JP3239456B2 - Method for dissolving liposome membrane and immunoassay using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for dissolving liposome membranes utilizing the reaction between avidin and biotin, and a novel method for quantifying antigens, antibodies, sugar chains, lectins, nucleic acids, etc. using the dissolution method. About the method.

[0002]

BACKGROUND OF THE INVENTION An immunoassay is an assay utilizing an antigen-antibody reaction, such as protein, hormone,
It is widely used as one of the methods for specifically measuring biological components such as active peptides, autakoids, tumor markers, and immunoglobulins, and trace components such as drugs such as digoxin, phenytoin, and phenobarbital. Examples of immunoassays that are currently generally used include, for example, a radioimmunoassay (RIA) and an enzyme immunoassay (EIA). Each of these methods is a method capable of quantitatively measuring a trace component in a specimen, but each has its own problems. That is, in the RIA method, radioisotopes must be used, so that special equipment is required, and the problem of waste disposal becomes a problem. However, there are problems in that the measurement takes a relatively long time and that application to an automatic analyzer is difficult.

Therefore, recently, as an immunoassay method which does not have these problems, an immunoassay method using liposomes (hereinafter abbreviated as liposome immunoassay method) has been proposed and attracted attention. A representative example of this method is the method described in JP-A-56-132564. In this method, a liposome in which a substance to be measured is immobilized on a surface and a labeling substance (eg, an enzyme or the like) is held, a sample, and an antibody to the substance to be measured are mixed, and an antigen-antibody reaction is performed. Upon addition, the complement activated by the antigen-antibody complex formed on the surface of the liposome dissolves the liposome membrane, allowing the labeling substance in the liposome to elute, and measuring the amount in the sample from the amount. This is a method of measuring the amount of a target substance. Liposome immunoassay using this complement (hereinafter abbreviated as complement immunoassay).
Is a measurement method that has attracted attention because it does not have the above-mentioned problems of the RIA method or the EIA method and can perform a series of reactions in a homogeneous reaction system, so that measurement can be performed easily and in a short time. is there.

However, in the complement immunoassay, there is a problem in the stability of the measuring reagent because complement itself is an unstable substance, and the complement reacts non-specifically with the measuring reagent to convert complement. Dissolution of liposome membranes other than complement can be caused by the presence of a substance in the sample that has the effect of activating and destroying part of the liposome to dislodge the labeling substance, causing measurement errors. A liposome immunoassay using an active substance (hereinafter abbreviated as a membrane lysing agent) was then developed.

That is, a method using cytolysin represented by melittin, which is a main component of bee venom, reported by Richfield et al. This method prepares a complex of a melittin molecule and a substance to be measured, and takes advantage of the fact that the membrane lytic activity of melittin is lost when the complex binds to an antibody against the substance to be measured. This is a method for quantifying a substance to be measured by reacting the body and a free substance to be measured with the antibody competitively, and then measuring the liposome membrane dissolving activity of the remaining melittin (WJ Litchfield et al., Clin. Chem., Volume 30, 14
41, 1984). However, this method also has problems that it is difficult to obtain melittin and that sufficient liposome membrane dissolving activity cannot be obtained unless melittin is used at a high concentration. I can not say.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and provides a new liposome membrane dissolving method instead of a method using complement or cytolysin, and an antigen, antibody, It is an object of the present invention to provide a novel method for quantifying sugar chains, lectins, nucleic acids, and the like.

[0007]

The present invention provides a method for dissolving a liposome membrane and a method for dissolving a surfactant and avidin, wherein a surfactant and avidin are allowed to act on liposomes having biotin immobilized on the membrane surface in advance. A liposome membrane solubilizer comprising biotin immobilized on the membrane surface in advance, and a liposome comprising a surfactant and avidin for the liposome in which biotin is immobilized on the membrane surface in advance and a label is retained inside Membrane lytic activity is reduced by using a reaction between a substance to be measured and a substance having a property of specifically binding thereto, and the amount of a labeling substance that migrates from the liposome is measured according to the degree of the reduction. This is an invention of a quantification method characterized by quantifying the amount of a substance to be measured.

That is, the inventors of the present invention have intensively studied to find a new method for dissolving a liposome membrane instead of a method using complement or cytolysine. In the course of intensive research, liposomes having biotin immobilized on the membrane surface in advance (hereinafter referred to as biotin liposome) When a surfactant is present in the reaction of avidin, the liposome membrane is unexpectedly dissolved, and the degree of the dissolution corresponds to the amount of avidin to be reacted. And the reaction between a substance to be measured, such as an antigen, an antibody, a sugar chain, a lectin, or a nucleic acid, and a substance having a property capable of specifically binding to the substance. The present invention has been completed.

The biotin used in the present invention is not particularly limited as long as it has an ability to bind to avidin, and is derived from animal liver, meat, milk and eggs. It may be any of those derived from microorganisms, those derived from cereals or vegetables, and those synthesized.

[0010] The biotin liposome according to the present invention is not particularly limited as long as biotin is immobilized on the membrane surface of the liposome. Examples of the method for producing such biotin liposome include cross-linking. Liposomes are prepared by immobilizing biotin on the liposome membrane surface by a conventional method such as a lipid activation method or a lipid to which biotin has been covalently bonded in advance (hereinafter abbreviated as biotin-bound lipid). By incorporating biotin into the liposome membrane (Bayer et al., BBA, vol.550,
464, 1979, etc.). Among them, a method of incorporating biotin into a liposome membrane by preparing a liposome using a biotin-bound lipid is more preferable.
The amount of biotin immobilized on the biotin liposome membrane surface is not particularly limited as long as the liposome membrane is dissolved in the presence of avidin and a surfactant.
Usually, the range is 10 to 400 pmol, preferably 60 to 350 pmol.

As the biotin liposome containing a labeled substance therein (hereinafter, abbreviated as labeled biotin liposome) used in the present invention, the labeled substance is retained inside the biotin liposome obtained as described above. Any of them may be used. The method for preparing the liposome itself used in the present invention includes vortex swing method, ultrasonic method, surfactant removal method, reverse phase evaporation method (REV method), ethanol injection method, ether injection method, and the like. Pre-Vesicle method, French Press Extrusion
Method, Ca2 + fusion method, annealing (Annealing) method, freeze-thaw fusion method, freeze-drying method, W / O / W emulsion method, and recently, SMGruner et al. [Biochemistry, 24, 2833 (198
5)] Stable Plurilamellar Vesicle reported by
And the method of using lipopolysaccharide as a part of the membrane component reported by some of the present inventors (JP-A-63-107742). The main membrane components include natural lecithin (eg, egg yolk lecithin, soybean lecithin, etc.), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl, which are used as membrane materials in the preparation of ordinary liposomes. Phosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPS), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), One kind of phospholipid such as palmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidic acid (DMPA), egg yolk phosphatidyl glycerol, or glycolipid such as ganglioside glycolipid, glycosphingolipid, glyceroglycolipid, etc. Or two or more, or these and cholesterol Mixed system, or more like a combination of lipopolysaccharide etc. thereof all.

