JP3407076B2 - Method for stabilizing liposome membrane and method for measuring hapten 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 stabilizing a liposome membrane and a novel method for quantifying hapten using the stabilizing method.

[0002]

BACKGROUND OF THE INVENTION The immunoassay is an assay utilizing an antigen-antibody reaction, for example, biological components such as proteins, hormones, active peptides, autacoids, tumor markers, immunoglobulins present in body fluids such as digoxin. , Phenytoin, phenobarbital, etc. are widely used as one of the methods for specifically measuring trace components such as drugs.

As an immunoassay method which is widely used at present, for example, a radioimmunoassay method (RIA method) is used.
And an enzyme immunoassay method (EIA method) and the like. Although these methods are methods capable of quantitatively measuring a trace component in a sample, they also have problems. That is, in the RIA method, since radioactive isotopes must be used, special equipment is required, and waste disposal becomes a problem.
In the IA method, there are problems such as a relatively long time required for measurement and a difficulty in application to an automatic analyzer.

Therefore, recently, as an immunoassay method which does not have these problems, an immunoactivity assay method using liposomes (hereinafter, abbreviated as liposome immunoassay method) has been proposed and has been attracting attention. A typical example of this method is the method described in JP-A-56-132564. This method comprises a liposome, a sample, which has a target substance immobilized on its surface and which holds a labeling substance (eg, enzyme) inside,
And, after the antigen-antibody reaction is carried out by mixing the antibody against the measurement target and the complement is added, the complement activated by the antigen-antibody complex formed on the surface of the liposome will cause the liposome membrane to react. It is a method of dissolving and allowing the labeling substance in the liposome to migrate, and measuring the amount of the measurement target substance in the sample from the amount. The liposome immunoassay using this complement (hereinafter abbreviated as complement immunoassay) is RI.
This measurement method is attracting attention because it does not have the above-mentioned problems of the A method and EIA method and can perform a series of reactions in a homogeneous reaction system, so that the measurement can be carried out easily and in a short time.

However, in the immunoassay method for complement, since the complement itself is an unstable substance, there is a problem in the stability of the measuring reagent, and the complement reacts nonspecifically with the measuring reagent to detect complement. Dissolution of liposome membranes other than complement is a problem because there are problems such as measurement errors due to the presence of substances in the sample that have the effect of activating and destroying some liposomes and causing the labeled substances to migrate. A liposome immunoassay using an active substance (hereinafter, abbreviated as a membrane lysing agent) was then developed.

That is, it is a method using a membrane-dissolving agent typified 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 measurement target, and utilizes the fact that the membrane-dissolving activity of melittin is lost when the complex binds to an antibody against the measurement target. The body and the free measurement target are reacted with the antibody in a competitive manner, and then the liposomal membrane lytic activity of the remaining melittin is measured to quantify the measurement target (WJ Litchfield et al., Clin. Chem., 30, 1441, 198.
Four). However, this method also has problems such as difficulty in obtaining melittin and sufficient liposome membrane-dissolving activity not being obtained unless melittin is used at a high concentration, and it cannot be said that this method is always practical. .

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a new method for stabilizing a liposome membrane and a new method for quantifying a hapten, which is an alternative to the method using complement, which utilizes this membrane stabilizing method. The purpose is to provide.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is characterized in that a liposome having a hapten immobilized on the surface of the membrane is reacted with an antibody against the hapten , the liposome membrane for a membrane-dissolving agent.
Method of stabilizing, and a sample containing the hapten to be measured, and liposomes hold a labeling substance therein beforehand membrane immobilized hapten to be measured on the surface and, after reacting the antibody against the hapten, further A method for quantifying hapten, which comprises reacting a membrane-dissolving agent, then measuring the degree of increase in stability of the liposome membrane to the membrane-dissolving agent, and quantifying the hapten in the sample from the degree of increase. It is an invention.

That is, the inventors of the present invention have been earnestly researching in order to find a new measuring method using a liposome, which is an alternative to the method using a complement, and a liposome having a hapten immobilized on the membrane surface in advance (hereinafter referred to as hapten liposome The reaction of the antibody against the hapten increases the resistance of the liposome membrane to the membrane-dissolving agent, in other words, the reaction increases the stability of the liposome membrane to the membrane-dissolving agent. Further, they have found that quantification of hapten can be performed by using this membrane stabilization method and a hapten liposome having a labeling substance held therein, and have completed the present invention.

