JPH02108969A - Immunoassay using liposome - Google Patents

Abstract

PURPOSE:To make stable and exact measurement by incorporating such a high-polymer material which does not activate a complement system into a salts soln. CONSTITUTION:The liposome which is sealed internally with a labeling material and is subjected to a film damaging effect by complement activity and the complement are used and the complement is acted on the liposome. The labeling material liberated from the liposome is measured according to the complement activity or the antigen quantity, by which the complement or antigen existing in the sample to be measured is measured. Such a high-polymer material which does not activate the complement system is incorporated into the salts soln. for diluting and preserving the liposome or the sample to be inspected at this time. The liposome and complement are protected and stabilized and the liberation of the nonspecific labeling material which does not depend on the complement activity and the antigen quantity and the rapid deactivation of the complement are prevented. The stable and exact measurement of the complement or antigen is thus made.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immunoassay method using liposomes, and more specifically, a method for qualitatively or quantitatively measuring complement or antigen present in a test sample. This invention relates to an immunoassay method using liposomes.

[Prior art and problems]

The complement system is contained in the fresh blood and lu of animals such as humans.
It is composed of nearly 0 types of proteins and is an important reaction system involved in various immune reactions such as defense against infection in living organisms. The complement system plays an indispensable role in biological defense, and since abnormalities in the complement system are observed in various diseases, measurement of complement activity, that is, complement value, is used for clinical diagnosis. Particularly in the case of diseases where the complement value decreases, such as systemic lupus erythematosus, measurement of the complement value is important.

Currently, the most common complement value measurement method is the 50% hemolysis method using biologically derived red blood cells [for example, [
Useful Immunology Experimental Methods J, edited by Nishioka, Shimada, and Masaki, Kodansha Scientific (1984), pp. 99-104]
. This 50% hemolysis method measures the amount of complement required to hemolyze half of a set number of red blood cells to activate the complement system under optimal conditions, and is extremely complicated. It has problems such as requiring operations and taking a long time for measurement, living body-derived red blood cells are unstable and cannot be stored for a long period of time, and requires centrifugation, making automation difficult.

On the other hand, in recent years, complement measurement methods are being developed that use liposomes (liposome-e), which are artificial lipid membrane vesicles, instead of biologically derived red blood cells. Complement measurement method using liposomes is a method that uses liposomes that contain a labeling substance and are prepared to damage the membrane through complement activation to interact with the complement present in the test sample to improve the complement system. This method measures the activity of complement or complement components by activating liposome M to cause a damage reaction that corresponds to the complement activity in the test sample, and measuring the labeling substance released from the liposome as a result. be.

Compared to the 50% hemolysis method, this liposome-based complement measurement method uses liposomes, which are more stable than biologically derived red blood cells, is simple and requires a short measurement time, and does not require separation operations such as centrifugation. It has the advantage that it is a measurement method suitable for automation because it does not require any additional measurement.

On the other hand, immunoassay methods, which utilize antigen-antibody reactions to detect trace amounts of biological components such as protein hormones, active peptides, autacoids, and tumor markers, as well as drugs, etc. present in the blood, are widely applied in medical diagnosis. There is.

Conventionally, this immunoassay method generally uses a radioimmunoassay (R
1) and enzyme-linked immunosorbent assay (EIA), which uses antigens or antibodies labeled with enzymes, etc., have been used. However, R1 is difficult to handle and disposal becomes a problem.
In addition, EIA has the drawback that it requires some method to separate antigen or antibody that is not bound to the antigen-antibody complex, and that the operation is complicated and is not suitable for automation.

In contrast, in recent years, immunoassay methods using liposomes, like complement assays, have been developed as homogeneous assay methods that do not require separation of antigen-antibody bound substances and free substances. This is an antigen-antibody combination that is formed on the liposome membrane by adding a liposome containing an antibody against the antigen in the test sample and encapsulating a labeling substance to the test sample, and then adding complement. activates the complement system, causing a series of complement-dependent liposome membrane damage reactions depending on the amount of antigen in the test sample, and as a result, the labeled substance encapsulated within the liposome is released, so the amount can be reduced. It is something to be measured.

Another measurement method involves reacting an antibody-bound liposome with an antigen, then adding an antibody specific to the antigen and allowing the reaction to occur again, and activating the complement system with the secondary antigen-antibody conjugate. has also been developed.

This immunoassay method using liposomes is a homogeneous measurement that does not require separation of antigen-antibody bound substances and free substances.
It has the advantages of being easy to operate, requiring only a short time, and being suitable for automation.