The method for preparing the biotin liposome (or labeled biotin liposome) according to the present invention will be described in detail below, taking as an example a case where the biotin liposome is prepared by a surfactant removing method using a biotin-bound lipid as one of the raw materials. I do. That is, first, for example, the above-mentioned known literature (Bayer et al., BBA, vol.5)
50, 464, 1979), dissolving a biotin-bound lipid and cholesterol prepared according to the method described in an appropriate organic solvent (eg, chloroform, ethers, alcohols, etc.) and concentrating to dryness under reduced pressure. After solidification, the mixture is sufficiently dried in a desiccator under reduced pressure. Next, an aqueous surfactant solution (20 to 100 mM) is added to the prepared lipid film, and the resultant is uniformly dispersed. Examples of the surfactant used here include cholic acid, polyoxyethylene octyl phenyl ether, and coxy acid, which are conventionally used in this field.
Octyl glucoside and the like can be mentioned, and a surfactant having a high critical micelle concentration (CMC) such as octyl glucoside is preferable. Next, if necessary, the lipopolysaccharides are added as a powder or as a solution, and if necessary, a solution containing an appropriate labeling substance is added, followed by sufficient stirring and mixing.
After this, it is most desirable to immediately remove the surfactant, and examples thereof include methods known per se, such as dialysis, gel filtration, and resin adsorption. The treatment conditions are 1 hour to 1 day and night, and the temperature varies slightly depending on the membrane components of the liposome, etc., but may be appropriately selected within the range of about 0 to 70 ° C. In particular, it is advantageous to remove the surfactant by performing an operation such as gel filtration or centrifugation using Sepharose 4B (trade name of Pharmacia) as a method for removing the surfactant, because free labeling substances and the like can be removed at the same time. The biotin liposome (or labeled biotin liposome) thus obtained is used or stored after being concentrated to a predetermined concentration by ultrafiltration or the like. Regarding the homogenization of the size of biotin liposomes (or labeled biotin liposomes), a method using a commonly used polycarbonate membrane may be used, but a gel filtration method [for example, using Sephacryl S-1000 (trade name of Pharmacia)] is used. It is also effective to do so.

[0013] The biotin liposome (or labeled biotin liposome) of the present invention by a method other than the surfactant removal method
In the case of preparing the liposome, it may be similarly performed according to a method known per se or another liposome preparation method.

The labeling substance retained in the labeled biotin liposome may be any labeling substance which is usually used in liposome immunoassay using complement and which can be detected when it has migrated out of the liposome. Examples thereof include, but are not limited to, enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase, and β-galactosidase, for example, 4-methylumbelliferyl β-D
-Galactoside, p-hydroxyphenylpropionic acid,
4-methylumbelliferyl phosphate, glucose
Substrates for enzymes such as -6-phosphate, for example, fluorescent substances such as carboxyfluorescein, fluorescein isothiocyanate, fluorescein isocyanate, tetrarhodamine isothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride, for example, arsenazo III, 4- (2
Dyes such as -pyridylazo) resorcinol, 2- (5-bromo-2-pyridylazo) -5- (N-propyl-N-sulfopropyl) aminophenol sodium salt, for example, luminol,
Isorluminol, luciferin, eosin Y, auramine O, bis (2,4,6-trichlorophenyl) oxalate,
Luminescent substances such as N-methylacridinium ester, for example, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMP
Typical examples thereof include spin labeling agents represented by O) and the like. Further, the amount of the labeling substance retained in the liposome is different depending on the type of the labeling substance and is not particularly limited. It is sufficient that the labeling substance is retained in such an amount that a sufficiently detectable difference before and after the liposome membrane is destroyed is retained. Taking the case of glucose-6-phosphate dehydrogenase as an example, an enzyme solution having a concentration of usually 1,000 to 5,000 U / ml, preferably 2,000 to 3,000 U / ml is used as a solution containing a labeling substance used in preparing liposomes. I just need.

The origin of avidin used in the present invention is not particularly limited as long as it has a binding ability to biotin, but usually, it is preferably derived from egg white. It is needless to say that those having the same action as avidin, such as streptavidin derived from microorganisms, can be used in the present invention in the same manner as avidin.

The surfactant used in the present invention includes, for example, polyoxyethylene alkyl ethers such as polyoxyethylene cetyl ether and polyoxyethylene lauryl ether, and polyoxyethylene alkyl such as polyoxyethylene octyl phenyl ether. Phenyl ethers, for example, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene alkyl esters such as polyoxyethylene sorbitan triolate, for example, octanoyl-N-methyl Methylglucamide derivatives such as glucamide, nonanoyl-N-methylglucamide, decanoyl-N-methylglucamide, and alkyl sugar derivatives such as n-octyl-β-D-glucoside Nonionic surfactants like, for example, sodium dodecyl sulfate (SDS), lauryl benzenesulfonic acid, deoxycholic acid, cholic acid, tris (hydroxymethyl) aminomethane dodecyl Sulfate (T
anionic surfactants such as ris DS), for example, alkylamine salts such as octadecylamine acetate, tetradecylamine acetate, stearylamine acetate, laurylamine acetate, lauryldiethanolamine acetate, for example, octadecyltrimethylammonium chloride, dodecyl chloride Trimethylammonium, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, lauryltrimethylammonium chloride, allyltrimethylammonium methylsulfate, benzalkonium chloride, tetradecyldimethylbenzylammonium chloride, octadecyldimethylbenzylammonium chloride, lauryldimethylbenzylammonium chloride, etc. Quaternary ammonium salts such as laurylpyridinium chloride, stearylamidomethylpyridinium chloride, etc. Cationic surfactants such as alkylpyridinium salts, 3-[(3-cholamidoamidopropyl) dimethylammonio] -1-propanesulfonate, 3-[(3-cholamidoamidopropyl) dimethylammonio] -2- Examples of the surfactant include amphoteric surfactants such as hydroxy-1-propanesulfonate, and natural surfactants such as saponin (derived from soybean) and digitonin. The concentration of these used in the dissolution method of the present invention varies depending on the membrane composition and the preparation method of the biotin liposome (or labeled biotin liposome) to be used, or the type of the surfactant. Then, the concentration of the biotin liposome (or labeled biotin liposome) or the concentration of the biotin liposome (or labeled biotin liposome) to be dissolved only when avidin coexists was dissolved without dissolving the biotin liposome (or labeled biotin liposome) membrane. As long as the concentration is such that the difference is sufficiently large compared to the case where avidin is dissolved in the coexistence, it may be any concentration, usually as the final concentration in the solution containing liposomes, 0.001 to 1.0 V / V
%, Preferably in the range of 0.02 to 0.5 V / V%.

When the amount of the biotin liposome (or labeled biotin liposome) used in the present invention is indicated by the concentration in the final reaction solution, the amount of the phospholipid contained in the liposome is usually 1 to 500 nmol / ml, Preferably 5-100n
mol / ml.

The dissolution method of the present invention can be easily carried out, for example, as follows. That is, if a surfactant is added to a solution containing biotin liposomes (or labeled biotin liposomes) so as to have a concentration as described above and an appropriate amount of avidin is added, the liposome membrane is added to the added avidin. Dissolved according to volume. The amount of avidin to be added at this time is not particularly limited as long as it can bind to biotin on the biotin liposome (or labeled biotin liposome) and dissolve the liposome membrane.
The concentration in the final reaction solution is usually 0.5 to 6 μg / ml, preferably 1.0 to 2.5 μg / ml.

