JPH04303770A - Analyzing method for immunity of antigen - Google Patents

Abstract

PURPOSE:To provide a new immunity analyzing method, of a high detection sensitivity, to destroy an immunoriposome and detect indirectly an antigen, after catching the multivalent antigen between an immobilized primary antibody and the immunoriposome. CONSTITUTION:A primary antibody immobilized to the solid phase of a microwell, etc., and a immunoriposome to enclose a detecting element of an enzyme reagent, etc., within the reposome and to combine the secondary antibody in its surface are used, and by stages, a multivalent antigen is in a sandwich state caught between these primary and secondary antibodies, and an immunity complex is formed. The quantity of the antigen is measured by destroying the immunoriposome, with which the secondary antibody taken part in this complex formation combines, with a lipase, etc., and measuring the quantity reacted by a discharged detecting element.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immunoassay of antigens using immunoliposomes. More specifically, the primary antibody is immobilized on a solid phase such as a plastic tube, microwell, or membrane surface, and the secondary antibody is immobilized on the liposome surface. The present invention relates to a method of separating and detecting specific antigens from a test liquid by sandwiching compounds having two or more antibody recognition sites as antigens.

0002

[Prior Art] Immunoassay is based on various hormones (thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, thyroxine, prolactin, chorionic gonadotropin, etc.) in the blood and urine, tumor markers (α-fetoprotein,
carcinoembryonic antigen, ferritin, elastase, neuron-specific enolase, etc.), viruses (measles virus, rubella virus, herpes simplex virus, cytomegalovirus, adult T-cell leukemia virus, AIDS virus, EB)
Viruses, etc.), pathogenic microorganisms (Chlamydia, Phosphorus bacteria, Streptococcus, Mycoplasma pneumoniae, Aspergillus, Histoplasma, Coccidioides, Toxoplasma, etc.), leukocyte antigens (platelet surface antigens, HLA-A, C, DR, etc.), autoantibodies (antinuclear antibodies, anti-DNA antibodies, anti-histone nuclear protein antibodies, anti-centromere antibodies, pancreatic islet cell membrane antibodies, anti-mitochondrial antibodies, etc.), as well as contaminating pathogenic bacteria in the environment such as food and sewage (Salmonella, Enterobacter, Staphylococcus, Escherichia coli). This is currently the most frequently used method for detecting trace components of enterotoxins, various enterotoxins, etc.

[0003] Various methods are known for immunoassay, but among them, when the protein, polysaccharide, etc. to be detected is a so-called multivalent antigen having a high molecular weight and two or more antibody recognition sites. There is a method of capturing and detecting an antigen by using two or more types of antibodies with different specific antigen recognition sites and sandwiching the antigen between them.

[0004] In this method, an immune complex is first formed with a large excess of a solid-phase immobilized antibody (primary antibody) and an antigen in a sample, and then an excess amount of an enzyme-labeled secondary antibody is injected into the first antibody. After binding to different antibody recognition sites on the immune complex, unreacted labeled antibodies are removed by washing, and information resulting from the enzyme of the secondary antibody label is measured by absorbance, etc., and finally a concentration range of It is an immunoassay method that attempts to detect antigens, and is a sandwich type enzyme-linked immunosor.
bent assay method (Sandwich-ELI
SA method) [References: (1) Edited by Ishikawa et al., "Enzyme immunoassay method 2nd edition", Igaku Shoin (1982), (2) Yumiko Imada, Food Hygiene Research, Vol. 38, No. 5, pp.
39-54, (3) Addison, G. M. ，
Horm. Metab. Res. , 3, 59 (
1971)]. At present, this method is commonly used as one of the highly sensitive methods for detecting antigens.

