JP2516359B2 - Immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay method, and more particularly to an immunoassay method for analyzing an antigen or an antibody in a sample using liposomes.

[Conventional technology]

The immunoassay method utilizing the antigen-antibody reaction is indispensably important in the clinical diagnosis of various endocrine diseases, and this method can be roughly classified into a labeling method and a non-labeling method.

Among them, various labeling substances such as radioisotopes, fluorescent substances, enzymes or enzyme-related substances have been used to carry out a labeled immunoassay which is superior in sensitivity. Radioisotopes have been widely used as labeling substances from the viewpoint of sensitivity, but radioisotope reagents have a half-life and are unstable, causing problems with radioactivity and the use of expensive facilities. , The measurement method using a fluorescent substance or enzyme as a label has been attracting more attention in cooperation with the research on the improvement of the sensitivity.

This immunoassay using a fluorescent substance or an enzyme as a labeling substance is currently a heterogeneous system that requires a step of separating the bound substance and the unbound substance in the labeled substance from the antigen-antibody reaction. And a method using a homogeneous system that does not require such a separation step.

As an immunoassay using a heterogeneous system, a two-antibody method and a solid-phase method, which are also used in radioisotope-labeled immunoassays, are known. The separation is essential, and the separation step is complicated, and thus there is a defect that it is difficult to speed up the quantification. on the other hand,
The immunoassay method using a homogeneous system was proposed for the purpose of simplifying and speeding up the quantification by eliminating the need for a separation step, but in reality, it has low sensitivity and a narrow measurement range. In reality, it has not been put to practical use for quantitative determination of antigens or antibodies.

The present inventors, as a result of conducting research to eliminate the drawbacks of the immunoassay method using a homogeneous system, the above-mentioned problems, encapsulating a marker or preparing liposomes to this antigen or antibody After sensitization, the sensitized liposome and the specimen are allowed to coexist, and the antigen or antibody bound to the liposome is reacted with the antibody or antigen in the specimen to form an antigen-antibody complex, and this complex is formed. It was previously found that the problem can be solved by specifically destroying the liposome and measuring a marker flowing out from the liposome.

[Problems to be solved by the invention]

In the above means, as a marker to be encapsulated in the liposome, a fluorescent dye, an enzyme substrate, a coenzyme, an enzyme, an ionic compound or the like is used, but the fluorescent dye is a special instrument for measurement (fluorescent photometer). ) Is required, and an enzyme substrate, coenzyme, and enzyme can be measured using an ordinary spectrophotometer, but when an enzyme substrate and coenzyme are used, the measurement sensitivity is low and the enzyme is used. If the enzyme is encapsulated in the liposome, it can be measured with high sensitivity.
It has a problem that its activity cannot be maintained. Also, when an ionic compound is used, the measurement sensitivity is low and it is difficult to stably retain it in the liposome. Therefore, it is desirable that the marker to be encapsulated in the liposome is a highly sensitive substance that does not require a special measuring instrument, is capable of colorimetric analysis, and is stable for a long time in the state of an aqueous solution. Was there.

[Means for solving problems]

Therefore, the present inventors have conducted various studies on the marker substance to be encapsulated in the liposome in the above method, and as a result, found that the above problems can be solved by using an electron carrier involved in a redox reaction as a marker, The present invention has been completed.

That is, according to the present invention, a liposome formed by binding an antigen or antibody on the surface and encapsulating an electronic carrier involved in a redox reaction, and a sample and complement containing the antibody or antigen were mixed and destroyed. The present invention provides a method for analyzing an antigen or antibody in a sample, which comprises measuring the amount of the electron carrier that has flowed out from a liposome.

In the present invention, the antigen or antibody bound to the surface of the liposome needs to correspond to the antibody or antigen to be detected in the sample. For example, when the substance to be detected in the sample is anti-human IgG, the substance bound to the surface of the liposome is human IgG which is an antigen for the substance.
Must.