The lysis method of the present invention can be used to specifically bind to a substance to be measured, such as an antigen-antibody reaction, a reaction between a sugar chain and a lectin, or a reaction between a nucleic acid and a polynucleotide complementary thereto. When combined with a reaction with a substance having the following properties (hereinafter, abbreviated as a binding ability substance), the quantification of various substances can be performed effectively. The quantification method of the present invention utilizes the dissolution method of the present invention effectively. That is, the quantification method of the present invention is a method for immobilizing biotin on a membrane surface in advance, and dissolving the liposome membrane dissolving activity of a surfactant and avidin for a liposome holding a labeling substance therein, with respect to the measurement target substance and the The amount is reduced by utilizing a reaction with a substance having a property of specifically binding, and the amount of a substance to be measured is determined by measuring the amount of a labeling substance that migrates from the liposome according to the degree of the reduction. It is a quantification method. The main configuration of the measurement system when the quantification method of the present invention is carried out comprises a combination of any one of the following groups (A), (B), (C) and (D). (A) Liposomes (i) Labeled biotin liposomes. (ii) A labeled biotin liposome in which a substance to be measured and biotin are both immobilized on a membrane surface. (iii) A labeled biotin liposome in which a binding substance and biotin are both immobilized on a membrane surface. (B) Surfactant (C) Reagents containing avidin (i) Combined reagent of avidin bound to a substance to be measured and a binding substance. (ii) Avidin bound to a binding substance. (iii) a combination of a substance to be measured bound to biotin, a substance to be measured and avidin. (iv) A combination reagent of avidin and a binding substance that has been bound to biotin. (v) A combination reagent of avidin and a binding substance. (vi) Avidin. (D) Sample (contains the substance to be measured) However, among the combinations of the constituent elements (A) to (D),
Combinations of the liposomes (A) and the reagents containing avidin (C) are limited to the following. When the liposomes of (A) are (i) labeled biotin liposomes, (C)
The reagents containing avidin are any of (i) to (iv).
When the liposomes (A) are (ii) labeled biotin liposomes in which a substance to be measured and biotin are immobilized on the membrane surface, the reagents containing avidin (C) are limited to (v). Further, when the liposomes of (A) are (iii) labeled biotin liposomes having both a binding substance and biotin immobilized on the membrane surface, the reagents containing avidin of (C) are:
Limited to (vi). Representative quantification methods of the present invention comprising the above various combinations are shown in the following schematic diagrams, but it goes without saying that the quantification method of the present invention is not limited to these.

(Equation 1)

(Equation 2)

(Equation 3)

(Equation 4)

(Equation 5)

(Equation 6) Table 1 shows typical examples of the combination of the substance to be measured and the binding substance in the above-described quantification method.

[Table 1] The above quantification method will be described in more detail below, taking the case where the measurement target is an antigen or an antibody as an example.

<Method-1> (Reagent)-Labeled biotin liposome in which biotin and an antigen to be measured are both immobilized on the membrane surface (hereinafter abbreviated as antigen-immobilized liposome-1)-Avidin-Above Antibody to antigen ・ Surfactant (Principle and operation method) A solution containing the antigen to be measured, avidin, and a solution containing the antibody to the antigen are added to a solution containing the antigen-immobilized liposome-1 and the surfactant. Upon addition and reaction, an antigen-antibody reaction occurs between the antibody and the antigen in the sample, or the antibody and the antigen immobilized on the liposome membrane surface, and at the same time, immobilizes avidin and the liposome membrane surface. An avidin-biotin binding reaction occurs between the biotin and the immobilized biotin. On the other hand, when an antigen-antibody reaction occurs on the liposome membrane surface, the antibody immobilized on the liposome membrane surface sterically inhibits the avidin-biotin binding reaction. Therefore, if the amount of the antigen in the sample is large, the amount of the antibody immobilized on the surface of the liposome membrane is small, so that the rate of inhibition of the avidin-biotin binding reaction is reduced, and the labeling substance retained by the liposome is reduced. There occurs a phenomenon that the amount of transmigration increases, and conversely, if the amount of the antigen in the sample is small, the amount of transmigration of the labeling substance decreases. Therefore, the amount of the labeling substance leached as a result of the above reaction was measured by a method according to its properties, and the measured values obtained were prepared by performing the same reaction using the antigen solution having a known concentration in advance as a sample. When applied to a calibration curve representing the relationship between the antigen concentration and the amount of the labeled substance that is leached, the amount of the antigen in the sample can be measured.

<Method-2> (Reagent) A labeled biotin liposome (hereinafter, referred to as "labeled biotin liposome") in which biotin and an antigen for an antibody to be measured are both immobilized on a membrane surface.
Abbreviated as antigen-immobilized liposome-2. -Avidin-Surfactant (Principle and operation method) When a sample containing an antibody to be measured and a solution containing avidin are added to a solution containing antigen-immobilized liposome-2 and a surfactant, and reacted. At the same time, an antigen-antibody reaction occurs between the antibody in the sample and the antigen immobilized on the liposome membrane surface, and at the same time, an avidin-biotin binding reaction is caused by the added avidin and the biotin immobilized on the liposome membrane surface. Occur. On the other hand, when an antigen-antibody reaction occurs on the liposome membrane surface, the antibody immobilized on the liposome membrane surface eventually becomes avidin-
Sterically inhibits biotin binding reaction. Therefore, if the amount of the antibody in the sample is large, the amount of the antibody immobilized on the surface of the liposome membrane is large, so that the rate of inhibition of the avidin-biotin binding reaction is increased, and the labeling substance held by the liposome is increased. Phenomenon that the amount of migration of
On the contrary, if the amount of the antibody in the sample is small, a phenomenon occurs in which the amount of the labeled substance translocating increases. Therefore, the amount of the label substance that was leached as a result of the above reaction was measured by a method according to its properties, and the measured values obtained were prepared by performing the same reaction using the antibody solution having a known concentration in advance as a sample. When applied to a calibration curve representing the relationship between the concentration of the antibody and the amount of the labeled substance to be leached, the amount of the antibody in the sample can be measured.

<Method-3> (Reagent)-Labeled biotin liposome-Avidin on which the antigen to be measured is immobilized-Antibody to the above-mentioned antigen-Surfactant (Principle and operation method) Labeling biotin liposome and surfactant The solution containing the antigen to be measured, a sample containing the antigen to which the antigen is immobilized, a solution containing avidin having the antigen immobilized thereon, and a solution containing an antibody to the antigen are added and reacted. Alternatively, an antigen-antibody reaction occurs between the antibody and the antigen immobilized on avidin, and at the same time, an avidin-biotin binding reaction occurs between the avidin and biotin immobilized on the liposome membrane surface. On the other hand, when an antigen-antibody reaction occurs on the avidin, the antibody immobilized on the avidin sterically inhibits the avidin-biotin binding reaction. Therefore, if the amount of the antigen in the sample is large, the amount of the antibody immobilized on the avidin will be small, and the rate of inhibiting the avidin-biotin binding reaction will be reduced, so that the labeled substance retained in the liposome will play a role. A phenomenon occurs in which the amount of output increases, and a phenomenon in which, if the amount of the antigen in the sample is small, the amount of migration of the labeling substance decreases. Therefore, the amount of the label substance that has been leached as a result of the above reaction is measured by a method according to its properties,
If the obtained measured value is applied to a calibration curve representing the relationship between the antigen concentration and the amount of label substance to be eluted prepared by performing the same reaction using the antigen solution having a known concentration in advance as a sample,
It becomes possible to measure the amount of the antigen in the sample.