The hapten according to the present invention is a low molecular weight compound having a molecular weight of 10,000 or less and does not have an antigenicity by itself, but is bound to a high molecular weight compound having an antigenicity such as a protein or a polysaccharide. It means a substance that shows antigenicity.
More specifically, for example, physiologically active components such as thyroxine (T ₄ ) and triiodothyronine (T ₃ ) contained in biological fluids such as plasma, serum, urine, and spinal fluid, and metabolites thereof, for example, Preferable examples thereof include drugs administered to the living body such as digoxin, phenytoin, morphine, nicotine, phenobarbital and metabolites thereof.

The hapten liposome according to the present invention is not particularly limited as long as the hapten is immobilized on the membrane surface of the liposome, and a method for producing such a hapten liposome is, for example, crosslinking. Method, lipid activation method and the like to immobilize the hapten on the surface of the liposome membrane, or a lipid prepared by covalently binding the hapten in advance (hereinafter abbreviated as hapten-bound lipid) is used to prepare liposomes. Thus, a method of incorporating a hapten into a liposome membrane (Japanese Patent Laid-Open No. 62-501800) and the like can be mentioned, and among them, a method of incorporating a hapten into a liposome membrane by preparing a liposome using a hapten-bound lipid is more preferable.

The hapten liposome having a labeling substance retained therein (hereinafter abbreviated as a labeled hapten liposome) used in the present invention has the labeling substance retained inside the hapten liposome obtained as described above. It may be any as long as it is one.

As the method for preparing the liposome itself used in the present invention, there are known vortex swing method, ultrasonic method, surfactant removal method, reverse phase evaporation method (REV method), ethanol injection method, ether. Injection method, Pre-Vesicle method, French Press Extrusion method, Ca ²⁺ fusion method, Annealing method, freeze-thaw fusion method, freeze-drying method, W / O / W Methods such as the emulsion method and, as a relatively new method, SMGruner et al. [Biochemistry, 24, 283
3 (1985)] by Stable Plurilamellar Ves
The icle method (SPLV method) and the method reported by some of the inventors of the present invention such that lipopolysaccharide is used as a part of the membrane constituent (Japanese Patent Laid-Open No. 63-107742). In addition, as its main membrane constituents, natural lecithin (eg, egg yolk lecithin, soybean lecithin, etc.) and dipalmitoylphosphatidylcholine (DPPC), which are used as membrane materials in the preparation of ordinary liposomes,
Dimyristoylphosphatidylcholine (DMPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPS), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE) , Phospholipids such as dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidic acid (DMPA) and egg yolk phosphatidylglycerol, or glycolipids such as ganglioside glycolipids, glycosphingolipids and glyceroglycolipids Examples thereof include one type or two or more types, or a mixed system of these with cholesterols, or a system in which these are further combined with lipopolysaccharide and the like.

The method for preparing the hapten liposomes (or labeled hapten liposomes) according to the present invention will be described in detail below, taking as an example the case where the hapten-bonded lipid is used as one of the raw materials and prepared by the surfactant removal method. To do. That is, first, for example, a hapten-bound lipid and cholesterols prepared according to the method described in the above-mentioned known document (JP-A-62-501800) are mixed with a suitable organic solvent (for example,
It is dissolved in chloroform, ethers, alcohols, etc.), concentrated to dryness under reduced pressure, and dried under reduced pressure in a desiccator. Then, an aqueous surfactant solution (20 to 100 mM) is added to the prepared lipid film to disperse it uniformly. Examples of the surfactant used here include cholic acid, polyoxyethylene octyl phenyl ether, octyl glucoside and the like which have been often used in this field from the past. Desirably, octyl glucoside such as octyl glucoside is used. A surfactant with a high critical micelle concentration (CMC) 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 and thoroughly mixed with stirring. After this,
It is most desirable to immediately remove the surfactant, and examples of the method include dialysis method, gel filtration method, resin adsorption method and the like known per se. The treatment condition is 1 hour to 1 day and night, and the temperature may be selected appropriately in the range of 0 to 70 ° C., although it varies slightly depending on the membrane constituent components of the liposome and the like. Especially, as a method for removing the surfactant, Sephar
It is advantageous to carry out operations such as gel filtration and centrifugation using ose 4B (trade name of Pharmacia Co.), since free labeling substances and the like can be removed at the same time. The thus-obtained hapten liposome (or labeled hapten liposome) is used or stored after being concentrated to a predetermined concentration by ultrafiltration or the like. Regarding the homogenization of the size of hapten liposomes (or labeled hapten liposomes), a method using a commonly used polycarbonate membrane may be used, but a gel filtration method [for example, Sephacryl S-1000 (trade name of Pharmacia) is used]. It is also effective to do.