[0005] The solid phase for immobilizing the primary antibody in this immunoassay is generally made of plastic microwells such as polystyrene, various plastic tubes, latex beads, polystyrene balls [Yoshihiri et al.
o Kasai, Clin. Chem. Enzy
m. Comms. , Vol. 2, pp137-1
43 (1990)], magnetic particles
[Smith, K. O. : J. Intect.
Disease, 136, 329 (1977)
] etc., and both the primary antibody and the secondary antibody immobilized on such various solid phases are monoclonal antibodies or polyclonal antibodies. In addition to enzymes, fluorescent substances, luminescent substances, radioactive isotopes, etc. are used to label the secondary antibodies, and each label differs depending on the labeling substance used.
Immunofluorometric assay
(IFMA) method, Immunoluminometri
c Assay (ILMA) method, Immunora
It is called the diometric assay (IRMA) method [Translated by Koichiro Kawashima, "Introduction to Immunoassay",
Nanzando (1987)].

[0006] Specifically, these methods involve sandwiching an antigen between antibodies as described above, and then performing ELIS.
In method A, a secondary antibody-labeled enzyme, such as HRP (Hors
(eradishPeroxidase) Enzyme activity can be measured by color development, or IRMA method can be used to measure I-125 enzyme activity.
This is a method for detecting antigens by measuring the radioactivity of labeled radioisotopes, such as, using a liquid scintillation counter. In this sandwich-type immunoassay method, the detection sensitivity of the antigen varies greatly depending on the labeling substance of the secondary antibody as mentioned above. It has many advantages and disadvantages. However, due to convenience, sensitivity, speed, accuracy, and historical aspects, the IFMA method and the IRMA method are
method is currently the most commonly used.

[0007] However, various problems have been pointed out regarding immunoassay methods using such various labels. For example, the method that uses radioisotopes to label antibodies is currently the most sensitive (several
l) Regarding the detection method, (1) the purity of the labeled compound is a problem, (2) sensitivity above a certain antigen concentration is difficult due to the fundamental limit of decay of the nuclide, and (3) isotope can be used. The problem is that it can only be used under certain facilities. The method of using a fluorescent substance as a label is (1)
Non-specific adsorption during washing removal of unreacted fluorescent labeling substances is a problem; (2) fluorescence may weaken due to interaction with other molecules under fluorescent conditions; (3) absorption spectra and fluorescence spectra. (4) It is difficult to simplify the equipment; (4) it is difficult to simplify the equipment;
5) There is a problem that the fluorescence emission time is limited. Methods using luminescent substances as labels include (1) the reagents used are often expensive; (2) when bioluminescence is used,
Since two types of enzymes are used, it is expensive and the operation is complicated, and (3) the luminescence time is relatively short. On the other hand, the method using an enzyme as a label does not have the above-mentioned problems, but rather (1) the reagent is cheap;
2) The measurement device is simple, (3) Color development continues for a relatively long time as time passes, so if non-specific adsorption is eliminated, higher sensitivity can be expected, and (4) It is safe to handle. .

[0008] As described above, the enzyme labeling method is found to be useful in terms of economy, operability, and safety compared to other labeling methods. In addition, there are reports that it has achieved higher sensitivity (less than amol) compared to radiolabeling methods [Eiji Ishikawa, Pathophysiology, Vol. 3. No.
8 (1984:8) pp654-660].

On the other hand, several attempts have been made to utilize liposomes in immunoassays. For example, in a so-called homogeneous solution system (homogenous solution system) in which the primary antibody is not immobilized on a solid phase,
genetic system, heterogeneous system when the primary antibody is immobilized on a solid phase
sandwich method [Literatures: (1) JP-A-60-138466, (2) JP-A-61-269070, (3) JP-A-63-1
18658], and the antigen in the sample and the liposome-labeled antigen as a reagent are competitively bound to the primary antibody immobilized on a solid phase, and after removing the unreacted liposome reagent, only the bound liposomes are destroyed. Competitive Immunization Method
oasay method) [J. P. O. Connell,
Clin. Chem. , 31, 1424 (1
985)] is known. Comparing the sandwich method and the competitive method, the sandwich method is generally considered to be more sensitive with less noise in low-concentration antigen regions; In other words, in the case of haptens where the only antibody recognition site is a low-molecular antigen, a competitive method is used.

