JPH07107536B2 - Highly sensitive immunoassay using liposome - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunological assay method, and more particularly, to a method for rapidly and accurately measuring a trace amount of a biological component using an antigen-antibody reaction.

[Prior Art] An immunological assay method for detecting a small amount of hormones, enzymes, etc. present in blood or the like by utilizing an antigen-antibody reaction is widely applied to medical diagnosis and the like.

Conventionally, as the immunological assay method, a method utilizing a precipitation reaction such as an overlay method, an immunodiffusion method, a capillary method, a laser nephelometry method or a labeled antibody such as an immunofluorescence method, an enzyme immunoassay method and a radioimmunoassay method is used. There is one that uses. In particular, radioimmunoassay, which uses a reagent in which a radioactive isotope is labeled on an antigen or an antibody, is widely used. On the other hand, methods that do not use radioactive substances, that is, methods that use enzymes, bacteriophage free radicals, chemiluminescent substances, fluorescent substances, etc. as labels have also been studied,
Some have been put to practical use. In such a method, a sandwich immunoassay method is applied in order to enhance the measurement sensitivity. For example, recently, a method in which the labeling substance is a fluorescent substance using an antibody immobilized on a carrier such as a porous membrane (special feature JP-A-60-57257 and 61-272659) and phosphorescence method (see JP-A-61-275656) have been reported.

[Problems to be Solved by the Invention] However, in the method utilizing the precipitation reaction, since a large amount of antigen and antibody are required to form a lattice-like bound product, the measurement sensitivity is poor, and on the carrier. In the method using the labeled antibody in which the antigen to be measured is reacted with the antibody immobilized on the antibody, and then the generated antigen-antibody complex and the labeled antibody are reacted, the detection sensitivity of the labeling substance is poor, and the antibody and the antigen are There are many problems such as insufficient detection sensitivity for a small amount of antigen unless the affinity with and the purity of the antibody are increased. Therefore, it has been strongly desired to develop a rapid and accurate measuring method capable of detecting even a small amount of antigen with high sensitivity.

As a result of intensive studies on an immunoassay having high detection sensitivity, the present inventors have found that a liposome (liposomes) in which an antibody is bound to the surface and a labeling substance is encapsulated therein
It was found that by reacting (me) with an antigen in a sample in the presence of complement, it is possible to detect a very small amount of antigen with high sensitivity,
The present invention has been completed.

[Means for Solving Problems] In the present invention, the presence of an antigen is qualitatively determined by reacting an antibody-bound unilamellar liposome having a labeling substance encapsulated therein with an antigen in a sample in the presence of complement. Alternatively, the present invention relates to a highly sensitive immunoassay using liposomes, which is characterized by quantitative measurement.

That is, the present invention is an immunological assay method using a unilamellar liposome, which is different from the conventional method utilizing a precipitation reaction and the sandwich immunoassay method utilizing an antibody immobilized on a solid phase. , Has the advantage of extremely high detection sensitivity.

[Examples] Liposomes refer to artificially synthesized lipid membrane vesicles, and include general multilamellar liposomes (hereinafter referred to as MLV) and unilamellar liposomes having a phospholipid membrane. A unilamellar liposome is used.

Examples of the phospholipid that can be used in the present invention include phospholipids widely present 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 bovine brain, soybean, etc. can also be applied.

The types of fatty acids in such phospholipids include various saturated or unsaturated fatty acids. For example, for phosphatidylcholine, diheptanoyl, dicaproyl,
Fatty acids such as didecanoyl, dilauroyl, diheptadecanoyl, dibehenoyl, dimyristoyl, and dipalmitoilouphosphatidylcholine are mentioned, and various other phospholipids mentioned above also include various saturated or unsaturated fatty acids.

As a method for preparing an MLV or a single-layered liposome in which a labeling substance is encapsulated, a conventionally known method for producing a liposome can be applied. For example, (1) application of phospholipid dissolved in an organic solvent into a container, removing the solvent under reduced pressure by blowing an evaporator or nitrogen gas to form a thin lipid film, and then including a labeling substance to be encapsulated in advance. A method of adding a salt solution or an aqueous solution in which a labeling substance is dissolved in advance and shaking the mixture (AD Bangam (ADBa
ngham) et al., Journal of Molecular Biology (J. Mol. Biol.), 13, 238 (1965) and D. Papahadjopoulo et al., Biochemical Biophysical. Actor (Biochim.Bi
ophys.Acta), 135, 639 (1967)) or a mixer method (K. Inoue, Biochemical Biophysical Actor, 339, 390 (1974) and S. Sea Kinsky
Et al., Biochemistry, Volume 8, 4149
Page (1969)), and the resulting MLV is sonicated with a water bath sonicator to prepare single-membrane liposomes (Die Papahajopoulos et al., Biochemical Biophysical Actor). , 311 page 310 (1973)), (2) a method of rapidly mixing phospholipids dissolved in an organic solvent with a salt solution containing a labeling substance (S. Batzri et al., Biochemical. Bio-Physical Actor, Vol. 298, page 1015 (1973)), (3) A method of slowly blowing out a phospholipid dissolved in ether to an aqueous solution in which a labeling substance in which the phospholipid is dissolved above the boiling point of ether is dissolved (D. W Deemer (DWDeamer), Annual New York Academy of Sciences (Ann.N.Acad.Sc)
i.), 308, p. 259 (1978)), (4) Phosphatidylserine alone or liposomes consisting of equal amounts of phosphatidylserine and cholesterol are ultrasonically treated in a water bath sonicator to prepare one-coated liposomes, and calcium-treated After that, a method of adding a salt solution containing a labeling substance and EDTA (see D. Papahajopoulos, Annual New York Academy of Science, 308, page 259 (1978)) and (5) reverse phase liposome A method in which an aqueous solution in which a labeling substance is dissolved is added and ultrasonic treatment is performed with a water bath sonicator, and then the solvent is removed with a rotary evaporator (F. Szoka, Jr.).
, Proceeding of National Academy of Sciences in USA (Proc.N
atl.Acad.Sci.USA.), Vol. 75, p. 4149 (1978)).