<Method-4> (Reagent)-Labeled biotin liposome-Avidin immobilized with an antigen for the antibody to be measured-Surfactant (Operation method) Measured in a solution containing labeled biotin liposome and surfactant When a solution containing avidin in which an antigen for the target antibody is immobilized and a sample containing the antibody to be measured are added and allowed to react, the antigen in the sample and the antigen immobilized on avidin react with the antigen-antibody. At the same time as the reaction occurs, an avidin-biotin binding reaction occurs between the avidin and the biotin immobilized on the liposome membrane surface.
On the other hand, when an antigen-antibody reaction occurs on the avidin, the antibody immobilized on the avidin sterically inhibits the avidin-biotin binding reaction. Therefore, if the amount of the antibody in the sample is large, the amount of the antibody immobilized on the avidin is large, so that the rate of inhibition of the avidin-biotin binding reaction is increased, and the amount of the labeled substance retained in the liposome is increased. A phenomenon occurs in which the amount of transmigration decreases, and a phenomenon occurs in which, if the amount of the antibody in the sample is small, the amount of transmigration of the labeling substance increases. Therefore, the amount of the label substance that was leached as a result of the above reaction was measured by a method according to its properties, and the measured values obtained were prepared by performing the same reaction using the antibody solution having a known concentration in advance as a sample. When applied to a calibration curve representing the relationship between the concentration of the antibody and the amount of the labeled substance to be leached, the amount of the antibody in the sample can be measured.

<Method-5> (Reagent) • Labeled biotin liposome • Antigen to be measured on which biotin is immobilized • Antibody against the above antigen • Avidin • Surfactant (Operation method) Contains labeled biotin liposome and surfactant To the solution, a sample containing the antigen to be measured, a solution containing the antigen to be measured with biotin immobilized thereon, and a solution containing an antibody to the antigen and avidin are added to react with each other. An antigen-antibody reaction occurs between the antigen in the sample or the antibody and the antigen on which biotin is immobilized, and at the same time, biotin and avidin on the biotin-immobilized antigen or immobilized on the liposome membrane surface An avidin-biotin binding reaction is caused by the biotin and avidin. On the other hand, when an antigen-antibody reaction occurs on the biotin-immobilized antigen, the antibody immobilized on the biotin-immobilized antigen results in steric inhibition of the avidin-biotin binding reaction. Therefore, if the amount of the antigen in the sample is large, the amount of the antibody immobilized on the biotin-immobilized antigen is reduced, so that the rate at which the reaction between biotin and avidin on the antigen is inhibited decreases, and In contrast, since the reaction between biotin and avidin immobilized on the liposome membrane surface is unlikely to occur, a phenomenon occurs in which the amount of the labeling substance retained in the liposome is reduced, and
If the amount of the antigen in the sample is small, a phenomenon occurs in which the amount of the labeled substance translocating increases. Therefore, the amount of the labeling substance leached as a result of the above reaction was measured by a method according to its properties, and the measured values obtained were prepared by performing the same reaction using the antigen solution having a known concentration in advance as a sample. When applied to a calibration curve representing the relationship between the antigen concentration and the amount of the labeled substance that is leached, the amount of the antigen in the sample can be measured.

<Method-6> (Reagent) Labeled biotin liposome Antigen to antibody to be measured, immobilized with biotin Avidin Surfactant (Operation method) Solution containing labeled biotin liposome and surfactant Then, a solution containing biotin-immobilized antigen containing the antibody to be measured, a solution containing avidin, and a sample containing the antibody to be measured are added and allowed to react, thereby immobilizing the antibody and biotin in the sample. At the same time as the antigen-antibody reaction occurs with the immobilized antigen, an avidin-biotin binding reaction occurs between biotin and avidin on the biotin-immobilized antigen or biotin and avidin immobilized on the liposome membrane surface. . On the other hand, when an antigen-antibody reaction occurs on the biotin-immobilized antigen, the antibody immobilized on the biotin-immobilized antigen sterically inhibits the avidin-biotin binding reaction. Therefore, if the amount of the antibody in the sample is large, the amount of the antibody immobilized on the antigen on which biotin is immobilized is increased, so that the rate at which the reaction between biotin and avidin on the antigen is inhibited increases, In contrast, a reaction between avidin and biotin immobilized on the liposome membrane surface is apt to occur, so that a phenomenon occurs in which the amount of the labeling substance retained in the liposome is increased, and the On the contrary, if the amount of the antibody is small, the phenomenon that the amount of the labeling substance translocated decreases will occur. Therefore, the amount of the label substance that was leached as a result of the above reaction was measured by a method according to its properties, and the measured values obtained were prepared by performing the same reaction using the antibody solution having a known concentration in advance as a sample. When applied to a calibration curve representing the relationship between the concentration of the antibody and the amount of the labeled substance to be leached, the amount of the antibody in the sample can be measured.

When the object to be measured is not an antigen or an antibody but a sugar chain, a lectin or a nucleic acid, a sugar chain and a lectin or a nucleic acid are used instead of the antigen and the antibody in the above-described method. The same operation may be performed using a polynucleotide complementary thereto.

The measuring reagents used for carrying out the above-mentioned quantification method are prepared as two or more reagents containing the respective reagents used in each of the above methods as appropriate. When deciding the combination of reagents to be contained in each test solution, avidin and biotin, antigen and antibody, sugar chain and lectin, or nucleic acid and its complementary polynucleotide, etc. Needless to say, it is necessary to consider that the reactants do not coexist in the same reagent solution. Preferred combinations of reagents include, for example, the following combinations.

I) When the measurement target is an antigen: The first test solution contains a labeled biotin liposome in which biotin and a measurement target substance (antigen) are both immobilized on the membrane surface.・ Second test solution Contains antibodies against the measurement target. -Third reagent solution Contains avidin and a surfactant.

Ii) When the object to be measured is an antibody • First test solution Contains a labeled biotin liposome in which biotin and an antigen for the antibody to be measured are both immobilized on the membrane surface. -Second reagent solution Contains avidin and a surfactant.

Each of the test liquids thus prepared was
The pH is not particularly limited as long as it does not inhibit the storage stability of the reagent and the reaction between avidin and biotin, as described above, but is usually appropriately selected from the range of usually 5 to 9, preferably 6 to 8, and In order to maintain the pH, the reagent may contain a buffer such as phosphate, borate, tris (hydroxymethyl) aminomethane, Good (Good) buffer and Veronal.

The main reagents used in the quantification method of the present invention may be appropriately selected from the various reagents used in the dissolution method of the present invention, and may be used as necessary. Various preservatives to be added and reagents necessary for measuring the labeling substance (eg, enzyme substrate) are appropriately selected from those usually used in a complement immunoassay known per se. And use it.
Also, the concentration range of the reagents and the like in the test solution for measurement may be determined by appropriately selecting from the concentration range ordinarily used in a complement immunoassay known per se.