0010

[Problems to be Solved by the Invention] Although the sandwich method using enzyme labels is generally an excellent method, it has been pointed out that the detection sensitivity is still insufficient and the time required for detection is long. There is. For example, currently, in the case of Salmonella, for example, the detection of pathogenic bacteria in foods is carried out using a sandwich-ELISA method using monoclonal or polyclonal antibodies against bacterial flagellar proteins. Is going. However, the number of bacteria per test is approximately 10 to the 4th to 5th power, and the detection method at this time is to first culture the extract from the food overnight in a nutrient medium as a pretreatment, and then to screen. In this method, the infected bacterial group is further cultured overnight in a suitable medium. With this method, it takes about two days to get test results, so it cannot be called a quick test.

[0011]Therefore, it is desired to develop a measurement method that can further shorten the inspection time. For this purpose, it is necessary to increase antigen detection sensitivity, and it is desired that contaminating bacteria can be detected with just one night of preculture. The same thing is required for the detection of infectious pathogens such as Group A and B Streptococcus, and the reality is that reducing testing time by increasing sensitivity is an issue in this industry. .

[0012] Furthermore, as mentioned above, sandwich methods using liposomes are known, but as mentioned above, these are all so-called homogeneous solution systems, and the liposomes are destroyed using complement. Therefore, ■ complement activation must be performed at 37°C, ■ generation of noise due to contamination in the complement solution (serum) used, ■ length of reaction time, ■ instability of complement. However, it has been pointed out that this method has the problem of having to be stored at 4°C, and this method is still not satisfactory.

[0013]

[Means for Solving the Problems] In view of the above-mentioned problems, the present inventors encapsulated enzyme molecules in order to achieve a novel sandwich method with detection sensitivity as high as or higher than conventional methods. We focused on a method using immunoliposomes with secondary antibodies bound to the surface, and conducted repeated research. As a result, they discovered a method that allows detection with higher sensitivity than conventional methods in the low antigen concentration range, and completed the present invention.

That is, the gist of the present invention is to capture an antigen to be tested by sandwiching it between a primary antibody immobilized on a solid phase and an immunoliposome in which a secondary antibody is bound to the surface of the liposome encapsulating a detection element. The present invention also relates to an immunoassay method for antigens, which is characterized in that the liposomes of the formed immune complexes are destroyed and the released detection elements are measured.

[0015] Compared to the conventional method using liposomes in a homogeneous solution system as described above, in the sandwich method of the present invention, the primary antibody is immobilized on a solid phase, and the secondary antibody is bound to the surface of the liposome. to form immunoliposomes,
Furthermore, the liposome contains a detection element such as an enzyme. In the present invention, in particular, since the detection element is not directly bound to the antibody but encapsulated in the liposome, the antigen is bound between the primary antibody and the secondary antibody in a sandwich manner, and then the unreacted immunoliposome is removed. In this method, only the liposomes bound to the secondary antibodies that remain after forming a sandwich after being removed by a washing operation are destroyed, a detection element is released, and the amount of antigen is determined by measuring this detection element.

[0016] In this way, in the conventional method described above, the primary antibody is immobilized on the liposome, and the immune complex formation occurs on the liposome surface, so the reaction by the effluent material after liposome destruction involves the dispersed liposomes. It is thought that it occurs uniformly in solution. On the other hand, in the method of the present invention, since the primary antibody is immobilized on a solid phase such as plastic, it is thought that the color reaction after liposome disruption occurs mainly on the surface of the solid phase. Therefore, the advantage of the method of the present invention is that it is easy to develop color even in a very small amount of solution. In addition, since the amount of primary antibody adsorbed is proportional to the surface area of the solid phase, when binding the primary antibody onto liposomes using conventional methods, the amount of binding is limited due to the instability of the liposomes, but in the case of the method of the present invention, A large amount of primary antibody can be adsorbed onto the solid phase surface. Further, it is also possible to achieve even higher sensitivity by changing the solid phase surface state qualitatively and quantitatively.

