JPH08211062A - Reagent for measuring immunity and immunity measurement method - Google Patents

Abstract

PURPOSE: To easily, rapidly, and accurately perform measurement by using a reagent consisting of a laminar structure including liposome. CONSTITUTION: After adding solvent to phospholipid and/or glycolipid for reaction, the solvent is eliminated and is subjected to sucked and dried. After that, it is added to a solution of a specific labeled substance to obtain liposome where the labeled substance is sealed. By reacting it with modification antigen in a buffer liquid, antigen which is a measurement reagent and/or antibody sensitization liposome can be obtained. Finally, by mixing it with gel substance such as gelatine, laminar structure is formed. By dropping a sample to be inspected onto the laminar structure of the reagent for measuring immunity which is prepared in this manner, labeled substance is released from the inside of the liposome proportionally to the amount of reaction. Quantification is performed by the analysis method according to the labeled substance and the antibody, antigen, or the amount of complement in the sample is measured by a calibration curve which is created in advance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay reagent for quantitatively or qualitatively analyzing a specific antigen (or antibody) or complement present in a sample, and an immunoassay method using the reagent.

[0002]

2. Description of the Related Art Immunoassay methods for samples include cross immunoelectrophoresis (CIE), two-dimensional immunodiffusion (MO), passive hemagglutination (PHA), immunoadhesive hemagglutination (IAHA), and enzymes. Immunoassay (EIA), solid phase radioimmunoassay (RIA) and the like are known. At present, the enzyme immunoassay (EIA) is mainly used. Among them, CIE and MO have a long measurement time and low sensitivity. Further, it is difficult to prepare stable antigen-coated erythrocytes for PHA and IAHA, the reagent price is high, and the quantification can be performed only stepwise with the control as a comparison. Further, although EIA and RIA are 5,000 to 50,000 times higher than MO in terms of sensitivity, they have the drawback that the reagent price is high and the measurement takes several days.

In order to solve these problems, the inventors of the present invention, as described in JP-A-63-309864, have immobilized hydrophilic antibodies or antigens on the surface and have hydrophilic antibodies inside. A measurement method using a liposome reagent encapsulating a labeling substance (for example, a fluorescent substance) has been proposed. This method is LI
LA (Liposome Immunolysis Assay; Journal of Immunological Methods, Vol. 75, 351).
─360 page, 1984), and the liposome reagent having a composition of at least one of phospholipid and glycolipid is often used. This LIL
A is as follows. That is, a liposome reagent is added to a reagent in which an antigen and / or an antibody and / or complement are present, and a substance capable of invading complement or membrane is added to the reagent (this is not necessary in the case of complement measurement, When a complement component is contained, a complement may or may not be added separately), and an antigen-antibody reaction and accompanying activation of a substance capable of invading the complement or the membrane occur, and the complement or The liposome is destroyed by the membrane-damaging action of the substance capable of invading the membrane, and the encapsulated labeling substance flows out. Since there is a correlation between the amount of the labeled substance that has flowed out and the test substance in the sample, that is, the antibody or the antigen or the complement, the amount of the labeled substance that has flowed out can be quantified by a predetermined analysis method (for example, fluorescence analysis). By doing so, the test substance can be quantified. Therefore, this LILA solves the above problem.

However, since the LILA method is a reaction in an aqueous phase, it is necessary to sequentially add samples and reagents, and a large-scale dispenser is provided for automation. Requires automated equipment. As a measurement method that does not require such an automated device, a dedicated solid-phase reagent is used in biochemical tests (hemoglobin, total protein, total cholesterol, blood sugar, uric acid, potassium, GOT, GPT, ALP, LDH, amylase). A measurement method by dry chemistry has been established in which a sample is dropped and reacted and then measured by optical and electrical means. However, even in dry chemistry, in order to measure an antibody or an antigen, it is necessary to perform an operation (B / F separation) of separating only the antibody or the antigen to be measured, and it is not possible to realize with a layer structure of dry chemistry. It was not easy, and the B / F separation was insufficient, so that the background was extremely high.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a dry chemistry reagent for immunoassay having extremely high accuracy (large S / N ratio) of a test substance. The purpose is to do. It is another object of the present invention to provide an immunoassay method that enables simple, rapid and accurate measurement using this dry chemistry reagent.