The reaction conditions for carrying out the above-described quantification method include a binding reaction between avidin and biotin and a binding reaction between a substance to be measured and a binding ability substance without inactivating the detectable properties of the labeling substance. (Eg, antigen-antibody reaction, reaction between sugar chain and lectin, reaction between nucleic acid and polynucleotide complementary thereto) is not particularly limited as long as it does not inhibit the reaction. ~ 50
° C, preferably in the range of 25 to 40 ° C, and the reaction time is appropriately selected in the range of usually 5 to 60 minutes, preferably 5 to 30 minutes.

The surfactant used in carrying out the above quantification method includes a detectable property possessed by the labeling substance when used in the above-mentioned concentration range used in the dissolution method of the present invention. And a binding reaction between avidin and biotin and a binding reaction between a substance to be measured and a binding substance (for example, an antigen-antibody reaction, a reaction between a sugar chain and a lectin, or a nucleic acid and its complementary Needless to say, it must be appropriately selected from those which do not inhibit the reaction with the polynucleotide.

In the quantification method of the present invention, the antibody used when the substance to be measured is an antigen is not particularly limited as long as it is an antibody against the substance to be measured. That is, a conventional method, for example, "Introduction to immunological experiments, 2nd printing,
Polyclonal antibodies prepared by immunizing animals such as horses, cows, sheep, rabbits, goats, rats, mice and the like according to the method described in Nao Matsuhashi, Gakkai Shuppan Center, 1981, etc. However, or alternatively, according to the cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975), cells were previously immunized with cells from the tumor line of the mouse and the analyte. Any monoclonal antibody produced by a hybridoma obtained by fusing with mouse spleen cells may be used, and these may be used alone or in an appropriate combination thereof. Also, these antibodies
Needless to say, it may be digested with enzymes such as pepsin and papain and used as F (ab '), Fab' or Fab.

In the quantification method of the present invention, the antigen used when the substance to be measured is an antibody is not particularly limited as long as it is an antigen that binds to the substance to be measured.

In the quantification method of the present invention, the lectin used when the substance to be measured is a sugar chain is not particularly limited as long as it has a property capable of specifically binding to the sugar chain to be measured. Examples thereof include, but are not limited to, concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, Japanese bean lectin, Japanese bean lectin, peanut lectin, and wheat germ lectin.

In the quantification method of the present invention, when the substance to be measured is a lectin, any sugar chain that is naturally derived as long as it binds to the substance to be measured may be synthesized. It may be any one of them and is not particularly limited.

In the quantification method of the present invention, the polynucleotide complementary to the nucleic acid to be measured used when the substance to be measured is a nucleic acid has a property capable of specifically binding to the nucleic acid to be measured. As long as it has, it can be used without any particular limitation, whether it is of natural origin or synthesized.

As a method for measuring the amount of the labeling substance, for example, when the labeling substance is an enzyme, for example, "Enzyme-linked immunosorbent assay, Protein Nucleic Acid Enzyme Supplement No. 31, Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Ishikawa The labeling substance may be measured according to the method described in Eiji Edit, pp. 51-63, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987. For example, the labeling substance may be measured according to the method described in “Illustration, Fluorescent Antibody, Akira Kawao, First Edition, Soft Science Co., Ltd., 1983” or the like. In the case of, for example, "enzyme immunoassay,
Protein Nucleic Acid Enzyme Separate Volume No.31, Tsunehiro Kitagawa, Toshio Minamihara,
Edited by Akio Tsuji and Eiji Ishikawa, pages 252-263, Kyoritsu Shuppan Co., Ltd.
The labeling substance may be measured according to the method described in "September 10, 1987" or the like. When the labeling substance is a substance having properties as a spin labeling agent, for example, "enzyme" Immunoassay, Protein Nucleic Acid Enzyme Separate Volume No. 31, Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 264-271
Page, published by Kyoritsu Shuppan Co., Ltd., September 10, 1987 ”or the like.

The substance to be measured which can be measured by the quantification method of the present invention includes, but is not limited to, a substance having a binding ability to the substance. Examples include antibodies produced in the body, those containing a sugar chain having a property of specifically binding to a specific lectin, lectins, and nucleic acids, and specifically, in biological fluids such as serum, blood, plasma, and urine. Representative examples thereof include nucleic acids, proteins, lipids, hormones, active peptides, autakoids, drugs, antibodies to specific substances, lectins, and the like. More specifically, for example, α-fetoprotein (AFP), CA
19-9, cancer markers such as prostate specific antigen (PSA) and carcinoembryonic antigen (CEA) such as insulin, human chorionic gonadotropin (hCG), thyroxine (T4), and triiodothyronine (T
3), hormones such as prolactin and thyroid stimulating hormone (THS), such as histamine, serotonin, prostaglandin, anotachoids such as angiotensin, bradykinin, etc., such as digoxin, phenytoin, morphine, nicotine, dinitrophenol (DNP), phenobarbital Drugs such as (PB), for example, antibodies such as anti-toxoplasma antibody, for example, lectins such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, Hilochawan lectin, chickpea lectin, peanut lectin, wheat germ lectin, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), adult T-cell leukemia virus (HTLV), cytomegalovirus (CMV), human papillomavirus (HPV)
And oncogenes derived from cancer cells such as human gastric cancer, human leukemia, and human neuroblastoma.

The lysis method of the present invention can also be used for qualitative detection of a specific substance. That is, for example, avidin is labeled on a substance capable of binding to a substance to be detected (for example, an antibody to an antigen, a lectin to a specific sugar chain, a polynucleotide complementary to a nucleic acid, etc.), and is reacted with the substance to be detected. The complex of the substance to be detected and the substance capable of binding thereto is separated, and the labeled biotin liposome and a surfactant are allowed to act on the complex to allow the labeled substance to translocate. The presence or absence of the substance to be detected can be determined by measuring the migrated label substance by a method according to the property.