The solid phase used in the method of the present invention may be any commonly used material that can immobilize the primary antibody. Examples include plastic surfaces such as polystyrene tubes, microwells, beads, particles, padels, and fibers, natural fiber membranes, and synthetic polymer membranes.

[0018] The detection element used in the method of the present invention may be any substance that does not affect the antigen-antibody reaction and can be detected even if a reactant is present. It is not preferable to have a strong amount of water that leaks out of the membrane arbitrarily. Further, there is no particular restriction as long as the detection element itself or the product produced by a chemical reaction has high spectroscopic properties and can produce a product that can be detected with high sensitivity as secondary information. Examples include enzymes, antibodies, coenzymes, enzyme substrates, physiologically active substances such as peptides or glycosides, fluorescent substances, dyes, and drugs. Examples of enzymes include Horseradish Peroxidas
e, Alkaline Phosphatase, β
-Galactosidase, Glucose O
xidase, Glucose-6-Phosphat
As an antibody, a labeled antibody such as a biotinylated antibody or an avidinized antibody can be used, and a coenzyme can be, for example, NAD.
, NAD(P)H, FAD, biotin, FMN, ascorbic acid, etc., and enzyme substrates such as p-nito
rophenyl phosphate, o-phe
nylene diamine, o-nitrop
peptides such as avidin, lectin, protein A, cell surface antigens, etc., and glycosides such as digoxin, digoxigenin, cAMP, ATP, glucose derivatives, etc.
Examples of fluorescent substances include FITC, Calcein, R
hodamine (isothiocyanate),
Examples of dyes include Luminol, p-lodophenol, ac
ridium ester, etc., and drugs such as Ampicillin, Penicillin,
Examples include Chloramphenicol. Among these, enzymes are particularly preferred as detection elements in terms of substrate diversity and information amplification; for example, Ho
rseradish peroxidase is preferred.

The primary antibody and secondary antibody are not particularly limited as long as they can bind to the detection antigen in a sandwich-like manner. For example, a combination in which the primary antibody and secondary antibody are monoclonal antibodies that recognize different epitopes,
It may be a combination of polyclonal antibodies or a combination of monoclonal and polyclonal antibodies to this antigen. In the method of the present invention, both the primary and secondary antibodies are preferably polyclonal (purified using an affinity column).

[0020] Liposomes can be easily obtained by conventional methods for producing liposomes, and there are no particular limitations on the liposomes obtained. For example, Dimyristo
yl Phosphatidyl Choline (
DMPC), Dilauroyl Phosphat
idyl Ethanolamine (DLPE),
Liposomes using phospholipids such as Dicetylphosphate (DCP) and having a diameter of about 0.05 to 0.2 μm can be used. In particular, DMPC, DLPE
, DCP is the constituent phospholipid, and the liposome diameter is approximately 0.1
Preferably, the thickness is μm.

[0021] A method for encapsulating a detection element in a liposome is to suspend an enzyme aqueous solution in an organic solvent in which various lipid molecules are dissolved, and then evaporate the organic solvent at room temperature and under reduced pressure to form a sol-like lipid film. (reverse phase evaporation method) is preferred, and a method of forming uniform liposome particles by subsequent ultrasonication is exemplified. The liposome thus prepared consists of 10,000 to 100,000 molecules of phospholipid, preferably about 50,000 to 100,000 molecules, and contains about 100 molecules, preferably about 10 molecules of detection elements inside.

[0022] Immunoliposomes having a secondary antibody bound to the surface of the liposomes are produced by a general method for producing peptides. For example, water-soluble coupling reagents such as carbodiimides, N-hydroxysuccinimidy
l 3-(2-pyridyldithio)prop
Immunoliposomes can be obtained by creating a peptide bond between the amino group of the liposome-constituting phospholipid and the carboxyl group in the secondary antibody molecule using ionate (SPDP) or the like. In the thus prepared immunoliposome, 1 to 10 molecules, preferably about 5 molecules of secondary antibody are bound to the surface of each liposome.