The liposome is preferably composed of phospholipid or glycolipid and cholesterol. For example, when preparing liposomes from phospholipids and cholesterol, these can be combined arbitrarily, but the molar ratio of the
A ratio of about 1: 1 is preferable because stable liposomes can be obtained. Examples of phospholipids include dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and the like. Available. Furthermore, when introducing an amino group for binding to the liposome, phosphatidylethanolamine or the like is used, and when giving a charge, dicetyl phosphite, phosphatidic acid, stearylamine or the like is used. It can be used by mixing with phospholipids. The liposomes are multilamellar liposomes (MLV) obtained by stirring with a vortex mixer, and unilamellar liposomes (SUV, obtained by ultrasonic treatment, dialysis, etc.).
It doesn't matter if it is LUV).

To bind the liposome to the antigen or antibody, a bifunctional reagent such as N-hydroxysuccinimidyl-
3- (2-pyridyldithio) propionate (SPDP),
N-succinimidyl-2-dithiobenzothiazoyl propionate (BTNOS), N- (ε-maleimidocaproyloxy) succinimide (EMCS), N- (γ-maleimidobutyryloxy) succinimide (GMBS), and the like,
A liposome in which a conjugate with phosphatidylethanolamine is incorporated is prepared, and an antigen or an antibody is introduced with a reactive group if necessary, and then treated with a reducing agent (eg, dithiothreitol) to suspend these liposomes. The suspension may be reacted with an antigen or antibody having a reactive group in an appropriate buffer solution, and the antigen or antibody not bound to the liposome may be removed by centrifugation or gel filtration.

The electron carrier enclosed in the liposome is described in Biochemistry Dictionary (Imahori et al., Tokyo Kagaku Dojin, No. 852).
P., April 1, 1985), electron carrier
er), a substance capable of receiving an electron from an electron donor to be reduced in an organism's redox system and then passing the electron to another substance (electron acceptor) for oxidation. is there. That is, the electron carrier has the property of catalyzing the redox reaction in the living body. Examples of applications of the electron carrier to the measurement of biological components include those described in "Analytical Chemistry" [Kazuhiro Tabe et al., 36, 82-87, (1987)]. The greatest advantage of using an electron carrier in the present invention is that the electron carrier acts catalytically,
As long as there is a substance involved in the reaction, the reaction continues, and as a result, the detection sensitivity of the target substance can be improved. As such an electron carrier, 9-dimethylaminobenzo-α-phenazoxonium chloride,
Examples thereof include 1-methoxy-5-methylphenadium methylsulfate and phenazine methosulfate. Of these, 1-methoxy-5-methylphenazinium methylsulfate is particularly advantageous as a marker to be encapsulated in liposomes because it easily dissolves in water and its solution is stable for a long period of time.

The method of the present invention is carried out, for example, as follows. First, a liposome (hereinafter abbreviated as "carrier-encapsulated liposome") in which an antigen or an antibody corresponding to a test substance is bound to the surface and an electronic carrier is encapsulated inside thereof and a complement are added to a sample, and the reaction is performed. Let

The reaction is preferably carried out at 37 ° C. using an appropriate buffer solution (eg, gelatin-veronal buffer solution, Tris buffer solution, etc.).

In addition, when measuring an antigen in a sample using a liposome bound with an antibody, even if the antibody bound to the liposome reacts with the antigen in the sample to form an antigen-antibody complex, complement is not activated. However, in this case, if the antibody is added in a free state, an antibody-antigen-antibody type complex is formed on the liposome and it is possible to activate complement.

By the above reaction, the antigen in the carrier-encapsulated liposome and the antibody in the specimen or the carrier-encapsulated liposome antigen and the antibody in the specimen or the antibody in the carrier-encapsulated liposome and the antigen in the specimen form an antigen-antibody complex, and this complex is formed. The liposomes are specifically destroyed by complement, and the electron carriers flow out.