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

【Example】

Embodiment 1 Preparation of biotin-modified dipalmitoyl phosphatidylethanolamine Dipalmitoyl phosphatidylethanolamine (Sigma) (76 mg, 0.11 mmol) suspended in chloroform (5 ml) was mixed with N-methylmorpholine (Wako Pure Chemical Industries, Ltd.).
1) of a dimethylformamide (DMF) solution containing 24 μl (0.22 mmol) and 50 mg (0.11 mmol) of N-hydroxysuccinimidoaminocaproylbiotin (manufactured by Vector Laboratories) was added, and the mixture was stirred and reacted at 5 ° C. overnight. After completion of the reaction, the solvent was distilled off, and purification was performed using a silica gel column (eluent; mixed solvent of chloroform and methanol) to obtain 68 mg of biotin-modified dipalmitoylphosphatidylethanolamine (yield; 60%). Preparation of Labeled Biotin Liposome Solution Dimyristoyl phosphatidylcholine 48 mg (71 μmo
l), dimyristoyl phosphatidyl glycerol 5.5mg
(8 μmol), 31.75 mg (82 μmol) of cholesterol and 0.8 mg (0.79 μmol) of the biotin-modified dipalmitoylphosphatidylethanolamine prepared above were dissolved in 5 ml of chloroform, and the solvent was distilled off using a rotary evaporator. It was left overnight in a desiccator to form a thin film on the wall of the flask. 7.5 ml of an aqueous solution of glucose-6-phosphate dehydrogenase [2,500 U / ml, in
10mM Tris (hydroxymethyl) aminomethane (Tri
s) Buffer solution (pH 7.8)] and stirred with a vortex mixer until uniform to prepare a liposome suspension.
The obtained liposome suspension was passed through an extruder (registered trademark of Leipex Biomembrane's), twice with a Nuclepore membrane (registered trademark of Nuclelipore) having pore sizes of 2 μM, 1 μM and 0.6 μM, and 0.4 μM and 0.2 μM, respectively.
Sizing was performed three times with a μM Nuclepore membrane to obtain liposomes having an average particle size of 225 nm. The operation of transferring the obtained liposome suspension to a centrifuge tube and centrifuging at 4 ° C. and 36,000 rpm for 1 hour was repeated 5 times, and finally, 5 ml of 100 mM Tris
The suspension was suspended in a buffer (pH 7.8) to obtain a labeled biotin liposome solution. Dissolution of labeled biotin liposome membrane using avidin-biotin binding reaction (Sample) Contains a given concentration of a given surfactant and avidin
A 100 mM Tris buffer (pH 7.8) was prepared and used as a sample. (Enzyme substrate solution) 16.4 mM glucose-6-phosphate, 7.0 mM nicotinamide adenine dinucleotide (NAD), 3- (N
-Morpholino) -2-hydroxypropanesulfonic acid (MOPS
A solution containing 20 mM O) and 20 mM Tris was prepared and used as an enzyme substrate solution. (Operation method) A solution obtained by diluting the labeled biotin liposome solution prepared above with 400 times with 225 mM Tris buffer (pH 7.8).
50 μl of the sample was added to 100 μl, and reacted at 37 ° C. for 2.5 minutes. Next, 95 μl of the enzyme substrate solution was added thereto, and the mixture was reacted at 37 ° C. for 5 minutes. Then, the change in absorbance at 340 nm per 5 minutes (ΔE)
Was measured. FIG. 1 shows a calibration curve representing the relationship between the obtained ΔE and the avidin concentration in the sample. In FIG. 1,
-○-is 1.0% sodium cholate as a surfactant
The results obtained using the sample contained therein,-□-indicates Triton X-100 (Rohm and
The results obtained using samples containing 0.25% of Haas (trade name) are shown.

Comparative Example 1 ΔE was measured by using the same reagent as in Example 1 except that a solution containing a predetermined concentration of melittin was used as a sample. Δ obtained
A calibration curve representing the relationship between E and the concentration of melittin in the sample is also shown in FIG. 1 (− × −). As is clear from the results of FIG. 1, when the labeled biotin liposome membrane containing glucose-6-phosphate dehydrogenase as a labeling substance is dissolved by the dissolution method of the present invention, the liposome membrane coexists. It turns out that it is dissolved in proportion to the avidin concentration. Further, it can be seen that the membrane dissolving action by the dissolving method of the present invention is clearly higher than that of melittin used for the same purpose as before.

Experimental Example 1 (Avidin solution) 100 mM Tris-H containing avidin at a specified concentration
A Cl buffer solution (pH 7.8, containing 30 mM NaCl and 0.5 mM ethylenediaminetetraacetic acid / 2Na) was prepared and used as an avidin solution. (Surfactant solution) Contains a specified concentration of a specified surfactant 10
A 0 mM Tris-HCl buffer solution (pH 7.8, containing 30 mM NaCl and 0.5 mM ethylenediaminetetraacetic acid / 2Na) was prepared and used as a surfactant solution. (Enzyme substrate solution) glucose-6-phosphate at 15.4 mM, NA
A solution containing 7.0 mM of D, 20 mM of 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO) and 20 mM of Tris was prepared as an enzyme substrate solution. (Liposome test solution) The labeled biotin liposome solution prepared in Example 1 was diluted 400-fold with 225 mM Tris buffer (pH 7.8) to obtain a liposome test solution. (Operation method) Avidin solution 50μl, liposome test solution 100μl
And 20 µl of a surfactant solution, and reacted at 37 ° C for 5 minutes. Next, 95 μl of the enzyme substrate solution was added thereto and reacted at 37 ° C. for 5 minutes, and the change in absorbance (ΔE) at 340 nm per 5 minutes was measured. Calibration curves showing the relationship between the obtained ΔE and the avidin concentration in the avidin solution are shown in FIGS. FIG. 2 shows Triton X-100 as a surfactant.
(Rohm and Haas product name)
FIG. 3 shows a case where sodium cholate was used as a surfactant, and FIG. 4 shows a case where 3-[(3-cholamidoamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO) was used as a surfactant. ) (Manufactured by Dojindo Laboratories) is shown, and in each figure,-○-indicates that the surfactant concentration is 2.0% (the concentration in the surfactant solution).
same as below. ),-●-indicates that the surfactant concentration is
1.0%,-□-when the surfactant concentration is 0.5%,-△-when the surfactant concentration is 0.25%,-◇-when the surfactant concentration is 0.125%. And-◆-indicates the case where no surfactant was added.
As is clear from the results of FIGS. 2 to 4, it is understood that a surfactant is essential in the dissolution method of the present invention.