[0023] After the complex formation, the liposomes are destroyed using a nonionic surfactant, which is a general liposome destruction method, such as Triton X-10.
0, Tween 80 [(1) Anzai, K
．． , Chem. Pharm. Bull. , 28
(1980) 1762, (2) G. Eytan
, FEBS Letters, 57 (1975)
121-125], the use of ionic surfactants, e.g. sodium cholate [Javier Ruiz, B.
iochimica et Biophysica,
Acta, 937 (1988) 127-134]
can be used. In addition, the present inventor has already developed phospholipase A2 (PLA2) apart from these methods of destruction.
) has been found to efficiently destroy liposomes of this composition without causing deactivation of the encapsulated detection element (Japanese Patent Application No. 2-336363), and PLA2 can also be used.

[0024] In the case of enzymes, various substrates are added and the reaction products produced are detected spectroscopically, and in the case of antibodies, the detection elements released by the destruction of liposomes are detected using a label. By spectroscopically detecting the information resulting from the antibody label, in the case of a coenzyme, the activity of the enzyme generated by the reaction with the apoenzyme is measured, and in the case of an enzyme substrate, the activity of the enzyme generated by the reaction with the enzyme substrate can be measured. By adding enzymes,
By spectroscopically detecting the reaction product, in the case of a peptide, the labeled substance is detected spectroscopically after the antibody that reacts with the peptide undergoes a competitive reaction with the same labeled peptide compound. In the case of glycosides, the product is detected spectroscopically after an enzymatic reaction or competitive reaction with antibodies, and in the case of fluorescent substances, it is done by measuring the fluorescence emitted by the reactant. In the case of dyes, it is detected by spectroscopically measuring enzymatic reaction products or luminescent substances, and in the case of drugs, it is detected by measuring the spectroscopic characteristics of the drug itself.

[0025] In the method of the present invention, the antigen to be detected is not particularly limited as long as it is a so-called multivalent antigen having two or more antibody recognition sites. Components of blood, urine, food, waste fluids, etc., such as blood hormones essential for maintaining the homeostasis of living organisms, pathogenic bacteria, proteins derived from oncogenic viruses, contaminated bacteria in food, and specific compounds in waste fluids. It will be done.

[0026] There are no particular limitations on the manner in which the antigen reacts with the antibody in the present invention; for example, (1) a two-step method in which the antigen is reacted with a primary antibody and then reacted with a secondary antibody;
) Either a one-step method in which an antigen and a secondary antibody are reacted simultaneously in the presence of a primary antibody, or (3) a two-step method in which a secondary antibody is reacted with an antigen and then reacted with a primary antibody. .

The case where HRP is used as a detection element will be explained below as an example. That is, HRP enzyme is encapsulated and Di
myristoyl Phosphatidyl Ch
oline (DMPC), Dilauroyl P
Hosphatidyl Ethanolamine
(DLPE), Dicetylphosphate
(DCP) etc. as a constituent lipid, liposomes with a diameter of about 0.1 μm are formed and used for preparing immunoliposomes. The preparation of immunoliposomes is carried out by using carbodiimide, which is a water-soluble coupling reagent, to combine the amino group of DLPE, which is one of the liposome-constituting phospholipids, with the antigen and mouse Ig.
A goat anti-mouse IgG antibody is bound onto the liposome surface by forming a peptide bond between the carboxyl groups of a goat-produced anti-mouse IgG (secondary antibody) protein, which is a specific antibody for G. The immunoliposome prepared in this way consists of approximately 50,000 to 100,000 molecules of phospholipid, has approximately 10 molecules of HRP enzyme incorporated inside it, and has approximately 5 molecules of anti-mouse IgG bound to the surface. ing.

[0028] The method of the present invention using the immunoliposomes thus obtained is carried out using 96-well polystyrene microwells coated with polyclonal anti-mouse IgG as the primary antibody, and Aff as the antigen.
inityColumn Purified mouse IgG is used. In the sandwich reaction using immunoliposomes as secondary antibodies, immunoliposomes having molecules of about 10 to the 8th power to the 9th power are used for the reaction. To destroy the liposomes after the reaction, 2 uni of bovine pancreatic phospholipase A2 (PLA2) dissolved in phosphate buffer (pH 7.2) was added.
ts (U) can be used.