The hapten liposome (or labeled hapten liposome) according to the present invention by a method other than the method for removing a surfactant.
Also in the case of preparing, the same may be performed according to a method known per se or other liposome preparation method.

The labeling substance retained in the labeled hapten liposome may be any labeling substance which is usually used in the complement immunoassay and can be detected when it is migrated out of the liposome. Examples include, but are not limited to, alkaline phosphatase, glucose-6-
Enzymes such as phosphate dehydrogenase, β-galactosidase,
For example 4-methylumbelliferyl-β-D-galactoside,
Substrates for enzymes such as p-hydroxyphenylpropionic acid, 4-methylumbelliferyl phosphate, glucose-6-phosphate, etc., such as carboxyfluorescein, fluorescein isothiocyanate, fluorescein isocyanate, tetrarhodamine isothiocyanate, 5-dimethylamino-1 -Fluorescent substances such as naphthalene sulphonyl chloride, for example, arsenazo III, 4- (2-pyridylazo) resorcinol, 2- (5-bromo-2-pyridylazo) -5- (N-propyl-N-sulfopropylamino) phenol sodium Dyes such as salts, such as luminol, isoluminol, luciferin, eosin Y, auramine O, bis (2,4,6-
Typical examples are luminescent substances such as trichlorophenyl) oxalate and N-methylacridinium ester, and spin labeling agents such as 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). It can be cited as an example.
Further, the amount of the labeling substance retained in the liposome varies depending on the type of the labeling substance and is not particularly limited. For example, when the labeling substance is glucose-6-phosphate dehydrogenase, it is used during liposome preparation. The solution containing the labeling substance is usually 1000 to 5000 U / ml, preferably 2000 to 3000 U / ml.
An enzyme solution having a concentration of

Examples of the naturally-occurring membrane-dissolving agent in the present invention include proteins (or polypeptides) such as melittin, streptolysin O, polymyxin B, glycosides such as saponin and digitonin, and amphotericin B, for example. , Macrolides such as polyene antibiotics such as Philippines, nystatin and the like are preferable.

Examples of the synthetic film dissolving agent in the present invention include polyoxyethylene alkyl ethers such as polyoxyethylene cetyl ether and polyoxyethylene lauryl ether, and polyoxyethylene alkylphenyl such as polyoxyethylene octyl phenyl ether. Ethers such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate, and other polyoxyethylene alkyl esters such as octanoyl-N-methylgluca Methyl, nonanoyl-N-methylglucamide, decanoyl-N-methylglucamide and other methylglucamide derivatives, such as n-octyl-β-D-glucoside and other alkyl sugar derivatives On surfactants such as sodium dodecyl sulfate (SDS), lauryl benzenesulfonic acid, deoxycholic acid, cholic acid, tris (hydroxymethyl) aminomethane dodecyl Sulfate (Tris DS)
Anionic surfactants such as octadecylamine acetate, tetradecylamine acetate, stearylamine acetate, laurylamine acetate, lauryldiethanolamine acetate, and other alkylamine salts such as octadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride, Quaternary ammonium salts such as cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, allyltrimethylammonium methylsulfate, benzalkonium chloride, tetradecyldimethylbenzylammonium chloride, octadecyldimethylbenzylammonium chloride and lauryldimethylbenzylammonium chloride, eg chloride Cationic surface activity of alkylpyridinium salts such as laurylpyridinium and stearylamidomethylpyridinium chloride Agent, 3 - [(3-Kola bromide amidopropyl) dimethyl ammonio] -1-propanesulfonate Nate, 3-
[(3-Colamideamidopropyl) dimethylammonio] -2-
Preferable examples include surfactants such as amphoteric surfactants such as hydroxy-1-propane sulfonate, and cyclodextrin derivatives such as bis (3-carboxypropanoyl) -tritrityl-β-cyclodextrin.