Embodiment 2 FIG. The formal names of the abbreviations in the compound names used in this example are as follows. BOC: t-butoxycarbonyl group, Z: benzyloxycarbonyl group. Synthesis of Nα-biotinyl-Nε-dinitrophenyl-L-lysine dipalmitoylphosphatidylethanolamine condensate i) Synthesis of Nα-BOC-L-lysine dipalmitoylphosphatidylethanolamine condensate Nα-BOC-Nε-ZL-lysine (Manufactured by Wako Pure Chemical Industries, Ltd.) 440mg
(1.16 mmol) dissolved in 20 ml of chloroform was added to 254 μl of N-methylmorpholine (manufactured by Wako Pure Chemical Industries, Ltd.) (2.32 mm
ol) and isobutyl chloroformate (manufactured by Wako Pure Chemical Industries, Ltd.)
237 µl (1.74 mmol) was added, and the mixture was stirred and reacted at 0 ° C for 30 minutes. Then, dipalmitoyl phosphatidylethanolamine (manufactured by Sigma, hereinafter abbreviated as DPPE) 50 is added thereto.
0 mg (0.72 mmol) was added, and the mixture was reacted at room temperature with stirring overnight. After completion of the reaction, the solvent is distilled off, and the mixture is purified by a silica gel column (eluent; mixed solvent of chloroform and methanol), and Nα-BOC-Nε-ZL-lysine / DPPE condensate
592 mg (yield: 78%) were obtained. Obtained Nα-BOC-Nε-ZL
-Dissolve 285 mg (0.27 mmol) of lysine-DPPE condensate in 20 ml of a mixed solvent of methanol and ethanol, and add 5%
After adding 200 mg of palladium carbon (manufactured by Wako Pure Chemical Industries, Ltd.), hydrogenated catalytic reduction was performed. After completion of the reaction, the palladium carbon was filtered off, and the reaction solution was concentrated to obtain 240 mg of Nα-BOC-L-lysine / DPPE condensate (yield: 96%). ii) Nα-amino-Nε-dinitrophenyl-L-lysineDPPE
Synthesis of condensate Dinitrophenylaminobutyric acid (Sigma) 50mg
N-methylmorpholine (manufactured by Wako Pure Chemical Industries, Ltd.) 40.8 μl (0.38 m
mol) and isobutyl chloroformate (Wako Pure Chemical Industries, Ltd.)
36.1 μl (0.29 mmol) was added and reacted at 0 ° C. for 30 minutes with stirring. Next, 120 mg (0.13 mmol) of the Nα-BOC-L-lysine / DPPE condensate obtained in the above i) was added thereto, and the mixture was reacted at room temperature with stirring overnight. After completion of the reaction, the solvent was distilled off.
Purification was performed using a silica gel column (eluent; mixed solvent of chloroform and methanol), and 82 mg of Nα-BOC-Nε-dinitrophenyl-L-lysine / DPPE condensate (yield: 54%)
I got 82 mg (0.07 mmol) of the obtained Nα-BOC-Nε-dinitrophenyl-L-lysine / DPPE condensate is dissolved in a chloroform solution containing 40% of trifluoroacetic acid (TFA) cooled to 0 ° C. and stirred for 2 hours. Reacted. After completion of the reaction, the solvent was distilled off and the residue was purified with a silica gel column (eluent; mixed solvent of chloroform and methanol).
60 mg (yield: 80%) of -Nε-dinitrophenyl-L-lysine.DPPE condensate was obtained. iii) Nα-biotinyl-Nε-dinitrophenyl-L-lysine
Synthesis of DPPE condensate 60 mg (0.056 mmol) of Nα-amino-Nε-dinitrophenyl-L-lysine / DPPE condensate obtained in ii) above was mixed with 3 m of chloroform.
l-N-methylmorpholine (Wako Pure Chemical Industries, Ltd.)
A DMF solution containing 14.5 μl (0.132 mmol) and 30 mg (0.066 mmol) of N-hydroxysuccinimidoaminocaproylbiotin (manufactured by Vector Laboratories) was added, and reacted at 5 ° C. overnight with stirring. After the reaction is completed, the solvent is distilled off, and the residue is purified by a silica gel column (eluent; mixed solvent of chloroform and methanol), and Nα-biotinyl-Nε-dinitrophenyl-L-lysine / DPPE condensate
61 mg (yield: 77%) were obtained. Preparation of DNP-immobilized labeled biotin liposome solution The procedure was performed except that the Nα-biotinyl-Nε-dinitrophenyl-L-lysine / DPPE condensate (0.79 μmol) obtained above was used instead of the biotin-modified dipalmitoyl phosphatidylethanolamine. Using the same reagents as in Example 1, the same operation was performed to obtain a DNP-immobilized labeled biotin liposome solution having an average particle size of 223 nm. Dissolution of DNP-Immobilized Labeled Biotin Liposome Membrane Using Avidin-Biotin Binding Reaction Instead of the labeled biotin liposome solution, the DN obtained above was used.
Except that the P-immobilized labeled biotin liposome solution was used, the same operation was performed using the same reagent as in Example 1, and the DNP-immobilized labeled biotin liposome membrane was dissolved using an avidin-biotin binding reaction. . FIG. 5 shows a calibration curve representing the relationship between the obtained ΔE and the avidin concentration in the sample. In FIG. 5, -−- indicates the results obtained when a sample containing 1.0% of sodium cholate was used as a surfactant, and-□-indicates Triton X as a surfactant.
-100% (Rohm and Haas brand name) 0.25%
The results obtained when using the contained samples are shown below.

Comparative Example 2 ΔE was measured by using the same reagent as in Example 2 except that a solution containing a predetermined concentration of melittin was used as a sample. Δ obtained
A calibration curve showing the relationship between E and the melittin concentration in the sample is also shown in FIG. 5 (− × −). As is clear from the results shown in FIG. 5, the membrane dissolution method of the present invention effectively acts on liposomes having DNP immobilized on the surface together with biotin, and the degree of dissolution is proportional to the concentration of coexisting avidin. You can see. In addition, it can be seen that the membrane dissolving effect of the dissolving method of the present invention is clearly higher for DNP-immobilized labeled biotin liposomes than for melittin which has been used for the same purpose as before.

Embodiment 3 FIG. Measurement of DNP (Sample) 100 mM Tris buffer (p
H7.8) was used as a sample. (Enzyme substrate solution) 15.4 mM glucose-6-phosphate, NAD
A solution containing 7.0 mM, 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO) at 20 mM, Tris at 20 mM and an anti-DNP antibody (ICN Immunobiochemicals, Inc.) at 4 μg Ab / ml was prepared. A substrate solution was used. (Liposome test solution) The DNP-immobilized labeled biotin liposome solution prepared in Example 2 was mixed with a 225 mM Tris buffer solution (pH
What was diluted 500 times in 7.8) was used as a liposome test solution. (Avidin solution) 132 nM of avidin and Triton X-
0.25% for 100 (Rohm and Haas registered trademark)
The 100 mM Tris buffer (pH 7.8) was used as an avidin solution. (Operation method) Mix 10 μl of the sample with 100 μl of the enzyme substrate solution,
After incubating at 7 ° C for 2.5 minutes, liposome TS 95
μl was added, and the mixture was further incubated at 37 ° C. for 2.5 minutes.
Next, 50 μl of the avidin solution was added thereto, and the mixture was incubated at 37 ° C. for 5 minutes, and then the change in absorbance (ΔE) at 340 nm per 5 minutes was measured. The obtained ΔE and D in the sample
FIG. 6 shows a calibration curve representing the relationship with the NP concentration. As is clear from the results of FIG. 6, it can be seen that DNP can be measured with high sensitivity by the method of the present invention.