[0029] Unreacted immunoliposomes are removed by washing with a citrate buffer. Detection of immune complexes generated as a result of sandwich reactions using immunoliposomes against antigens captured by primary antibodies can be performed by measuring the enzymatic activity of HRP enzyme released upon destruction of immunoliposomes. To measure enzyme activity using this ELISA method, 2U of PLA2 solution and a large excess of enzyme substrate [H2
O2 and tetramethyl benzidine
(TMB) mixed solution] was added, and as time passed, 6
This can be done by measuring the absorbance at 50 nm, or by measuring the absorbance at 450 nm after stopping the reaction using an acid.

[0030]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples in any way. Preparation of immunoliposomes (1) Preparation of HRP enzyme-encapsulated liposomes: DMPC, DLPE, DCP, 13.0, 5.0, respectively, as phospholipids;
1.0mg of chloroform:diethyl ether=2:
It was dissolved in 1 volume of 3.0 ml of the mixed solution. Next, add 1.2 ml of HRP enzyme solution (1.0 mg/ml) to the obtained solution.
HEPES buffer, pH 7.2) and completely w/o
A mold emulsion was formed, and the solvent was evaporated from the emulsion solution under reduced pressure using a rotary evaporator until the odor of the organic solvent was completely eliminated. After this
Bath type ultrasonic transmitter for about 3 minutes, then Pr
Ultrasonication was performed for about 70 seconds using an obe type (TOMY, UD-201). The generated HRP enzyme-encapsulated liposomes were filtered and washed several times using a molecular sieve membrane with a molecular weight of 60,000 (4 to 5 times at 5000 rpm x 40 minutes), and it was confirmed that the filtrate had almost no HRP enzyme activity ( HR
6 minutes after adding a large excess of substrate when measuring P enzyme activity.
The absorbance at 50 nm is around 0.05). next,
Apply to a Sepharose 4B column (41.0 × 1.6 cm) equilibrated with HEPES buffer, and
The liposome fraction flowing out near the volume was collected.

(2) Confirmation of HRP enzyme-encapsulated liposomes To confirm enzyme-encapsulated liposomes, dilute each effluent fraction of the Sepharose 4B column 10 times with HEPES buffer, add 0.5 μl of the diluted liposome solution to 50 μl of each Aliquoted into microwells containing HEPES buffer. Next, 10 μl of 0.1% sodium deoxycholate (S
DC) aqueous solution was added to the microwell, and after about 1 minute, 50
μl of H2O2-TMB substrate solution (KPL 505
07600, TMB Microwell Pero
xidase Substrate System) was added, and after another minute, the absorbance at 650 nm was measured using a microplate reader (Molecular Devices).
, M-Vmax). The absorbance value at this time was compared with the absorbance value when enzyme activity was measured using the same liposome sample without adding 0.1% SDC.
If the absorbance value after treatment with %SDC was higher than that without treatment, it was determined that the liposomes were destroyed by SDC and activated by the released encapsulated enzyme. It was thus determined that the fraction activated by SDC contained enzyme-encapsulated liposomes. Similarly, measuring the absorbance at 280 nm of the column effluent fraction, Bartl
Phospholipids were also quantified by the ett method. In addition, the particle size of liposomes was measured using a laser particle size analyzer (OTSUKA E).
LECTRONICS, LPA-3100), and the diameter was about 0.1 μm.