[0006]

In order to achieve the above object, the first aspect of the present invention is to enclose a labeling substance inside a phospholipid and / or glycolipid as a main component, and at the same time, an antigen and / or an antibody. An immunoassay reagent characterized by comprising a layered structure encapsulating a liposome formed by binding
A second aspect of the present invention has an immunoassay method characterized by using the above reagent. In the layered structure encapsulating liposomes, the phase-matching reaction, which is a feature of the LILA method, reacts with the sample inside the layered structure, and the labeling substance diffuses from the liposome. / The background does not become high unlike the antibody measurement, and a sufficient S / N ratio can be obtained.

The present invention will be described in more detail below. In the present invention, the liposome may be any conventionally used one, as long as it has at least one of phospholipids and glycolipids as a main constituent. Examples of such phospholipids include phospholipids widely existing in cell membranes of animals and microorganisms, for example, various phospholipids such as phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, and sphingomyelins. Of course, natural egg yolk, phosphatidylcholine obtained from bovine brain, soybean, etc. can also be applied. Further, a natural extract or artificial synthetic product of a thermophilic fungal lipid belonging to archaea can also be applied.

The types of fatty acids in such phospholipids include various saturated or unsaturated fatty acids. For example, specific examples of phosphatidylcholine include diheptanoyl-, dicaproyl-, didecanoyl-, dilauroyl- and diheptadeca. Examples thereof include fatty acids such as noyl-, dibehenoyl-, dimyristoyl-, and dipalmitoyl-phosphatidylcholine, and the above-mentioned other phospholipids also include various saturated and unsaturated fatty acids. In the case of a phospholipid in which a carbon chain is ester-bonded to a glycerol portion, a stable liposome can be obtained by adding an appropriate amount of cholesterol to the lipid, for example, 10 to 500 mol%, if necessary.

The glycolipid is not particularly limited as long as it forms a liposome, and may be a glycosphingolipid or a glyceroglycolipid. Such glyceroglycolipids include mannosylphosphatidylinositols, galactosyldiacylglycerols, sulphoquinovosyldiacylglycerols, glycosyldiacylglycerols, and seminolipids, and sphingoglycolipids include mannosylglucosylceramides and mannosylinositolphosphorylceramides. These may be used alone or in combination of two or more.

The shape of the liposome used in the present invention is
Multilamellar vesicles (hereinafter, M)
LV), small unilamellar liposomes (small unilam)
ellar vesicle (hereinafter referred to as SUV), large unilamellar vesicle (hereinafter referred to as LU)
V)). Further, these liposomes can be produced by a known method.

As a method for preparing a liposome encapsulating a labeling substance, a conventionally known method for preparing a liposome can be applied. For example, (1) a phospholipid dissolved in an organic solvent is placed in a container, the solvent is removed under reduced pressure by blowing an evaporator or nitrogen gas to form a thin lipid film, and then a suitable labeling substance containing a labeling substance to be encapsulated in advance is added. A method of adding a salt solution or an aqueous solution in which the labeling substance is dissolved in advance and shaking [AD Bangham]
, Journal of Molecular Biology (J. Mol. Biol.), Vol. 13, 238 (1965) and D. Papahadjopoulos.
Biochemical Biophysical Actor (Bioc
hem. Biophys. Acta), 135, 639 (1967).
9)] or a method of shaking and agitating with a vortex mixer [K. Inoue, Biochemical Biophysical Actor, 339, 390 pages (197).
4) and SC Kinsky
Et al., Biochemistry, Volume 8, 41
P. 49 (1969)], (2) Method of producing SUV by ultrasonically treating MLV obtained by the method of (1) above with an ultrasonic oscillator [Die Papajopoulas et al., Biochemical Biophysical Actor]. 311 Volume 310
Page (1973)], (3) 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, Volume 298, page 1015 (1
973)], (4) a method of slowly blowing out a phospholipid dissolved in ether above the boiling point of ether into an aqueous solution in which the labeling substance is previously dissolved [D.
W Deamer, Annual
New York Academy of Sciences (Ann.
NY Acad. Sci.), 308, 259 (1978).
(5) Phosphatidylserine alone or liposomes consisting of equal amounts of phosphatidylserine and cholesterol were sonicated with an ultrasonic oscillator to prepare SUVs, and after calcium treatment, a salt solution containing a labeling substance and ED
A method of preparing LUV by adding TA [See D. Papajopoulas, Annual New York Academy of Sciences, Vol. 308, p. 259 (1978)], and (6) Aqueous solution in which a labeling substance is dissolved in an organic solvent containing lipid. After sonicating with an ultrasonic oscillating device, the organic solvent was removed by a rotary evaporator to obtain reverse-phase liposomes (F. Szoka. Jr.).
Et al., Proceeding of National Academy of Sciences in USA (Proc.
Natl. Acad. Sci. USA), 75, 4149 (197).
8), etc.] can be used.