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

The membrane stabilizing method of the present invention can be easily carried out as follows, for example. That is, by adding an antibody against the hapten immobilized on the liposome to a solution containing the hapten liposome (or labeled hapten liposome) to carry out the reaction, the stability of the liposome membrane against the membrane solubilizer is improved. It increases according to the amount of antibody added.

The amount of antibody to be added at this time is not particularly limited as long as it is capable of reacting with the hapten on the hapten liposome (or labeled hapten liposome) membrane to increase the stability of the liposome membrane against the membrane solubilizer. Although not limited, for example, it is appropriately selected from the range of usually 0.5 to 5 times, preferably 1 to 3 times, the amount of hapten contained in the liposome membrane (mol).

By utilizing the membrane stabilizing method of the present invention, various haptens can be effectively quantified.
This quantification method will be described in detail below. <Quantitative method> (Test solution) -Labeled hapten liposomes in which the hapten to be measured is immobilized on the membrane surface-Antibodies to the hapten to be measured-Membrane lysing agent (Principle and operating method) To a solution containing the antibody to the hapten, When a sample containing a hapten to be measured and a labeled hapten liposome are added and reacted, the antibody and the hapten in the sample, or the antibody and the hapten immobilized on the liposome membrane surface are antigen-antibody The reaction occurs competitively. Therefore, when a solution containing a membrane-dissolving agent is added here, the amount of antibody immobilized on the surface of the liposome membrane decreases as the amount of the hapten in the sample increases, so that the ratio of stabilizing the liposome membrane decreases. Then, a phenomenon occurs in which the amount of the labeled substance retained in the liposome increases, and when the amount of the hapten in the sample is small, a phenomenon in which the amount of the labeled substance migrated decreases. Therefore, the amount of the labeling substance that was migrated as a result of the above reaction was measured by a method according to the property, and the obtained measured value was prepared by carrying out a similar reaction using the previously known hapten solution as a sample. By fitting to a calibration curve showing the relationship between the concentration of hapten and the amount of labeled substance that is released, it becomes possible to measure the amount of hapten in a sample.

As the reagent solution for measurement used for carrying out the above-mentioned quantification method, the following combinations of three reagent solutions are preferably exemplified. -Contains an antibody against the hapten to be measured with the first reagent solution. -Contains a labeled hapten liposome having a hapten to be measured immobilized on the surface of the second reagent solution membrane.・ Contains the third reagent solution.

Each of the measurement test solutions thus prepared
The pH is not particularly limited as long as it does not inhibit the storage stability of the reagent as described above, the antigen-antibody reaction, etc., but is usually selected from the range of 5 to 9, preferably 6 to 8 as appropriate. May contain a buffering agent such as phosphate, borate, tris (hydroxymethyl) aminomethane, Good buffering agent, Veronal buffering agent to maintain the pH.

Reagents used in these quantification methods of the present invention, or other various antiseptics added as necessary, reagents necessary for measuring labeling substances (eg, enzyme substrates, etc.), It may be appropriately selected and used from among those commonly used in a known complement immunoassay, and the concentration range of these reagents in the reagent solution for measurement is also known in itself. It suffices to appropriately select and determine from the concentration range or the like which is usually used in the measuring method.

The reaction conditions for carrying out the above-mentioned quantification method are not particularly limited as long as they do not inactivate the detectable property of the labeling substance and do not inhibit the antigen-antibody reaction, but specifically, The reaction temperature is usually 20 to 50 ° C,
The reaction time is appropriately selected from the range of preferably 25 to 40 ° C., and the reaction time is usually 5 to 60 minutes, preferably 5 to 30 minutes.