Regarding the method of binding the antibody to the liposome, a conventionally known method,
For example, (1) a method of cross-linking phosphatidylethanolamine with an antibody by glutar (VPTorchilin et al., Biochemical Biophysical Research Communication (Biochem.Bi
ophys.Res.Comm.), Vol. 85, page 983 (1978)),
(2) Incorporation of phosphatidylethanolamine in which a reagent that reacts with the -SH group is covalently bonded is incorporated into a liposome,
A method in which the antibody is SH-modified or the S-S bond between the antibodies is removed to bind the SH-modified product (LDLeserman et al., Nature (Natu
re), 288, 602 (1980)), and (3) a method in which a glycolipid is incorporated into a liposome and the aldehyde group generated by the periodate treatment is reacted with an antibody (tee.
See TDHeath et al., Biochemical Biophysical Actor, 640, 66 (1981)).
Etc. can be used.

The antibody-bonded unilamellar vesicles thus prepared can be applied as they are, but the unilamellar vesicles are generally used in the carrier binding method, the crosslinking method,
It can also be used as an immobilized product obtained by an immobilization method such as an encapsulation method. As a carrier for the carrier binding method, a derivative of a polysaccharide such as cellulose, dextran, agarose, or starch, a porous synthetic resin, an inorganic substance such as a metal oxide, or the like, and a carrier for the encapsulation method is a synthetic polymer substance. Polyacrylamide, photo-curable resin (such as ENT film), urethane prepolymer and natural polymer materials such as alginic acid, carrageenan, starch and gelatin can be applied. The shape of the immobilizate varies depending on the type of immobilization method, but a film, bead, or other shape can be applied.

The antibody-bonded unilamellar vesicles thus prepared are destroyed by contact with the antigen in the presence of complement, and the encapsulated labeling substance flows out to the outside.
By analyzing this labeling substance, the antigen can be analyzed. The reason for the destruction of the liposome is not clear, but it is presumed that the complex of the antigen and the antibody activates complement, which is destroyed by a chain reaction.

Complement is a component contained in animal serum, and in the present invention, fresh serum of guinea pig, rabbit, pig, cow, horse, etc. or serum of any animal such as human serum can be used.

Labeling substances include radioisotopes, enzymes, fluorescent substances,
Any substance that can be encapsulated in a liposome, such as a luminescent substance, a metal, and a dye, is applicable. For example, radioisotopes such as ¹²⁵ I and ¹³¹ I used in radioimmunoassay, fluorescein isothiocyanate, fluorescein isocyanate, tetorhodamine isothiocyanate, 5-dimethylamino-1 used in immunofluorescence.
-Fluorescent substances such as naphthalene sulphonyl chloride and luminescent substances such as luminescent substances such as Eosin Y and auramine O can be used. In particular, β-D-galactosidase, peroxidase, used in enzyme immunoassay, etc.
Encapsulating an enzyme such as alkaline phosphatase, glucose-6-phosphate dehydratase, alcohol dehydratase, or a metal such as calcium ion in an antibody-bound single-membrane liposome allows the liposome to react in the reaction with an antigen in the presence of complement. 4-methylumbelliferyl β-D-galactoside, p-hydroxyphenylpropionic acid, 4-methylumbelliferyl phosphate, glucose-6- If phosphoric acid, ethanol, etc. are used, or if calcium-dependent enzymes for calcium ions, such as calpain, protein kinase C, transglutaminase, etc., are added to the reaction solution,
All of them are convenient because they constitute a biochemical amplification reaction system and respond with high sensitivity to an extremely small amount of antigen.