The labeling substance encapsulated in the liposome must be a substance that can be quantified when eluted outside the liposome. Such substances include, for example, radioisotopes such as ¹²⁵ I and ¹³¹ I used in radioimmunoassay; fluorescence such as carboxyfluorescein and fluorescein isothiocyanate which emit very strong fluorescence even in a low concentration range (10 ⁻³ M or less). Compounds; luminescent compounds such as luminol and luciferin that emit light by oxidation reaction outside the liposomes; light-absorbing compounds (water-soluble dyes, etc.) that have a specific absorption band in the visible or ultraviolet region; by the action of oxidase Sugars such as glucose and sucrose that are decomposed to cause oxygen consumption or hydrogen peroxide formation; relatively large ionic compounds such as tetrapentyl ammonium; metals such as calcium ions; supplements such as nicotinamide adenine dinucleotide (NAD) Enzymes; beginning with methylpiorogen Radical compounds; enzyme immunoassay used in such β- galactosidase, an enzyme such as peroxidase or the like is preferable. These are used alone or in combination of two or more.

When calcium ion is used as the labeling substance, a calcium-dependent enzyme (carbaine, protein kinase C, transglutamylase, etc.) is used, and when an enzyme is used, the substrate (the substrate for the above-mentioned enzyme is 4 -Methylumbelliferyl β-D-galactoside, p-hydroxyphenylpropionic acid, etc.), or conversely, when these substrates are used as labeling substances, enzymes, and in some cases coenzymes, are disrupted by liposome destruction. It must be used when the labeling substance flows out.

The antigen and / or antibody to be sensitized on the liposome is appropriately selected according to the purpose of the test. For example, A
anti-HBsAb, Anti-HBcAb, Anti
-HCV, Anti-HlVAb, human T-cell leukemia virus-type I (HTLV-I), human T-cell leukemia virus-type II (HTL)
Examples thereof include virus-related antibodies such as V-II), antigens against them, various hormones in blood, serum proteins such as lgG, and chemical substances that can be antigens such as haptens. These are used alone or in combination of two or more. The binding of the antigen and / or antibody to the liposome may be performed by a known method. For example, Les Le Mans (Le
srman) et al. (Nature, 288, 604-60)
6, 1980), the method of Martin et al. (Biochemistry, 20, 4229-4238, 1981) and the like.

The immunoassay reagent of the present invention is produced, for example, by the following method. First, phospholipids and / or glycolipids are placed in a flask, a solvent is added and reacted, then the solvent is distilled off and suction drying is performed. After that, an aqueous solution of a predetermined labeling substance is added into a flask having a thin film formed on the wall surface, the container is sealed and shaken to obtain a labeling substance-encapsulated liposome. Further, an operation for forming the SUV shape is added to this liposome. on the other hand,
Modification by reacting an antigen and / or antibody with a cross-linking agent in a buffer to introduce the cross-linking agent, and then reacting with a reagent that reduces the cross-linking group (eg, dithiothreitol; DTT), if necessary. Obtain the antigen (modified antibody or modified chemical) or modified portion thereof. When the Fab ′ fragment of the antibody is used, the modified antibody Fab ′ fragment can be obtained by reducing the (Fab ′) ₂ fragment without using a crosslinking agent. When an oligopeptide is used as the antigen, introduction of a cross-linking agent can be omitted by adding a Csy residue to the N-terminus or C-terminus in advance.

By reacting the labeled substance-encapsulated liposome with the modified antigen (modified antibody or modified chemical substance) in a buffer solution, an antigen- and / or antibody-sensitized liposome as the measuring reagent of the present invention can be obtained. Such a measuring reagent is usually
It is obtained as a liposome encapsulating a labeling substance and carrying an antigen and / or antibody bound to the surface.