The membrane-dissolving agent used in the quantification method of the present invention may be of natural origin or a synthetic membrane-dissolving agent such as a surfactant. The concentration of these used in the present quantification method varies depending on the membrane composition and preparation method of the liposome to be used, or the type of the membrane-dissolving agent, but liposome membrane dissolution by the membrane-dissolving agent was confirmed,
In addition, any concentration may be used as long as it is sufficiently observed that the stability of the liposome membrane against the membrane solubilizer is increased by adding the antibody against the hapten immobilized on the liposome membrane. The final concentration therein is appropriately selected within the range of 0.001 to 1.0 V / V%, preferably 0.005 to 0.5 V / V%.

The membrane-dissolving agent used in the above quantification method does not inactivate the detectable property of the labeling substance when used in the above concentration range and inhibits the antigen-antibody reaction. Needless to say, it must be appropriately selected from among those that are not performed.

The antibody used in the quantification method of the present invention is not particularly limited as long as it is an antibody to be measured. That is, a conventional method, for example, “Introduction to Immunological Experiments, 2nd edition, Nao Matsuhashi et al., Academic Publishing Center, 19
81 ”etc. according to the method described in“ Horse, cow, sheep, rabbit, goat,
A polyclonal antibody prepared by immunizing an animal to be measured with an animal such as rat or mouse, or according to a conventional method, that is, a cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975) , A monoclonal antibody produced by a hybridoma obtained by fusing cells from a mouse tumor line with splenocytes of a mouse previously immunized with a measurement target may be any of these, alone or Are used in an appropriate combination, and so on.

As a method for measuring the amount of the labeling substance, for example, when the labeling substance is an enzyme, for example, “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 31, Kitagawa Tsunehiro / Namihara Toshio / Tsuji Akio / Ishikawa is used. Edited by Eiji, pp. 51-63, Kyoritsu Shuppan Co., Ltd., published September 10, 1987, etc., and the labeled substance may be measured. When the labeled substance is a fluorescent substance, For example, the labeled substance may be measured according to the method described in, for example, “Illustrated Fluorescent Antibody, Akira Kawao, First Edition, Soft Science Co., Ltd., 1983”, and the labeled substance is a luminescent substance. 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 Publishing Co., Ltd.
The labeling substance may be measured according to the method described in “September 10, 1987”, etc. When the labeling substance is a substance having a property as a spin labeling agent, for example, “enzyme” may be used. Immunoassay, Protein / Nucleic Acid / Enzyme Separate Volume No.31, Tsunehiro Kitagawa / Toshio Minamihara / Akio Tsuji / Eiji Ishikawa, 264-271
Page, Kyoritsu Shuppan Co., Ltd., published September 10, 1987 ”, etc., and the labeled substance may be measured.

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

[0032]

【Example】

Reference example 1. Preparation of labeled hapten liposome solution Dimyristoylphosphatidylcholine 48 mg (71 μmo
l), 5.5 mg of dimyristoylphosphatidylglycerol
(8 μmol), cholesterol 31.75 mg (82 μmol), and dipalmitoylphosphatidylethanolamine 0.7 modified with a predetermined hapten prepared according to the method described in the above-mentioned known document (JP-A-62-501800).
After 9 μmol (1 mol% of the amount of phospholipid used) was dissolved in 5 ml of chloroform, the solvent was distilled off using a rotary evaporator and then left overnight in a vacuum desiccator to form a thin film on the wall surface of the flask. Glucose-6
-Add 7.5 ml of phosphate dehydrogenase aqueous solution [2,500 U / ml, 10 mM Tris (hydroxymethyl) aminomethane (Tris) buffer (pH 7.8) solution] and stir it with a vortex mixer until it becomes uniform and suspend the liposomes. A liquid was prepared. The obtained liposome suspension was subjected twice to an Nuclipor membrane (registered trademark of Nuclepore Co., Ltd.) having a pore size of 2 μm, 1 μm, and 0.6 μm using an extruder (registered trademark of Leipex Biomembranes Co., Ltd.), 0.4 μm and 0.2 μm. Sizing was performed three times with a Nuclepore membrane to obtain a liposome suspension. Transfer the obtained liposome suspension to a centrifuge tube, centrifuge at 36,000 rpm for 1 hour at 4 ° C, repeat 5 times, and finally suspend in 5 ml of 100 mM Tris buffer (pH 7.8) for labeling hapten liposome. It was a liquid. The structural formula of the hapten-modified dipalmitoylphosphatidylethanolamine (hereinafter abbreviated as H-DPPE) used is as follows. -Phenytoin (PHT) modified (hereinafter abbreviated as Formula 1)

[Formula 1] -Modified digoxin (Dx) (hereinafter abbreviated as formula 2)

[Formula 2] - triiodothyronine (T ₃₎ those that have been modified with A) (hereinafter, abbreviated as Equation 3.)