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

Example 1 A 50 ml pear-shaped flask was charged with 30 μmo in about 10 ml of chloroform.
After adding a solution containing le dipalmitoylphosphatidylcholine (DPPC) and 22.5 μmole cholesterol (Cho), and removing the solvent with a rotary evaporator,
Reverse-phase liposomes are prepared by dissolving the lipid film 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 separated phases are homogenized by sonication in a water bath sonicator for 10-30 minutes. After that, the solvent is completely removed by a rotary evaporator at a temperature of 20 to 30 ° C. The liposomes thus obtained were separated by centrifugation, and 10 mM acetic acid / 60 mM sodium chloride (pH
5.5). Next, after passing through a 0.2 μm polycarbonate filter, an equal volume of 10 mM acetic acid was added.
Add / 60 mM periodate and incubate at room temperature for about 30 minutes. The excess periodate is then added to Sephadex G.
Anti-human IgG containing fluorescein isothiocyanate after chromatographic separation with a -75 (trade name, manufactured by Pharmacia Fine Chemicals) column and the resulting liposomes.
Mix with 0.1 ml of antibody (rabbit) (100 mg / ml) in water. Next, recrystallized 10 mM NaBH ₃ CN is added and finally centrifuged in a dextran gradient (0 to 20%) to recover the antibody-bound unilamellar vesicles.

Human IgG as an antigen was added to a mixed solution containing 5 μ of the above-mentioned antibody-bound single-membrane liposomes and 120 μ of guinea pig complement serum cryopreserved at about −70 ° C. so as to have various concentrations shown in FIG. 1 ml of the reaction solution was reacted at a temperature of 37 ° C. for 0.5 to 2 hours to prepare a sample for measurement. The measurement was carried out by a known fluorescence intensity measuring method (excitation wavelength 495 nm, fluorescence wavelength 520 nm), and the results are shown in FIG. As is clear from FIG. 1, according to the present invention, it is possible to measure even an extremely low antigen concentration of 10 ⁻⁴ mg / dl or less.

Example 2 Reaction was carried out in the same manner as in Example 1 except that β-D-galactosidase was used instead of encapsulating fluorescein isothiocyanate. Then, a sodium phosphate buffer solution (pH 7.0) containing 4-methylumbellyphenyl-β-D-galactoside as a substrate is added to the obtained reaction solution to react, and after about 30 minutes, a glycine-NaOH buffer solution ( (pH 10.3)
Was added to stop the reaction, and a sample for measurement was prepared. The measurement is performed by a known fluorescence intensity measurement method (excitation wavelength 368 nm, fluorescence wavelength 4
49 nm) and the results are shown in FIG.

As is clear from FIG. 2, according to the present invention, 10 ⁻⁴ mg / d
It can be seen that even at an extremely low antigen concentration of 1 or less, it can be measured with high sensitivity by the biochemical amplification reaction.

Example 3 Instead of encapsulating fluorescein isothiocyanate
The reaction was performed in the same manner as in Example 1 except that a 0.01 M calcium ion solution was used. 200 μg of calcium-dependent protease and a known method (T. Sasaki et al., Journal of.
Biological Chemist (J. Biological Chemist
ry), 259, 12489 (1984)), a synthetic substrate 2 mM leucine and tyrosine peptide 4-methylcoumarin-7-amide (Suc-Leu-Tyr-MCA), 5 mM cysteine, 2.5 mM2- In a buffer solution (pH 7.3) containing mercaptoethanol and 4% dimethylsulfoxide, the temperature is 30 ° C.
Was reacted for 10 to 30 minutes to prepare a sample for measurement. The measurement is carried out by a known fluorescence intensity measuring method (excitation wavelength: 380 nm, fluorescence wavelength: 4
The results obtained by the measurement at 60 nm) are shown in FIG.

As is clear from FIG. 2, according to the present invention, 10 ⁻⁴ mg / d
It can be seen that even at an extremely low antigen concentration of 1 or less, it can be measured with high sensitivity by the biochemical amplification reaction.

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

Furthermore, by encapsulating an enzyme or calcium ion as a labeling substance in a liposome, a biochemical amplification reaction system is constituted, so that even a trace amount of antigen can be measured with high sensitivity.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the IgG concentration measured in Example 1 and the fluorescence intensity. FIG. 2 is a graph showing the relationship between the IgG concentration and fluorescence intensity measured in Examples 2 and 3.

Claims (4)

[Claims] 1. A method for qualitatively or quantitatively measuring the presence of an antigen by reacting an antibody-bound unilamellar liposome having a labeling substance encapsulated therein with anti-abdomen in a sample in the presence of complement. A highly sensitive immunoassay using a liposome. 2. The labeling substance is an enzyme or a metal that constitutes a biochemical amplification reaction system with another substance, and is used for the biochemical amplification reaction by destruction of liposomes by complement. The measuring method described in the first item of the range. 3. The method according to claim 1, wherein the phospholipid used in the liposome is phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine or sphingomyelin. 4. The measuring method according to claim 1, wherein an antibody-bound single-membrane liposome immobilized in the form of a film or a bead is used as the antibody-bound single-membrane liposome.