Examples of the cross-linking agent in the above production method include N-succinimidyl 3- (2-pyridyldithio).
Propionate (SPDP), N-succinimidyl 4
-(P-maleimidophenyl) butyrate (SMP
B), N-succinimidyl 4- (p-maleimidophenyl) acetate (SMPA), N-succinimidyl 4- (p-maleimidophenyl) propionate (SM
PP), N- (γ-maleimidobutyryloxy) succinimide (GMBS), N- (ε-maleimidocaproyloxy) succinimide (EMCS), dialdehydes such as glutaraldehyde and terephthalaldehyde, and the like. Alternatively, they may be used in combination of two or more.

Finally, a liposome encapsulating these labeling substances and carrying the antigen and / or antibody bound to the surface is formed into a layered structure. The layered structure may be formed by mixing the liposome with a gel substance such as gelatin, colloid, and agar to form a layer having an appropriate thickness on, for example, a slide glass. It may be enclosed and formed into a film. When forming into a film, if necessary, a plastic support may be formed at the same time.Furthermore, it functions as a diffusion layer for filtering blood into serum and selectively diffuses protein fractions such as antibodies. You may add a film that has the function of a diffusion layer, such as
It may be a multilayer structure. In addition, complement, substrate, enzyme,
If necessary, coenzymes and other chemicals may be included in the layered structure together with the liposome during molding.

In order to measure an antibody, an antigen or a complement in a subject using the immunoassay reagent composed of the layered structure thus prepared, the test sample is dropped onto the layered structure. Good. By this dropping, the labeling substance is released from the liposome in proportion to the reaction amount. Therefore, the labeling substance is quantified by an analysis method (for example, if the labeling substance is a fluorescent substance, a fluorescence analysis method), For example, the amount of antibody, antigen or complement in a sample can be measured by a calibration curve prepared in advance.

The endpoint method may be used as the measuring method, but needless to say, the rate method may be used, and the measuring method is not particularly limited. Furthermore, the reflected light from the upper and lower surfaces of the layered structure may be used,
Transmitted light may be used and is not particularly limited. The complement used in this measurement operation is not particularly limited, but usually,
Guinea pig serum is used. Rabbit, mouse, human, etc. serum may be used.

[0021]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 In this example, equine cytochrome C (hereinafter referred to as cytochrome C
Prepared as a gelatin layered structure, and using it, rabbit anti-horse cytochrome C was prepared.
(Hereinafter referred to as anti-cytochrome C antibody) was measured.

(1) Preparation of liposome (MLV) N-succinimidyl 4- (p-maleimidophenyl)
Butyrate (SMPB) 28.1 μmol and dipalmitoylphosphatidylethanolamine (DPPE) 20
μmol of chloroform 4.5 ml and anhydrous methanol 0.5
It was dissolved in a mixed solution with ml. Triethylamine 20μ
Mol was added, and the mixture was stirred and reacted at room temperature for 2 hours under nitrogen. After completion of the reaction, 3.5 ml of methanol and 2 ml of distilled water were added and shaken well. This was allowed to stand, the chloroform phase was taken, the solvent was evaporated, and about 5 ml of chloroform was added and dissolved. This solution was loaded on a silica gel (Wakogel C-100, manufactured by Wako Pure Chemical Industries, Ltd.) column having a capacity of 10 ml dried at 150 ° C. for 12 hours or more and equilibrated with chloroform, and chloroform-methanol (2
Wash with 40-50 ml (0: 1, v / v). Fractions eluted with chloroform-methanol (5: 1, v / v) were collected, the solvent was evaporated, and the residue was dissolved in 5 ml of chloroform. By the above operation, N- [4- (p-maleimidophenyl) butyryl] dipalmitoylphosphatidylethanolamine (MPB-DPPE) was prepared.

In a 100 ml eggplant type flask, Japanese Patent Laid-Open No. 3-2
1,2-di-o-phthalylglycero-3-phosphocholine (Phy) synthesized according to the method described in JP-A-62142.
PC) 75 μmol, dicetyl phosphate (DCP) 7.5 μ
mol, MPB-DPPE 7.5 μmol, and about 10
After dissolving in chloroform (ml) and removing the solvent with a rotary evaporator in an aqueous solution at 42 ° C. or higher, 0.2 M carboxyfluorescein (CF), which is a self-quenching fluorescent substance as a labeling substance aqueous solution, is applied to the remaining lipid thin film. ) 15
Add 1 ml with a Vortex mixer
MLV was prepared by shaking and stirring for 0 minutes. Then, in order to remove CF not encapsulated in the liposome, Biogel A-
15m (Medium) (trade name, manufactured by Bio-Rad)
Chromatographic separation by column, reactive liposomes (ML
V) was obtained.