[Formula 3] B) (hereinafter, abbreviated as Formula 4)

[Formula 4] Alternatively, C) (hereinafter, abbreviated as Formula 5).

[Formula 5]

Example 1. Stabilization of labeled hapten liposome membrane (sample) 100m so that a given antiserum is diluted to a given dilution ratio
A sample diluted with M Tris-HCl buffer (containing pH 7.8, 30 mM NaCl, 15 mM sodium azide and 0.5 mM ethylenediaminetetraacetic acid / 2Na) was used as a sample. The following antisera were used. -Anti-PHT antibody: Rabbit-derived, Wako Pure Chemical Industries, Ltd., IgG fraction. · Anti-T ₃ antibody: International Bio Co., Ltd., a monoclonal antibody. -Anti-Dx antibody: derived from rabbit, manufactured by Biodesign. (Liposome Reagent) Various labeled hapten liposome solutions prepared in Reference Example 1 were added to 225 mM Tris-HCl buffer (pH 7.8, 15 m).
M Contains sodium azide. ) Diluted 500 times to obtain a liposome reagent solution. (Membrane solubilizer solution) 100 mM containing a predetermined concentration of the membrane solubilizer
A Tris-HCl buffer solution (pH 7.8) was prepared and used as a membrane lysing agent solution. (Enzyme substrate solution) glucose-6-phosphate 15.4 mM, nicotinamide adenine dinucleotide (NAD) 7.0 mM, 3- (N
-Morpholino) -2-hydroxypropanesulfonic acid (MOPS
O) 20 mM, Tris 20 mM, sodium azide 15 mM and N
A solution containing 55 mM aCl was prepared and used as an enzyme substrate solution. (Operation method) 50 μl of a predetermined sample is added to 100 μl of a liposome reagent solution containing a labeled hapten liposome in which a hapten for the antibody in the sample is immobilized on a membrane, and the mixture is reacted at 37 ° C. for 2.5 minutes, and then a predetermined membrane lysing agent is added. Add 20 μl of solution and add 3 more
Incubated at 7 ° C for 2.5 minutes. Next, 95 μl of the enzyme substrate solution was added thereto and incubated at 37 ° C. for 10 minutes, and then the change in absorbance (ΔE) at 340 nm per 10 minutes was measured. The obtained results are shown in Tables 1-3.

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

In the above Tables 1 to 3, TX-100 is Triton X-100 (chemical name; polyoxyethylene (10) octyl phenyl ether, trade name of Rohm and Haas Company) and CHAPSO is 3- [. (3-colamidoamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (manufactured by Dojindo Laboratories) is shown respectively. As is clear from the results of Tables 1 to 3, in the labeled hapten liposome membrane, the amount of enzyme to be migrated is reduced by the addition of the corresponding antibody,
That is, it can be seen that the liposome membrane is stabilized against the membrane-dissolving agent by the reaction with the antibody. In addition, labeled hapten liposomes with PHT immobilized on the membrane surface were treated with an antibody against T ₃ or Dx.
When the antibody against E. coli was reacted, no decrease in the amount of translocating enzyme due to the addition of the antibody could be confirmed. Therefore, it is speculated that the stabilization of the liposome membrane according to the present invention is caused by the antigen-antibody reaction.