In this way, by using a liposome in which a labeling substance is encapsulated and which undergoes a membrane-damaging effect due to complement activity, and complement, complement is allowed to act on the liposome, and release is released from the liposome depending on the complement activity or the amount of antigen. The immunoassay method, which measures the complement or antigen present in the test sample by measuring the labeled substance, is expected to be a measurement method suitable for automation because of its slow operation. Although it is more stable than other substances, the structure of liposomes is maintained by non-bonding interactions between the lipid molecules that make up the liposomes.
When liposomes are made to interact with complement when measuring complement or antigen, in some cases, labeling substances may be released non-specifically from the liposome, independent of complement activity or antigen amount.
It has the disadvantage that accurate and quantitative measurements of complement and antigen cannot be performed.

As a result of intensive research to overcome the above-mentioned drawbacks, the present researchers used liposomes and complement, which have a labeling substance encapsulated inside and which undergoes membrane damage due to complement activation, and used complement to act on the liposomes. In an immunoassay method that measures complement or antigen present in a test sample by measuring the labeled substance released from the liposome according to complement activity or antigen amount, a saline solution containing the liposome is used. By containing a polymeric substance that does not activate the complement system,
It was discovered that stable and accurate measurements could be made, and the present invention was completed.

[Means for solving problems]

That is, the present invention uses a liposome in which a labeling substance is encapsulated inside and whose membrane is damaged by complement activity, and complement, and the liposome is made to act with complement, and the labeling substance is encapsulated inside the liposome and the membrane is damaged depending on the complement activity or the amount of antigen. In an immunoassay method that measures the complement or antigen present in a test sample by measuring the labeled substance released from the liposome, the salt solution containing the liposome contains a polymeric substance that does not activate the complement system. The subject matter is an immunoassay method using liposomes, which is characterized by the ability to increase blood pressure.

The immunoassay method using liposomes of the present invention involves activating the complement system in a salt solution used to dilute and preserve a liposome or a test sample that encapsulates a labeling substance and undergoes +19 damage due to complement activation. By containing a polymeric substance that does not cause oxidation and using it for measurement, the liposome and complement are protected and stabilized, and non-specific labeling substances that do not depend on complement activity or antigen amount are released from the liposome. As a result, stable and accurate complement or antigen measurements can be made.

Furthermore, if the liposomes are stored in a saline solution containing a polymeric substance that does not activate the complement system, the ability to retain the labeling substance encapsulated within the liposomes will be improved, and fusion between liposomes can be prevented, resulting in a long-term storage of the liposomes. It also has the advantage of being able to be stored stably for a long period of time.

As the polymeric substance that does not activate the complement system and is to be included in the salt i8 solution containing liposomes used in the present invention, any polymer substance that does not activate the complement system and is not adsorbed on the liposome membrane surface can be used. things are applicable.

Examples of such substances include gelatin, albumin, globulin, collagen, polyethylene glycol, polyvinyl alcohol, agar, agarose, sodium alginate, chitosan, carrageenan, starch, etc., and these may be used alone or in combination of two or more. 0.02-1.0% by weight in saline solution, preferably 0.05-0.2
It is used by containing about % by weight.

The liposome used in the immunoassay method of the present invention refers to an artificial lipid membrane vesicle that is obtained by dispersing phospholipids etc. in an aqueous solution and has an inner aqueous phase surrounded by a lipid bilayer membrane. Multilamellar liposomes (multilamellar liposomes)
ellar vesicle % or less, referred to as gate LV)
, small unit-a
llellar vesicle % or less, SUν), large unilamellar liposomes (large unil
amellar vesicle % or less, referred to as LtlV), all of which can be applied to the present invention.

Examples of lipids that can be used to prepare liposomes used in the immunoassay of the present invention include phospholipids, glycolipids, and cholesterol. Examples include various phospholipids such as phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and sphingomyelins. Of course, natural egg yolk, phosphatidylcholine obtained from cow brain, soybean, etc. can also be used.

Types of fatty acids in such phospholipids include various saturated or unsaturated fatty acids. Examples of phosphatidylcholine include diheptanoyl, cicabroyl, didecanoyl, dilauroyl, dihepkudecanoyl, dibehenoyl, and cimilistoyl. , dipalmitoyl-phosphatidylcholine, and the like, and the other phospholipids mentioned above also include various saturated and unsaturated fatty acids.

As a method for preparing a liposome encapsulating a labeling substance, a conventionally known liposome preparation method can be applied. For example, (1) put phospholipid dissolved in an organic solvent in a container,
Remove the solvent using an evaporator or blowing nitrogen gas under reduced pressure to form a thin lipid film, and then add an appropriate salt containing the labeling substance you want to encapsulate in advance. Method of adding liquid 8 or an aqueous solution in which the labeled substance is dissolved in advance and shaking [
N. D. Pangam (^, D. Rangha) et al., Journal of Molecular Biology (J.