(2) Preparation of liposome (SUV) The MLV obtained in (1) above was probed for 15 minutes with an ultrasonic oscillator (Nippon Seiki Seisakusho Ltd., US-300).
Was homogenized to obtain a uniform SUV. Then, in order to remove the CF due to the broken liposome, chromatography separation was performed using a Biogel A-15m (Medium) (trade name, manufactured by Bio-Rad) column to obtain a reactive liposome (SUV). The diameter measured by NICOMP370 particle size analyzer (trade name, manufactured by Nicomp) is 8
It was 0 nm.

(3) Sensitization of cytochrome C to liposomes In 1.5 ml of 0.5 mg / ml cytochrome C phosphate buffered saline solution, 40 mM N-succinimidyl-3- (2-
0.0323 ml of an ethanol solution of pyridyldithio) propionate (SPDP) was added, and the mixture was stirred at room temperature for 10 minutes for reaction. 20 mM M to remove unreacted SPDP
Chromatographic separation was performed using an Econopack P6 cartridge (trade name, manufactured by Bio-Rad) column equilibrated with an ES buffer (containing 0.15 M NaCl, pH 6.0). The first peak portion was collected, to which dithiothreitol (DTT) was added as a solid to 100 mM to dissolve it, and the mixture was allowed to stand at room temperature for 30 minutes under nitrogen. After completion of the reaction, the mixture was chromatographed on an Econopack P6 cartridge column equilibrated with phosphate buffered saline (PBS) (pH 7.4), and the first peak portion was collected. By the above operation, SH group was introduced into cytochrome C through SPDP to prepare SH group-containing cytochrome C. Next, the reactive liposome obtained in the above (2) and the SH group-introduced cytochrome C obtained by the above operation were 1: 1 (v /
v) were mixed, allowed to react at 4 ° C. for 24 hours under nitrogen inclusion, and then subjected to a chromatographic separation with a Biogel A-15m (Medirum) column to remove antigens not bound to liposomes, and each antigen-bound. Liposomes (SU
V) was obtained.

(4) Preparation of liposome-encapsulating layered structure The antigen-bound liposomes obtained in (3) above were treated with 0.5 mM Mg.
5% (w / v) gelatin containing Cl ₂ and 0.15 mM CaCl ₂ was mixed with the guinea pig whole complement so that the emission concentration of fluorescence emitted was appropriate, and the thickness was 0.5 mm on a slide glass. Was formed to obtain an immunoassay reagent.

(5) Measurement of anti-cytochrome C antibody On the layered structure of the above reagent, 5 μl of a double diluted buffer solution containing the anti-cytochrome C antibody was dropped on the layered structure on a slide glass at each concentration. Then, it was left to react at 37 ° C. for 60 minutes. The fluorescence intensity of each sample was measured with a spectrofluorometer F-2000 (manufactured by Hitachi, Ltd.) modified so as to emit excitation light with an optical fiber and measure fluorescence (excitation wavelength 490 nm, fluorescence wavelength 520 nm). The measured value was 5% -Triton X-100 on the measurement sample after the measurement was completed.
Fluorescence intensity after completely dropping the liposomes to completely destroy the liposomes and 5 μm of buffer solution instead of the anti-cytochrome C antibody buffer solution
The marker release rate (%) was obtained by relatively converting the fluorescence intensity of each test sample, with the difference between the added samples being 100%.
And The results are shown in Fig. 1.

[0029]

As described above, by using the immunoassay reagent comprising the layered structure of the present invention, the quantitative or qualitative analysis of the antigen, antibody or complement in the subject can be carried out by a simple operation.
It can be performed inexpensively and in a short time with high detection sensitivity and accuracy, and its automation is also extremely easy.

[Brief description of drawings]

FIG. 1 is a characteristic diagram showing the relationship between the concentration of anti-cytochrome C antibody and the relative release rate of a labeling substance when anti-cytochrome C antibody measurement is performed using cytochrome C-sensitized liposomes.

Claims (2)

[Claims] 1. A lamellar structure comprising phospholipids and / or glycolipids as main constituents, encapsulating a labeling substance therein, and encapsulating a liposome formed by binding an antigen and / or an antibody. A reagent for immunoassay. 2. An immunoassay method comprising using the reagent for immunoassay according to claim 1.