JPS63309864A - High-sensitivity immunological assay method by using liposome - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay method, and more particularly to a method for rapidly and accurately measuring trace biological components using antigen-antibody reactions.

[Prior Art] Immunological assay methods that utilize antigen-antibody reactions to detect trace amounts of hormones, enzymes, etc. present in blood and the like are widely applied in medical diagnosis and the like.

Conventionally, this immunoassay method uses precipitation reactions such as multilayer method, immunodiffusion method, capillary tube method, and laser nephelometry method, and labeled antibody method such as immunofluorescence method, enzyme immunoassay method, and radioimmunoassay method. There are some that use In particular, radioimmunoassay using a reagent in which an antigen or antibody is labeled with a radioactive isotope is widely used. On the other hand, methods that do not use radioactive substances, ie, methods that use enzymes, bacteriophages, free radicals, chemiluminescent substances, fluorescent substances, etc. as labels, are also being researched and some have been put into practical use. In such a method,
Sandwich immunoassay methods have been applied to increase measurement sensitivity, and recently, for example, methods in which antibodies immobilized on a carrier such as a porous membrane are used and the labeling substance is a fluorescent substance (Japanese Patent Application Laid-open No. 80-57257 and 61-272859) and the phosphorescence method (Japanese Patent Laid-Open No. 81-275)
(see Publication No. 858) have been reported.

[Problems to be Solved by the Invention] However, the method using precipitation reaction requires a large amount of antigen and antibody to form a lattice-like bond, resulting in poor measurement sensitivity and Methods using labeled antibodies in which the antigen to be measured is reacted with the antibody immobilized on There are many problems, such as insufficient detection sensitivity for trace amounts of antigen unless the affinity with the antigen and the purity of the antibody are improved. Therefore, there has been a strong desire to develop a rapid and accurate measurement method that can detect even trace amounts of antigen with high sensitivity.

As a result of intensive research on immunoassay methods with high detection sensitivity, the present inventors discovered that liposomes (11p
The present inventors have discovered that if a small amount of antigen (osome) is reacted with an antigen in a sample in the presence of complement, minute amounts of antigen can be detected with high sensitivity, and the present invention has been completed.

[Means for Solving the Problems] The present invention enables the presence of an antigen to be determined qualitatively or quantitatively by reacting an antibody-binding liposome in which a labeling substance is encapsulated with an antigen in a sample in the presence of complement. This invention relates to a high-sensitivity immunoassay method using liposomes, which is characterized in that it measures.

That is, the present invention is an immunoassay method using liposomes, and unlike conventional methods using precipitation reactions or sandwich immunoassay methods using antibodies immobilized on a solid phase, detection sensitivity is low. has extremely high advantages.

[Example] The liposomes used in the present invention refer to artificially synthesized lipid membrane vesicles, and include multilamellar liposomes (hereinafter referred to as MLV) and unilamellar liposomes (hereinafter referred to as SUv) can be applied.

Examples of phospholipids that can be used in the present invention include phospholipids that widely exist in cell membranes of animals and microorganisms, such as phosphatidylethanolamines, phosphatidylcholines,
Examples include various phospholipids such as phosphatidylserines and sphingomyelins. Of course, natural egg yolk, phosphatidylcholine obtained from cow brain, soybean, etc. can also be used.

= 4 - Types of fatty acids in such phospholipids include various saturated or unsaturated fatty acids; for example, for phosphatidylcholine, dihebutanoyl, cicabroyl,
Fatty acids such as didecanoyl, dilauroyl, diheptadecanoyl, dibehenoyl, cimilistoyl, and dipalmitoyl-phosphatidylcholine are mentioned, and various saturated or unsaturated fatty acids are similarly included in the other phospholipids mentioned above.

As a method for preparing MLVs and SUVs encapsulating labeling substances, conventionally known liposome production methods can be applied. For example, (1) phospholipids dissolved in an organic solvent are placed in a container and the solvent is removed under reduced pressure using an evaporator or by blowing nitrogen gas to form a thin lipid film. A method of adding a salt solution or an aqueous solution in which a labeling substance is dissolved in advance and shaking it (N.D. Bangam (A, D, Bang)
ham) et al., Journal of Molecular Biology (J, Mo1. Biol,), vol. 13, 238
(1985) and D. Papahadjopoulos (D.
, Papahadjopoulo et al., Biochemical and Biophysical φ Acta (Blochis, B.
iophys.

Acta), vol. 115, p. 639 (1987))
Method of stirring with a mixer or a mixer (K, I
Noue), Biochemical and Biophysical φ Acta, Vol. 339, p. 390 (1974) and Nis-C, K1n5ky et al., Biochemistry, Vol. 8, 414.
9 (1989)), (2) The MLV obtained by the method (1) above was further sonicated with a water bath sonicator to obtain SU.
Method for producing V (D. Papahajjiboulos et al., Biochemical Biophysical Acta, Vol. 311,
31O (197B)), (3) A method in which phospholipids dissolved in an organic solvent are rapidly mixed with a salt solution containing a labeling substance (Batzrl, S. et al., Biophysical Acta in Biochemicals). , 298
Vol., p. 1015 (197B)), (4) A method in which a phospholipid dissolved in ether is slowly blown out into an aqueous solution in which a labeling substance, which has been heated to a temperature higher than the boiling point of the ether, is dissolved (D. ,
W, Deas+er), Annual New York Academy of Science (Ann.