Mo1. Biol, ), 13S, p. 238 (1965
) and Dei Papajoboulas (D, Papah)
adjop.

Biochim, Biophys, Ac
La), Vol. 135, p. 639 (1967) and the method of shaking and stirring with a portex mixer C. Inouuni (K.
, Inoua), Biochemical Biophysical Acta, Vol. 339, p. 390 (1974) and Niss C. Kinski (S, C, K1n5ky) et al., Biochemistry (Bioche + m1stry).
, Vol. 8, p. 4149 (1969)], (2) A method of producing SUv by further sonicating the MLV obtained by the method (1) above with an ultrasonic oscillator [Dei-Papajoboulas et al., Biochemical・Refer to Biophysical Acta, Vol. 31, Vol. 1, p. 310 (1973)], (3) A method in which phospholipids dissolved in an organic solvent are rapidly mixed with a salt solution containing a labeling substance [Nis Batzili (S, Bat)
zri) et al., Biochemical Biophysical Acta, Vol. 298, p. 1015 (I973)], Ku4)
A method in which a phospholipid dissolved in ether is slowly blown out into an aqueous solution in which a labeling substance has been dissolved in advance at a temperature higher than the boiling point of ether (DW Daymer (D, W)).

Deamer, Annual New York Academy
・Of Science (Ann, N, Y, Acad
, 5ci), 308S, p. 259 (1978))
, (5) Liposomes consisting of phosphatidylserine alone or equal amounts of phosphatidylserine and cholesterol are sonicated with an ultrasonic oscillator to create SUv, and after calcium treatment, 4! saline solution containing a chemical substance and EOT
How to create LIJV by adding A [see Day Papajoboulas, Annual New York Academy of Sciences, 308S, p. 259 (1978)]
, and (6) A method of adding an aqueous solution in which a labeling substance is dissolved to an organic solvent containing lipids and subjecting it to ultrasonication using an ultrasonic oscillator, and then removing the organic solvent using a rotary evaporator to obtain a reversed phase liposome.C.F. Zuoka Jr. (F, 5zo
Ka.

Jr. et al., Proceedings of the National Academy of Sciences in Nis Nis Nis (Proc, Natl, Acad, Sci, US
A), Vol. 75, p. 4149 (1978)].

In addition, polymerizable functional groups such as acrylic groups, methacrylic groups, diacetylene groups, and diene groups are introduced into lipids, sialgyl phosphates, dialkyl ammonium salts, etc., and based on the above method, at least a portion of these polymerizable lipids, etc. A method in which a liposome containing a liposome is prepared, and then the liposome is stabilized by causing polymerization in the liposome membrane by irradiating it with ultraviolet rays or adding a polymerization initiator [for example, H, 0hno] et al., Macromolecules (Macro++olecules),
20! , p. 929 (1987), and E. Tsuchida (
E, Tsuchid-a) et al., Macromolecular
Hemy (Makromol.

Chem, ), p. 187, p. 1351 (1986)] can also be used. Polymerization of liposomes increases the physical strength of the membrane, improves the ability to retain labeling substances encapsulated within the liposomes, and prevents fusion of liposomes, allowing for stable storage for long periods of time. This is extremely advantageous because it allows for high measurement sensitivity. In the case of mixed liposomes consisting of polymerizable compounds and non-polymerizable lipids, it is possible to select compatible liposomes and non-compatible liposomes that cause phase separation within the liposome membrane. Furthermore, the physical strength and stability of the membrane can be controlled by changing the composition ratio of polymerizable compounds and non-polymerizable lipids. In the case of mixed liposomes in which phase separation occurs within the liposome membrane, when complement or antigen is present above a certain concentration, the labeling substance is released in a stepwise manner due to the action of complement. It is also possible to use this method to easily determine the presence of complement or antigen qualitatively.

The labeling substance to be encapsulated in the liposome may be any substance that is hydrophilic and can be quantified when released outside the liposome, such as radioisotopes, enzymes, fluorescent substances, metals, and pigments that can be encapsulated in the liposome. Things are applicable. For example, 1 to 5 used in radioimmunoassay
Radioactive isotopes such as 1 and 1fill, fluorescent substances such as carboxyfluorescein, fluorescein isothiocyanate, fluorescein isocyanate, tetrarhodamine isothiocyanate, 5-dimethylamino-1-naphthalenesulfonyl chloride used in fluorescence immunoassay, and eosin Y, Luminescent substances such as auramine 0, luminol, and luciferin can be used.

