml as a substrate solution. Add 100 μl of the dissolved solution to each well and
The reaction was performed for 5 minutes. Further, 100 μl of 0.5 N sodium hydroxide aqueous solution was added to each well to stop the enzyme reaction, and ImmunoReader NJ-2001 was added.
The absorbance (ΔE) at 405 nm was measured by (manufactured by Intermed). (3) Results The results of streptolidine O measurement by this method are shown in FIG.
From the results of FIG. 5, it was shown that the cholesterol-immobilized carrier formed according to the present invention has good storage stability.

[0077]

INDUSTRIAL APPLICABILITY According to the present invention, by using a carrier on which a steroid compound capable of binding streptolidine O is immobilized, streptolysin O can be specifically determined by an immunological method. Moreover, a stable and inexpensive determination method can be provided without requiring unstable red blood cells. The present invention exerts a remarkable effect in the above points as compared with the conventional method.

[Brief description of drawings]

FIG. 1 shows an electrophoretic pattern of a recovered solution after contacting a cholesterol-immobilized carrier with a solution containing streptolysin O according to Example 1 (3-1).

FIG. 2 shows an electrophoretic pattern of components bound to a carrier as a result of contacting a cholesterol-immobilized carrier with a solution containing streptolysin O according to Example 1 (3-2).

FIG. 3 is a diagram showing Streptolidine O in Example 2.
The calibration curve by the determination method is shown.

FIG. 4 shows the binding stability of streptolysin O bound to the carrier in Example 3.

FIG. 5 shows the storage stability of the cholesterol-immobilized carrier in Example 4.

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[Procedure amendment]

[Submission date] March 22, 1995

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

FIG.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

[Fig. 2]

Claims (5)

[Claims] 1. A method for immunologically determining streptolysin O in a test solution, comprising the following general formula (1): (In the formula, R1 is a substituted or unsubstituted, unsaturated or saturated cholesterol side chain group or cholic acid side chain group, R2 is a hydrogen atom or a hydroxyl group, and a broken line site is a saturated or double bond site. The method for determining streptolysin O is characterized by using a carrier on which one or more steroid compounds represented by the formula (1) are immobilized. 2. The steroid compound is 250 nmol /
The method according to claim 1, wherein the amount of binding per unit area of the carrier is from cm ^{2 to} 0.025 nmol / cm ² . 3. The steroid compound is 25 nmol / c
The method according to claim 1, wherein the binding amount is m ^{2 to} 0.25 nmol / cm ² per unit area of the carrier. 4. The steroid compound in the general formula (1) is cholesterol, 7-dehydrocholesterol, cholestanol, coprostanol, Δ ⁷ -cholestenol, Δ ⁷ -coprostenol, β-sitosterol, 7
- dehydro -β- sitosterol, stigmasterol, 7-dehydro loss stigmasterol, campesterol, 7-dehydrocholesterol campesterol, stigmastanol, delta ⁷ - stigmastanol mass tenor, 11Arufa- hydroxycholesterol, delta ²² - Suchigumasu Tenol, α-spinasterol, campestanol, Δ ⁷ -campestanol, 20α-hydroxycholesterol, brassicasterol, ergosterol, cerebrosterol, 7-
Dehydro cerebrosterol, cerebrostanol, Δ
⁷ - Celebrity loss tenor, desmosterol, 7-dehydrocholesterol desmosterol, 3.beta .- hydroxy cholanic acid or 3.beta .- hydroxy - [delta ⁵ - The method of claim 1 wherein the cholenic acid. 5. The method according to claim 1, wherein the carrier is a microtiter plate, latex particles, beads, balls, test tubes or membranes.