Next, the amount of this electronic carrier that has flowed out is measured.
As a method for measuring the electronic carrier, for example, a formazan dye is generated by using a tetrazolium salt and NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphite). , There is a colorimetric analysis. Coexistence of a tetrazolium salt with NAD ⁺ (oxidized nicotinamide adenine dinucleotide) or NADP ⁺ (oxidized nicotinamide adenine dinucleotide phosphate), dehydrogenase and its substrate also produces a formazan dye. , Colorimetric analysis of electronic carriers is possible.

In this way, the electronic carrier amount is obtained, and the amount of the antigen or antibody in the sample can be easily obtained from the electronic carrier amount.

〔Example〕

 Next, the present invention will be described in more detail with reference to examples.

Example 1 Encapsulation of 1-methoxy-5-methylphenazinium sulphate in liposomes: dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), dithiopyridylated dipalmitoylphosphatidylethanolamine (DTP). -DPPE) and dipalmitoyl phosphatidic acid (DPPA) in chloroform were added to 10 ml eggplant-shaped flasks so as to have the following compositions (1) to (4).

(1) DPPC: 1 μmol, Chol: 1 μmol (2) DPPC: 1 μmol, Chol: 1 μmol, DPPA: 0.05 μmol (3) DPPC: 1 μmol, Chol: 1 μmol, DTP-DPPE: 0.05 μmol (4) DPPC: 1 μmol, Chol : 1 μmol, DTP-DPPE: 0.05 μmol,
DPPA: 0.05 μmol From these, the solvent chloroform was volatilized by a rotary evaporator to form a lipid thin film on the inner wall of the flask, and further dried under reduced pressure for 1 hour in a desiccator. Add 250μ of 0.05M 1-methoxy-5 to each flask.
-Methylphenazinium sulphate-containing 0.1 M Tris-HCl buffer pH 7.4 was added, and the mixture was heated at 55 ° C for 1 to 2 minutes and then vigorously stirred with a vortex mixer for 10 minutes. Then
Transfer the contents of the flask to a centrifuge tube, add 0.05M Tris-HCl buffer pH7.4 (TBS) containing 0.15M sodium chloride and 0.02% sodium azide, centrifuge at 12,000 xg for 15 minutes, and remove the supernatant with an aspirator. did. After repeating this operation 3 times, the precipitate was suspended in 1.0 ml of TBS to prepare 4 types of 1-methoxy-5-methylphenazinium sulphate-encapsulating liposomes.

These liposome suspensions, diluted 500-fold with TBS, 10
0 µ and 5% Triton X-100 solution or TBS 100 µ were mixed and heated at 37 ° C for 5 minutes. Next, 0.5 ml of TBS containing 0.5 mM nitrotetrazolium blue and 1 mM NADH was added, and after heating at 37 ° C for 10 minutes, 1.0 ml of 0.2 N hydrochloric acid and 0.2% Triton X-100 was added to stop the reaction, and the wavelength was 570 nm. The absorbance was measured with. As a result, as shown in Table 1, 1-methoxy-5-methylphenazinium sulphate was encapsulated in the liposome, and when the liposome membrane was dissolved with Triton X-100, it was leaked to the outside and subjected to colorimetric analysis. Was confirmed to be possible.

Example 2 Preparation of goat IgG-sensitized liposome and measurement of anti-goat IgG antibody: 1-methoxy- according to the method described in Example 1 with the composition of DPPC: 1 μmol, Chol: 1 μmol, DTP-DPPE: 0.05 μmol.
A liposome encapsulating 5-methylphenazinium sulphate was prepared and suspended in 0.5 ml of HEPES buffer solution (pH 7.4) containing 0.15 M sodium chloride.