In particular, if enzymes such as β-D galactosidase, peroxidase, alkaline phosphatase, and glucose-6-phosphate dehydrogenase, which are used in enzyme-linked immunosorbent assay, or metals such as calcium ions are encapsulated in liposomes, they can be activated by the action of complement. When these substances leak out due to liposome destruction, 4-methylumbelliferyl β- is used as a substrate for each enzyme.
D-galactoside, p-hydroxyphenylpropionic acid, 4-methylumbelliferyl phosphate, glucose-6-phosphate, ethanol, etc. are used, and calcium-dependent enzymes for calcium ions, such as calpain and protein kinase, are used. C.) It is advantageous to include lance glutaminase in the reaction solution because it constitutes a biochemical amplification reaction and responds with high sensitivity to extremely small amounts of antigen.

The above-mentioned liposomes are prepared with appropriate functions for measuring complement or antigen. Liposomes for complement measurement are prepared to undergo a membrane damage response in response to complement activity, for example by incorporating into their membrane or binding on their membrane surface a substance that activates the complement system. be done. Liposomes for measuring antigens, for example, have an antibody against the antigen to be measured bound on their membrane surface.The method of the present invention is thus provided with functions corresponding to these for measuring complement or antigen. It is applicable to any liposomes prepared by

Liposomes prepared as described above can of course be used as they are, but they can also be used as immobilized products obtained by using commonly used immobilization methods such as carrier binding, crosslinking, and entrapping methods. You can also use it. Supports for the carrier binding method include polysaccharide derivatives such as cellulose, dextran, agarose, and starch, porous synthetic resins, and inorganic substances such as metal oxides, and carriers for the comprehensive method include synthetic polymer materials. polyacrylamide, photocurable resin (ENT film, etc.), urethane prepolymer and natural polymeric material alginic acid,
Applicable materials include carrageenan, starch, gelatin, and chitosan. Further, the shape of the immobilized material varies depending on the type of immobilization method, but may be film-like, bead-like, or other shapes.

〔Example〕

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

Example 1 Preparation of Rabbit T-globulin Binding Liposomes Two N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) 28.1μ+*ol and dipalmitoylphosphatidylethanolamine (DPPE)
20 μmol was dissolved in 4.5 ml of chloroform and 0.5 ml of absolute methanol. To this was added 20 μl 1 ol of triethylamine, and the mixture was stirred and reacted at room temperature under nitrogen for 2 hours. After the reaction was completed, 3.51 d of methanol and 2 ml of distilled water were added, and the mixture was thoroughly shaken. This was allowed to stand, the chloroform phase was removed, the solvent was evaporated, and about 5-chloroform was added and dissolved. 8 liquids were dried at 150°C for 12 hours or more and equilibrated with chloroform.
-100, manufactured by Wako Pure Chemical Industries, Ltd.) column, and chloroform-methanol (20:l V/V) 40-50-
Washed with.

Both fractions eluted with chloroform-methanol (5: 1.V/V) were collected, and after evaporating the solvent, a new 5.
- dissolved in chloroform. By the above operation, N-
(4-<p-maleimidophenyl)butyryl]dipalmitoyl phosphatidylethanolamine (MPB-DP
PE) was prepared.

In a 100 ml eggplant flask, add 75 μmol of dipalmitoylphosphatidylcholine (DPPC), 75 μmol of cholesterol, and dicetyl phosphate (CDCP).
7.5/Jmol, MPB-DPPE7.5μ
Take a mol and dissolve it in about 101R1 chloroform,
After removing the solvent with a rotary evaporator in a water bath at 42° C. or higher, 0.2 M carboxyfluorescein (CF) 15-, a self-quenching fluorescent substance, was added to the remaining lipid thin film as a labeling substance solution, and the mixture was mixed with portex ( V
MLV was prepared by stirring with shaking for 10 minutes using an (ortex) mixer.

This was subjected to ultrasonic treatment for 15 minutes using a probe-type ultrasonic oscillator (Nippon Seiki Seisaku Seiki, US-300) to homogenize it and make it SUv. Then, 5ephadexG-2 was added to remove CF that was not encapsulated in the liposomes.
5 (trade name, manufactured by Pharmacia) column to obtain reactive liposomes.