Example 2. Measurement of PHT (Sample) A 100 mM Tris-HCl buffer solution (pH 7.8) containing a predetermined concentration of PHT was used as a sample. (Enzyme substrate solution) Glucose-6-phosphate 15.4 mM, NAD
Anti-PHT diluted with a solution containing 7.0 mM, 3- (N-morpholino) -2-hydroxypropanesulfonic acid (MOPSO) 20 mM, Tris 20 mM, sodium azide 15 mM, and NaCl 55 mM 100 times.
An antibody (IgG fraction, manufactured by Wako Pure Chemical Industries, Ltd.) solution was used as an enzyme substrate solution. (Liposome Reagent) The PHT-immobilized labeled liposome solution prepared in Reference Example 1 was diluted with 225 mM Tris-HCl buffer (pH 7.8) to 500
What was diluted twice was used as the liposome reagent solution. (Membrane solubilizer solution) 100 mM containing a predetermined concentration of the membrane solubilizer
A Tris-HCl buffer solution (pH 7.8) was prepared and used as a membrane lysing agent solution. (Procedure) Mix 5 μl of sample with 100 μl of enzyme substrate solution,
After incubating for 2.5 minutes at ℃, liposome reagent solution 95
μl was added, and the mixture was further incubated at 37 ° C. for 2.5 minutes.
Then, 50 μl of the membrane lysing agent solution was added thereto and incubated at 37 ° C. for 5 minutes, and then the change in absorbance (ΔE) at 340 nm per 5 minutes was measured. Obtained ΔE and PH in the sample
A calibration curve showing the relationship with T concentration is shown in FIGS. still,
Figure 1 shows the results obtained when a 2.0% solution of TX-100 was used as the membrane lysing agent solution, and Figure 2 shows CHAP as the membrane lysing agent solution.
The results obtained when using a 0.8% SO solution are shown in FIG. 3 as 0.02 of melittin (manufactured by SIGMA) as a membrane solubilizer solution.
The results obtained with the 5% solution are shown respectively. Figure 1
As is clear from the results of ~ 3, PHT was obtained by the method of the present invention.
It can be seen that can be measured with high sensitivity.

[0039]

INDUSTRIAL APPLICABILITY As described above, the present invention utilizes a reaction between a hapten previously immobilized on a liposome membrane and an antibody against the hapten to stabilize the liposome membrane against a membrane-dissolving agent . Since this method provides a method and has a characteristic that the degree of stabilization corresponds to the amount of coexisting antibody, it is possible to quantify various haptens by utilizing this membrane stabilizing effect. It is a great invention that contributes to the industry.

[Brief description of drawings]

FIG. 1 shows a case where a 2.0% solution of Triton X-100 (chemical name; polyoxyethylene (10) octylphenyl ether, trade name of Rohm and Haas Co.) is used as a membrane solubilizer solution in Example 2. The obtained calibration curve is shown.

[Fig. 2] In Example 2, as a membrane lysing agent solution, 3-[(3-
The calibration curve obtained when a 0.8% solution of coramidoamidopropyl) dimethylammonio] -2-hydroxy-1-propane sulfonate (CHAPSO) is used is shown.

FIG. 3 shows a calibration curve obtained when a 0.025% solution of melittin (manufactured by SIGMA) was used as a solution of the membrane solubilizer in Example 2.

─────────────────────────────────────────────────── --- Continuation of the front page (56) References JP-A-2-162260 (JP, A) JP-A-3-73855 (JP, A) JP-A-63-151858 (JP, A) JP-A-1- 155271 (JP, A) JP-A-5-273213 (JP, A) Special table Sho 62-501800 (JP, A) Special table 3-502576 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/544 A61K 9/127 B01J 13/02

Claims (3)

(57) [Claims] 1. A substance having a liposomal membrane-dissolving activity other than complement , characterized in that a liposome having a hapten immobilized on the membrane surface is reacted with an antibody against the hapten .
Quality (hereinafter abbreviated as membrane solubilizer) liposome membrane
Method of stabilizing. 2. A sample containing a hapten to be measured, a liposome having a hapten to be measured previously immobilized on the surface of a membrane and holding a labeling substance therein, and an antibody against the hapten are reacted, and then the membrane is further reacted. A method for quantifying a hapten, which comprises reacting a lysing agent , then measuring the degree of increase in stability of the liposome membrane to a membrane lysing agent, and quantifying the hapten in the sample from the degree of increase. 3. The quantification method according to claim 2, wherein the antibody against the hapten is used in an amount of 0.5 to 5 times the amount (mol) of the hapten contained in the liposome membrane.