N, Acad, Sci, ), vol. 308, p. 259 (
197g)), (5) Liposomes consisting of phosphatidylserine alone or equal amounts of phosphatidylserine and cholesterol are sonicated with a water bath sonicator to form S
A method of preparing Uv and adding a saline solution containing a labeling substance and EDTA after calcium treatment (see D. Babaha Joboulos, Annual Φ New York Academy of Science, Vol. 308, p. 259 (1978)). and (6) adding an aqueous solution containing a labeling substance to the reversed-phase liposome and subjecting it to ultrasonication using a water bath sonicator;
How to remove solvent with a rotary evaporator
Jr.) et al., Proceedings of the National Academy of Sciences φin (Proc, Natl, Acad, Se1.USA,)
, Vol. 75, p. 4149 (197g)) can be used.

Regarding the antibody liposome binding method, conventionally known methods,
For example, (1) a method of cross-linking phosphatidylethanolamine and an antibody with geltar (V,P Torcbilin et al., Biochemical and Biophysical Research Communication (Biochom, Biophys).

Res, Co5m, ), vol. 85, p. 983 (1978)
(Reference), C2J phosphatidylethanolamine covalently bonded with a reagent that reacts with the 18H group is incorporated into a liposome, and an antibody is converted to 811 or the S-8 bond between the antibodies is removed to form 811. How to combine things (L, D, Le
serman) et al., Nature S2
88, p. 602 (1980)) and (3) a method in which glycolipids are incorporated into liposomes and the aldehyde groups generated by periodic acid treatment are reacted with antibodies (T.
(see T. D. Heath et al., Biochemical Biophysical Acta, Vol. 640, p. 6B (1981)), etc. can be used.

Of course, antibody-bound MLVs and SUVs prepared in this way can be applied as they are, but MLVs and SUVs can also be obtained by commonly used immobilization methods such as carrier binding methods, crosslinking methods, and entrapment methods. It can also be used as a fixed product.

Supports for the carrier binding method include polysaccharide derivatives such as cellulose, dextran, agarose, and starch, porous synthetic resins, and inorganic materials such as metal oxides, and carriers for the comprehensive method include synthetic polymer materials. Applicable materials include polyacrylamide, photocurable resins (ENT film, etc.), urethane prepolymers, and natural polymeric substances such as alginic acid, carrageenan, starch, and gelatin. Further, the shape of the immobilized material varies depending on the type of immobilization method, but film-like, bead-like, or other shapes can be applied.

When the antibody-bound MLV or SUV produced in this way comes into contact with an antigen in the presence of complement, the liposome will be destroyed and the encapsulated labeling substance will flow out, so this labeling substance cannot be removed. By analyzing the antigen, it becomes possible to analyze the antigen. Although the reason for the destruction of liposomes is not clear, it is presumed that the antigen-antibody complex activates complement, which causes a chain reaction, leading to destruction.

Complement is a component contained in animal serum, and the present invention can use any animal serum, such as fresh serum from commonly used guinea pigs, rabbits, pigs, cows, horses, etc., or human serum.

Labeling substances include radioactive isotopes, enzymes, fluorescent substances,
Anything that can be encapsulated in liposomes, such as luminescent substances, metals, and pigments, can be used. For example, radioisotopes such as I and 1 used in radioimmunoassay, fluorescent substances such as fluorescein isothiocyanate, fluorescein isocyanate, tetrarhodamine isothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride, and eosin used in immunofluorescence. Luminescent substances such as Y and auramine O can be used.

In particular, if enzymes such as β-D-galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydratase, and alcohol dehydratase, which are used in enzyme immunoassays, or metals such as calcium ions are encapsulated in antibody-bound liposomes, When liposomes are destroyed in the reaction with antigens in the presence of complement and leaked out, they act as substrates for each enzyme.
4-Methylumbelliferyl β-D-galactoside, p
-Hydroxyphenylpropionic acid, 4-methylumbelliferyl phosphate, glucose-6-phosphate,
By using ethanol, etc., or by adding a calcium-dependent enzyme for calcium ions, such as calpain, protein kinase C1 transglutaminase, etc. to the reaction solution, both constitute a biochemical amplification reaction system and a trace amount of antigen can be extracted. This is advantageous because it responds with high sensitivity to

Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

Example 1 A solution containing 30 μl 1 ole of dipalmitoylphosphatidylcholine (DPPC) and 22.5 μmol of cholesterol (Ch, ) in about 10 ml of chloroform was placed in a 50 ml pear-shaped flask, and after removing the solvent with a rotary evaporator, a lipid thin film was formed. A reversed phase liposome is prepared by dissolving the following in 3 ml of isopropyl ether and 3 ml of chloroform. at this point
An aqueous solution containing ganglioside (Gan) is added, and the two phases separated are homogenized by ultrasonication using a water bath sonicator for 10 to 80 minutes. After that, 20
The solvent is completely removed on a rotary evaporator at a temperature of ~30°C. The liposomes obtained in this way were separated by centrifugation, and 10+nM acetic acid/BO+nM
Suspend in common salt (pH 5.5). Next, 0.
After passing through a double polycarbonate filter,
Add an equal volume of 11011I acetate/60mM periodate and incubate for approximately 30 minutes at room temperature. Excess periodate was then chromatographically separated using a column of Sephadex G-75 (trade name, manufactured by Pharmacia Fine Chemicals), and the resulting liposomes were treated with an anti-human IgG antibody (rabbit) containing fluorescein inthiocyanate. (
100mg/ml) mixed together with 0.1ml of aqueous solution. Next, recrystallized 10+nM NaBHs
CN was added and finally a dextran gradient (0
Antibody-conjugated liposomes are collected by centrifugation in ˜20%).

Reaction Solution 1: Human IgG was added as an antigen to a mixture containing 5 μg of the above antibody-bound liposomes and 120 μg of guinea pig complement serum stored frozen at approximately -70°C at various concentrations shown in Figure 1. ml at a temperature of 37℃
．． The mixture was reacted for 5 to 2 hours and used as a sample for measurement. The measurement was carried out by a known fluorescence intensity measurement method (excitation wavelength: 495 ni, fluorescence wavelength: 520 nm), and the results are shown in FIG. As is clear from FIG. 1, according to the present invention, 10'mg
It can be seen that even extremely low antigen concentrations below /dρ can be measured.

Example 2 The reaction was carried out in the same manner as in Example 1 except that β-D-galactosidase was used instead of encapsulating fluorescein isothiocyanate.

Next, a sodium phosphate buffer (pH 7,0) containing 4-methylumbelliphenyl-β-D-galactoside as a substrate was added to the resulting reaction solution for reaction, and after about 30 minutes, a glycine-Na011 buffer ( pH 10.3) was added to stop the reaction and used as a sample for measurement.

Measurement was performed using a known fluorescence intensity measurement method (excitation wavelength 388 nm).

The results are shown in FIG. 2.

As is clear from FIG. 2, according to the present invention, 10'
It can be seen that even extremely low antigen concentrations below mg/dρ can be measured with high sensitivity by biochemical amplification reactions.

Example 3 The reaction was carried out in the same manner as in Example 1 except that a 0.01M calcium ion solution was used instead of encapsulating fluorescein isothiocyanate. To the resulting reaction solution,
200 μg of calcium-dependent protease and a known method (T, 5asak1 et al., Journal of Biological Chemistry (j, Biological Chen+1stry))
The leucine and tyrosine peptide 4-methylcoumarin 7-amide (Sue-L
eu-Tyr-MCA), 5 mM cysteine, 2
．． in a buffer (pH 7.3) containing 5mM 2-mercaptoethanol, 4% dimethyl sulfoxide at a temperature of 30°C.
The mixture was reacted at ℃ for 10 to 30 minutes to prepare a sample for measurement. The measurement was performed using a known fluorescence intensity measurement method (excitation wavelength 380 nm).
, fluorescence wavelength 4BOnm), and the results are shown in FIG.

As is clear from FIG. 2, according to the present invention, IN'm
It can be seen that even extremely low antigen concentrations of g/dj2 or less can be measured with high sensitivity by biochemical amplification reactions.

[Effects of the Invention] According to the present invention, an antibody-bound liposome encapsulating a labeling substance is subjected to an antigen-antibody reaction in the presence of complement, and the labeling substance flowing out is analyzed.
can quickly and accurately measure trace components present in almost all biological substances and drugs.

Furthermore, if an enzyme or calcium ion is encapsulated as a labeling substance in a liposome, a biochemical amplification reaction system will be constructed, so that even trace amounts of antigen can be measured with high sensitivity.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between IgG concentration and fluorescence intensity measured in Example 1. FIG. 2 is a graph showing the relationship between IgG concentration and fluorescence intensity measured in Example 2 and Example 3. Patent applicant Kanebuchi Chemical Industry Co., Ltd. Figure 21, Section 2 IgG (mg/dl >

Claims (1)

[Claims] 1. The present invention is characterized by qualitatively or quantitatively measuring the presence of an antigen by reacting an antibody-bound liposome in which a labeling substance is encapsulated with an antigen in a sample in the presence of complement. A highly sensitive immunoassay method using liposomes. 2. Claim 1, wherein the labeling substance is an enzyme or metal that constitutes a biochemical amplification reaction system with other substances, and is subjected to the biochemical amplification reaction by destruction of liposomes by complement. Described measurement method. 3. The measuring method according to claim 1, wherein the phospholipid used in the liposome is a phosphatidylcholine, a phosphatidylserine, a phosphatidylethanolamine, or a sphingomyelin. 4 Claim 1 which uses an antibody-binding liposome immobilized in a membrane or bead shape as the antibody-binding liposome
Measurement method described in section.