1 QdN-succinimidyl 3 to 5 nv/rat rabbit T-globulin phosphate-buffered saline solution 5-
-(2-pyridyldithio)propionate (SP[lP
) 0.1 d of ethanol solution was added, and the mixture was stirred at room temperature for 15 minutes to react. In order to remove unreacted 5PDP etc., 0.
1M acetic acid-sodium acetate buffer + 0°15M Na
5ephadex G equilibrated with C1 (pH 4,5)
Chromatographic separation was performed on a 25 column. The first peak portion was collected, and dithiothreitol (DTT) was added and dissolved as a solid to a concentration of 50 IIIM, and the solution was left for 30 minutes at room temperature under nitrogen. After the completion of the reaction, phosphate buffered saline (PBS) was added. ) (pH 7,4) equilibrated with 5epha
Chromatography was performed using dex G-25, and the first peak portion was collected. Through the above operations, an SR group was introduced into rabbit γ-globulin through 5PDP, and SHI-introduced T-globulin was prepared.

Next, a two-fold dilution of the reactive liposome and a two-fold dilution of the SH group-introduced γ-globulin obtained in step E were mixed at a ratio of 1:1 (V/V), and nitrogen was added. After being allowed to react for 12 hours under encapsulation at 4°C, 5eph was added to remove γ-globulin that did not bind to the liposomes.
Chromatography was performed using an acryl S-1000 (trade name, manufactured by Pharmacia) column to obtain a rabbit γ-globulin-binding liposome.

Measurement of complement activity: The rabbit r-globulin-binding liposomes were mixed with gelatin at a concentration of 0.0% so that the initial fluorescence intensity was appropriate.
1 wt% containing 0.5 mM MgCIz and 0.15d
Phosphate buffered saline (pH 7) containing CaC1z
The test sample was 0, 2x guinea pig complete complement (manufactured by Hosei Pharmaceutical Co., Ltd., manufactured by Kiku) diluted with PBS to a known concentration.
sl was added and allowed to react at 37°C for 60 minutes.The fluorescence intensity of each sample was measured using a spectrofluorometer (■Shimadzu Corporation, R
F-5000) (excitation wavelength: 490 nm, fluorescence wavelength 518 nm), and the measured values were obtained by adding 1-propatool (n-propyl alcohol) to the measurement sample after the measurement was completed.
．． 2 d was added to double the fluorescence intensity after completely destroying the liposomes (because it was diluted 2 times with 1-propatool), and PBS-t-0,2 was added instead of guinea pig total complement.
- Labeling substance release rate (%) is the relative conversion of the fluorescence intensity of each test sample, assuming that the difference in the added amount is 100%.
And so. The results are shown in Figure 1.

As can be seen from the figure, release of the labeling substance depending on the complement concentration is observed from low to high concentrations. This shows that by using the line shown in FIG. 1 as a calibration curve, complement can be stably measured from low to high concentrations.

Comparative Example 1 A rabbit γ-globulin-binding liposome obtained in the same manner as in Example 1 was mixed with 0.511 MMgc1. and O, 15mM
Guinea pig total complement (isolated pharmaceuticals) diluted with PBS to a known concentration was added to 4-4 diluted with phosphate buffered saline (pH 7.4) containing CaC11.
After adding 0.2- (manufactured by) and allowing it to react at 37° C. for 60 minutes, the fluorescence intensity was measured in the same manner as in Example 1. The results are shown in Figure 1. Note that the labeling substance release rate was displayed in accordance with Example 1.

m, 41, a labeling substance that does not depend on complement concentration, was observed at low complement concentrations, and release of labeling substances that depended on complement concentration from low to high concentrations, as seen in Example 1, was observed. I couldn't.

〔Effect of the invention〕

According to the invention, +! Using liposomes and complement, which are encapsulated inside a woven material and undergo membrane damage due to complement activation,
In an immunoassay method that measures complement or antigen present in a test sample by applying complement to the liposome and measuring the labeled substance released from the liposome depending on complement activity or antigen amount. , the liposome and the complement are protected and stabilized by containing a polymeric substance that does not activate the complement system in the salt solution containing the liposome,
The release of a non-specific labeling substance independent of complement activity or antigen amount from the liposome and the rapid deactivation of complement are prevented,
Stable and accurate complement or antigen measurement becomes possible.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between guinea pig complement concentration and labeling substance release rate measured in Example 1 and Comparative Example 1. Kare'3 Furengai S+. (CHBo/4IQ:]

Claims (1)

[Claims] 1. Using a liposome in which a labeling substance is encapsulated and which undergoes a membrane-damaging effect due to complement activity, and complement, complement is applied to the liposome, and the labeling substance released from the liposome is determined according to the complement activity or the amount of antigen. By measuring labeled substances,
An immunoassay method for measuring complement or antigen present in a test sample using liposomes, characterized in that a salt solution containing the liposomes contains a polymeric substance that does not activate the complement system.