Embodiment 4 FIG. Determination of phenobarbital Preparation of phenobarbital-bound avidin solution Avidin 16 mg (0.24 μmol) was added to 0.16 M borate buffer (0.
Contains 13M NaCl. 4-hydroxyazobenzene-2-carboxylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in 8 ml of pH 7.9).
0.1M M borate buffer (containing 0.13M NaCl. P
After adding 2 ml of H7.9), a DMF solution containing 6.5 mg of N-hydroxysuccinimide ester of 1- (4-carboxybutyl) phenobarbital (0.24 μmol × 63) was added, and the mixture was stirred at 5 ° C. overnight. Reacted. After completion of the reaction, the reaction solution was purified by gel filtration using a Sephadex G-25 column which had been equilibrated with a 0.16 M borate buffer (containing 0.13 M NaCl, pH 7.9), and phenobarbital (hereinafter referred to as PB). This was abbreviated as). 35 ml of bound avidin solution was prepared. Dissolution of labeled biotin liposome membrane using PB-conjugated avidin (Sample) The PB-conjugated avidin solution obtained above was dissolved in 100 mM Tris
A sample appropriately diluted with a buffer (pH 7.8) was used as a sample. (Enzyme substrate solution) The same one as in Example 1 was used. (Liposome Test Solution) The labeled biotin liposome solution prepared in Example 1 was diluted 300 times with 225 mM Tris buffer (pH 7.8) to obtain a liposome test solution. (Surfactant solution) A 100 mM Tris buffer (pH 7.8) containing 0.25% of Triton X-100 (Rohm and Haas®) was used as a surfactant solution. (Operation method) After mixing 50 µl of the sample and 100 µl of the enzyme substrate solution and incubating at 37 ° C for 2.5 minutes, 50 µl of a surfactant solution was added, and the mixture was further incubated at 37 ° C for 2.5 minutes. Next, 95 μl of the liposome test solution was added thereto, and the mixture was added at 37 ° C.
After incubating for 10 minutes at 340nm per 10 minutes
Was measured for absorbance change (ΔE). ΔE and PB obtained
FIG. 7 shows a calibration curve representing the relationship with the dilution ratio of the bound avidin solution. As is clear from the results in FIG. 7, the membrane of the labeled biotin liposome is dissolved in proportion to the concentration of the coexisting PB-bound avidin. Quantification of PB using PB-bound avidin (Sample) 100 mM Tris buffer (pH
7.8) was used as a sample. (Enzyme substrate solution) glucose-6-phosphate at 15.4 mM, NA
A solution containing 7.0 mM of D, 20 mM of 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), 20 mM of Tris and 5 μg Ab / ml of anti-PB antibody (manufactured by Wako Pure Chemical Industries, Ltd.). It was prepared and used as an enzyme substrate solution. (Liposome Test Solution) The labeled biotin liposome solution prepared in Example 1 was diluted 300 times with 225 mM Tris buffer (pH 7.8) to obtain a liposome test solution. (PB-conjugated avidin solution) A 100 mM Tris buffer (pH 7.8) containing 0.25% of Triton X-100 (registered trademark of Rohm and Haas Co.) with the PB-conjugated avidin solution obtained above.
And diluted 20-fold to give a PB-bound avidin solution. (Operation method) After mixing 10 µl of the sample and 100 µl of the enzyme substrate solution and incubating at 37 ° C for 2.5 minutes, 50 µl of a PB-conjugated avidin solution was added, and the mixture was further incubated at 37 ° C for 2.5 minutes. Next, 95 μl of the liposome test solution was added thereto, and the mixture was incubated at 37 ° C. for 10 minutes.
The change in absorbance at 340 nm (ΔE) was measured. ΔE obtained
FIG. 8 shows a calibration curve representing the relationship between the PB concentration and the PB concentration in the sample. As is clear from the results of FIG. 8, it can be seen that PB can be measured by the method of the present invention.

Embodiment 5 FIG. Determination of fetal bovine serum albumin (Sample) A 100 mM Tris buffer (pH 7.8) containing a predetermined concentration of fetal bovine serum albumin (BSA) was used as a sample. (Enzyme substrate solution) glucose-6-phosphate at 15.4 mM, NA
A solution containing 7.0 mM of D, 20 mM of 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO), 20 mM of Tris, and 75 μg Protein / ml of anti-BSA antibody (manufactured by Binding Site) was prepared. A substrate solution was used. (Liposome test solution) A biotin-modified BSA (manufactured by Sigma) was diluted to 1/400 with a 225 mM Tris buffer solution (pH 7.8), and 1 µg / ml of the labeled biotin liposome solution prepared in Example 1 was added thereto. did. (Avidin solution) A 100 mM Tris buffer (pH 7.8) containing 132 nM of avidin and 0.5% of sodium cholate was used as an avidin solution. (Operation method) After mixing 20 µl of the sample and 100 µl of the enzyme substrate solution and incubating at 37 ° C for 2.5 minutes, 95 µl of a liposome test solution was added, and the mixture was further incubated at 37 ° C for 2.5 minutes. Then add 50 μl of the avidin solution and add
After incubating for 0 minutes, the change in absorbance (ΔE) at 340 nm per 10 minutes was measured. FIG. 9 shows a calibration curve representing the relationship between the obtained ΔE and the BSA concentration. As is clear from FIG. 9, it can be seen that BSA can be measured with high sensitivity by the method of the present invention.

[0051]

As described above, the present invention provides a new method for dissolving a liposome membrane utilizing the reaction between avidin and biotin. It has the characteristic that it is more powerful and its action corresponds to the amount of coexisting avidin, so it has a remarkable effect in that quantification of various substances becomes possible by utilizing this membrane dissolving action. Playing,
It is a great invention that contributes to the industry.

[0052]

[Brief description of the drawings]

FIG. 1 shows the calibration curves obtained in Example 1 and Comparative Example 1.

FIG. 2 shows a calibration curve obtained in Experimental Example 1.

FIG. 3 shows a calibration curve obtained in Experimental Example 1.

FIG. 4 shows a calibration curve obtained in Experimental Example 1.

FIG. 5 shows calibration curves obtained in Example 2 and Comparative Example 2.

FIG. 6 shows a calibration curve obtained in Example 3.

FIG. 7 shows a calibration curve obtained in Example 4.

FIG. 8 shows a calibration curve obtained in Example 4.

FIG. 9 shows a calibration curve obtained in Example 5.

[0053]

[Explanation of symbols]

In FIGS. 1 and 5, -.largecircle. Represents the result obtained from a sample containing 1.0% of sodium cholate as a surfactant, and -.quadrature. Represents Triton X-100 as a surfactant.
(Rohm and Haas Co., Ltd.) contains 0.25% of the sample, and-×-shows the result obtained by using a melittin solution having a predetermined concentration as the sample. In FIGS. 2 to 4, -−- indicates that the surfactant concentration is 2.0.
% (Indicating the concentration in the surfactant solution; the same applies hereinafter),-●-indicates that the surfactant concentration is 1.0%, and-□-indicates that the surfactant concentration is 0.5%. If-
Δ- indicates the case where the surfactant concentration is 0.25%,-◇-indicates the case where the surfactant concentration is 0.125%, and-◆-indicates the case where no surfactant is added.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-61-53568 (JP, A) Noriaki Kato et al. “Research on recognition and association of polymer liposomes” Proceedings of the Society of Polymer Science, 38, 1989, pp. 2854- 2856 Alexander L. Klibanov et al, "Biotin-bearing pH-Sensitive Liposomes: High-Affinity Binding to Avidin Layer", Journal of Liposomal. 1, No. 2, 1989, pp233-244 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/544 G01N 33/53

Claims (7)

(57) [Claims] 1. A method for dissolving a liposome membrane, wherein a surfactant and avidin are allowed to act on liposomes having biotin immobilized on the membrane surface in advance. 2. The method for dissolving a liposome membrane according to claim 1, wherein the liposome is a liposome having biotin immobilized on the membrane surface and a label substance retained therein. 3. A liposome membrane-dissolving agent comprising biotin immobilized on a membrane surface in advance, comprising a surfactant and avidin. 4. A liposome membrane dissolving activity of a surfactant and avidin for a liposome having biotin immobilized on a membrane surface in advance and holding a labeling substance therein is specifically bound to a substance to be measured and the liposome membrane-dissolving activity. Quantifying the amount of a substance to be measured by measuring the amount of a labeling substance that migrates from the liposome in accordance with the degree of the reduction by utilizing a reaction with a substance having the property of causing . 5. The substance to be measured is an antigen (including a hapten).
5. The method according to claim 4, wherein the substance that is an antibody and has a property of specifically binding thereto is an antibody or an antigen (including a hapten). 6. The substance to be measured is a sugar chain or a lectin,
The method according to claim 4, wherein the substance having the property of specifically binding thereto is a lectin or a sugar chain. 7. The method according to claim 4, wherein the substance to be measured is a nucleic acid, and the substance having a property of specifically binding thereto is a polynucleotide complementary thereto.