(3) Preparation and confirmation of immunoliposomes (1)
0.2 ml of HRP-encapsulated liposome solution prepared and purified by
Add 2.6 mg of goat anti-mouse IgG and HEPES buffer to 1.5 ml of HEPES buffer at pH 4.8.
It was prepared as follows. While stirring gently at room temperature, add 20 mg (/
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC
) The solution was added dropwise and reacted for 1.5 hours. After this, p
Adjust to H7.2 and use a molecular sieve with a molecular weight of 60,000 (
5000 rpm x 30 minutes) Remove the enzyme and precipitate derived from the destroyed liposomes, and heat the remaining liposome solution with HE.
It was applied to a Sepharose 4B column equilibrated with PES buffer, and the enzyme activity of the effluent fraction was measured in the same manner as in (2) above.

[0033] The encapsulated enzyme unit was determined by the enzyme activity from the calibration curve using standard HRP enzyme, and the number of bound IgG was determined by Lo
Estimated from protein quantification using the wry method, the number was about 5 per liposome. Destruction and measurement of enzyme activity in confirmation of immune liposomes were carried out in almost the same way as enzyme activity measurement of enzyme-encapsulated liposomes, but instead of 0.1% SDC, approximately 2.0U/5μl of PLA2 derived from bovine pancreas was used. After destroying the liposome membrane using the enzyme, an enzyme substrate was added and the activity was measured. The immunoliposomes encapsulating the enzyme prepared and purified in this way were used in experiments within 2 to 3 days, and those that were used beyond that time were not used.

Preparation of primary antibodies (applying goat anti-mouse IgG to microwells) 0.25, 0.5, and 0.5, respectively, prepared with carbonate buffer pH 9.5
Polyclonal goat anti-mouse IgG solutions of 1.0, 2.0, and 4.0 μg/50 μl were dispensed into each well of a 96-well polystyrene tube in two rows in descending order of concentration from the right end to the left end. However, only the buffer solution was added to the leftmost two columns. Place the microwell in a mixer (T
AIYO MICRO MIXER, Taiyo Kagaku Kogyo)
After mixing at room temperature for 6 hours, the solution was removed by suction. Then add 100 μl of 1.0% Bovin to each well.
e Add Serum Albumin (BSA) aqueous solution, leave it for 1 hour, throw away the solution, and add 0.1
% Tween 20 - Physiological saline phosphate buffer (
Washed three times with PBS solution). For washing, a microplate washing device (IntiCorp., INTIWA
SH Immunoassay Washer) was used. Goat anti-mouse Ig prepared in this way at various concentrations
The G-coated microwells were stored at a humidity of 30% or less for more than 1.5 days until used as a primary antibody.

Example 1 Sandwich using immunoliposomes as secondary antibodies
Detection of antigen mouse IgG by ELISA method Using 4 μg of goat anti-mouse IgG polyclonal antibody obtained by the above method as the primary antibody, 2.0 μg of each as the antigen
, 4.0, and 6.0 μg of mouse IgG, and 30 μl of immunoliposomes obtained by the above method as a secondary antibody, sandwich-ELISA was performed. That is, the following procedure was followed. Affinity column-purified mouse IgG (ZYMED LABORATORIES,
INC. Mouse IgG 02-6502)
Add 2.0, 4.0, 6.0 μg of
The volume was adjusted to 0 μl. The antigen was adsorbed for about 20 minutes while being gently stirred with a microplate mixer. Thereafter, the plate was washed twice with 0.1% Tween-PBS solution, and then 30 μl of immunoliposome solution and PBS buffer were added to prepare a total volume of 50 μl. The mixture was stirred gently for the first few minutes, and then allowed to stand for about 20 minutes. After 20 minutes, it was washed three times with citrate buffer, pH 6.9. Then 50 μl of PB
Add S solution and 2.5 U/5 μl of PLA2 solution,
After about 1 minute, 100 μl of HRP enzyme substrate solution was added, and the absorbance at 650 nm was measured at 10 minute intervals. To stop the reaction, 50 μl of 3N phosphoric acid was added to the reaction solution, and the absorbance at 450 nm was measured.