In addition, to 2.0 ml of goat IgG (2.0 mg / ml) solution dissolved in HEPES buffer containing 0.15 M sodium chloride, 7 μ of 30 mM N-
Hydroxysuccinimidyl-3- (2-pyridyldithio) propionate-ethanol solution was added and at room temperature.
Allowed to react for 30 minutes. Then, using a Sephadex G-25 column, 0.1M acetate buffer containing 0.15M sodium chloride (pH
In 4.5), dithiopyridylated goat IgG was isolated. Furthermore, 15 mg of dithiothreitol was added to 2.0 ml of the dithiopyridylated antibody solution, and the mixture was reacted at room temperature for 20 minutes. Then, using Sephadex G-25, the thiolated antibody was separated from the reaction solution with HEPES buffer (pH 7.4) containing 0.15 M sodium chloride. 0.5 ml of this thiolated antibody (1.5 mg / ml) was added to 0.5 ml of the liposome suspension prepared above, and the mixture was cooled (4-10
After reacting at 20 ° C. for 20 hours, the mixture was centrifuged at 12,000 × g for 20 minutes to remove unbound goat IgG, and further suspended in 1.0 ml of 0.1% gelatin-containing TBS (GTBS ⁻ ).

This suspension, 0.5 mM magnesium chloride and 0.15mM calcium chloride content GTBS ^- liposome dilutions were diluted 300-fold with (GTBS ⁺⁺⁾ 100μ, Morumotsuto complement (10CH ₅₀₎ 200
μ and anti-goat IgG antibody (rabbit IgG fraction) with GTBS ⁺⁺
Sample 10 with a 2-fold dilution series than that diluted 1000 times
The mixture was mixed with 0 μ and reacted at 37 ° C. for 30 minutes. Then 0.5 mM
Nitrotetrazolium blue, 1 mM NADH containing GTBS ^- 0.5
After adding 20 ml and reacting at 37 ℃ for 20 minutes, 0.2N hydrochloric acid, 0.2%
The reaction was stopped by adding 1.0 ml of Triton X-100, and the wavelength was 570n.
Absorbance was measured at m. As a result, as shown in FIG. 1, it was shown that the absorbance increased with an increase in the amount of anti-goat IgG antibody.

Example 3 Measurement of electronic carrier flowing out of liposome conjugated with dehydrogenase: DPPC: 1 μmol, Chol: 1 μmo by the method described in Example 1.
A 1-methoxy-5-methylphenazinium sulphate-encapsulating liposome consisting of 1 was prepared and suspended in GTBS ^- 1.0 ml.

GTBS this liposome suspension 500 times, 1000 times, 2000 times ^-
100μ each diluted with 5% Triton X-100
Or GTBS ^- and 100μ were mixed and heated for 5 minutes at 37 ° C.. This was then mixed with 0.5 mM nitrotetrazolium blue, 10 mM glucose-6-phosphate, 0.02 mM NADP and 1 mM.
After addition of 0.5 ml, 150 U / ml of glucose-6-phosphate dehydrogenase -GTBS ^{- -} EDTA containing GTBS solution 50μ was added, 37
Warmed at 20 ° C for 20 minutes. Then, 1.0 ml of 0.2N hydrochloric acid and 0.2% Triton X-100 was added to stop the reaction, and the absorbance was measured at a wavelength of 570 nm.

As a result, as shown in Table 2, dehydrogenase (glucose-6-phosphate dehydrogenase) and its substrate (glucose-
6-phosphate) and NADP by conjugating
It was shown that it is possible to measure 1-methoxy-5-methylphenazinium sulphate flowing out from the liposome.

[Effects of the Invention] According to the immunoassay method of the present invention, by using an electronic carrier as a marker to be encapsulated in liposomes,
Quantitative analysis of various antigens or antibodies in a sample can be performed by a simple and highly sensitive measurement system for measuring absorbance. Therefore, the method of the present invention is useful in the fields of medical care and clinical examination.

[Brief description of drawings]

Fig. 1 shows anti-goat IgG using goat IgG-sensitized liposomes.
It is a figure which shows the relationship between anti-goat IgG antibody and light absorbency when measuring an antibody.

Claims (1)

(57) [Claims] 1. A liposome which has been disrupted by mixing a liposome having an antigen or antibody bound to its surface and encapsulating an electron carrier involved in a redox reaction, and a sample and complement containing the antibody or antigen. A method for analyzing an antigen or antibody in a sample, which comprises measuring the amount of the electron carrier flowing out from the sample.