The obtained results are shown in FIG. 1, and each point in the figure represents the enzyme activity value (N
SB value) is calculated by subtracting the enzyme activity value when the activity is measured using each concentration of antigen. In one experiment, each point was taken as the average value of at least four data points. N
SB values were at most <0.09 at absorbance at 650 nm. As a result, as shown in FIG. 1, the absorbance showed a linear change depending on the antigen concentration, indicating that the immunoliposome acted as a secondary antibody depending on the antigen amount. From this, it was found that the method of the present invention is effective as an antigen detection method similar to the conventional method.

Example 2 Comparison of the method of the present invention using immunoliposomes and the conventional method using a secondary antibody directly labeled with an enzyme In the sandwich-ELISA method, (A) secondary A comparison was made of antigen detection sensitivity when immunoliposomes were used as the antibody and (B) when a secondary antibody directly labeled with HRP enzyme was used. That is, (A)
was carried out in the same manner as in Example 1, the amount of primary antibody was 2.0 μg of anti-mouse IgG, and the amount of immunoliposome was 10 μl (approximately 2 to 5 x 109 liposome molecules).
, the amount of PLA2 was 2.0 U, and the enzyme reaction time was 80
It was a minute. On the other hand, in (B), an antibody directly labeled with HRP enzyme was used as the secondary antibody, and 50 μl (approximately 2 x 109
(number of labeled antibody molecules) were added and allowed to react for the same time as in (A). The washing method was citrate buffer for (A) and 0.1% Tween- for (B).
It was PBS. The results are shown in FIG. 2, and it was found that the method (A) of the present invention had higher antigen detection sensitivity than the conventional method in the low antigen concentration region of around 0.2 μg.

[0038]

[Effects of the Invention] A sandwich reaction using immunoliposomes was performed using the method of the present invention, and the antigen detection sensitivity was measured using an enzyme reaction. Compared to enzyme activity measurements,
In the low concentration range, particularly at antigen concentrations around pmol to fmol, the method using the immunoliposome of the present invention yielded higher enzyme activity values (approximately 2 to 5 times).

[0039] As described above, the method of the present invention is capable of detecting an antigen in a low concentration range, and is a method with a high antigen detection sensitivity equivalent to or higher than that of the conventional sandwich method. This shows that the sandwich ELISA method has been applied in the past for the above-mentioned bacterial tests, etc., but the method of the present invention can be fully applied to these tests, etc., and is highly sensitive. Detection of contaminating bacteria and the like can be performed only by pre-culturing, and testing time can be shortened.

[Brief explanation of the drawing]

FIG. 1 shows sandwich-ELI in Example 1.
It is a figure showing the result of SA method.

FIG. 2 shows sandwich-ELI in Example 2.
It is a figure showing the result of SA method.

[Explanation of symbols]

(A)...When immunoliposomes are used as the secondary antibody (B)...When a secondary antibody directly labeled with HRP enzyme is used

Claims (5)

[Claims] Claim 1: An immune complex formed by sandwiching and capturing an antigen to be detected between a primary antibody immobilized on a solid phase and an immunoliposome in which a secondary antibody is bound to the surface of the liposome encapsulating a detection element. An antigen immunoassay method characterized by measuring the detection element released by destroying liposomes in the body. 2. The detection element according to claim 1 is any one of a biologically active substance such as an enzyme, an antibody, a coenzyme, an enzyme substrate, a peptide or a glycoside, a fluorescent substance, a dye, or a drug. immunoassay method. 3. The immunoassay method according to claim 2, wherein the enzyme as the detection element according to claim 2 is Horseradish Peroxidase. 4. The immunoassay method according to claim 1, wherein the primary antibody and secondary antibody according to claim 1 are monoclonal antibodies that recognize different epitopes of the antigen to be detected or polyclonal antibodies directed against the antigen. 5. The reaction mode of the antigen and antibody consists of (1) a two-step method in which the antigen is reacted with a primary antibody and then reacted with a secondary antibody; (2) a method in which the antigen and the secondary antibody are reacted in the presence of the primary antibody; A one-step method of simultaneously reacting with antibodies, or (
3) The immunoassay method according to claim 1, which is a two-step method in which the secondary antibody is reacted with the antigen and then the antigen is reacted with the primary antibody.