JPH07113639B2 - Immunoassay reagent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay reagent, more specifically, an immunoassay capable of sensitively measuring a specific antigen present in a sample by a simple operation. Related reagents.

[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 these, various labeling substances (markers) such as radioisotopes, fluorescent substances, oxygen- or enzyme-related substances and the like have been used to carry out a labeled immunoassay which is superior in sensitivity. As a labeling substance, radioisotopes have been widely used in the past from the viewpoint of sensitivity, but radioisotope reagents have problems with radioactivity and the use of expensive facilities because of their half-life and instability. Measurement methods using fluorescent substances and enzymes as labels have attracted more attention in cooperation with studies on the improvement of their sensitivity.

In immunoassays that use a fluorescent substance or enzyme as a labeling substance, currently, a heterogeneous system that requires a step of separating those bound and not bound by the antigen-antibody reaction among the labeling substances is used. There are a method of using and a method of using a homogeneous system which does not require such a separation step.

Immunoassays that use a heterogeneous system include the two-antibody method and the solid-phase method that are also used in radioisotope-labeled immunoassays, but these methods require the separation of unreacted and reacted substances. Is essential, and the separation step is complicated to perform, and thus there is a defect that it is difficult to speed up the quantification. On the other hand, an 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, the sensitivity is low and the measurement range is low. Due to its small size, it has not been put to practical use for quantitative determination of antigens or antibodies.

As a method for overcoming such a problem, in recent years, a liposome encapsulating a marker is prepared, and an antigen or an antibody is sensitized to the liposome, and the sensitized liposome is coexistent with a sample and covalently bound to the liposome. Alternatively, an antibody is reacted with an antibody or an antigen in a sample to form an antigen-antibody complex, and the complex is specifically destroyed to measure a marker flowing out from the liposome. Coexist with a known amount of antigen or antibody, and measure the outflow marker in the same manner as above to prepare a standard calibration curve in advance,
A method for quantifying an antibody or an antigen by providing the above-mentioned measurement result of a sample against the above-mentioned standard calibration curve was provided.

[Problems to be solved by the invention]

However, even in the immunoassay method using this liposome, there are various problems in quantifying the antigen in the sample.

When an antigen-sensitized liposome is used, it is performed by a competitive method. In this competitive method, free antigen in a sample is allowed to compete with an antigen-sensitized liposome in the presence of a specific amount of specific antibody,
The amount of the antigen in the sample is measured from the outflow amount of the marker released from the liposome by the addition of guinea pig complement. Therefore, it is necessary to fully consider the amount of antigen and the amount of specific antibody that are sensitized to the liposome.

When an antibody-sensitized liposome is used, the liposome was sensitized with a polyclonal antibody obtained by immunizing a rabbit or goat with an antigen. However, when a liposome-sensitized antibody (IgG fraction) obtained from a rabbit was used, non-specific marker release was observed from the liposome (non-specific marker release by addition of guinea pig complement). However, it was unsuitable for actual measurement. When a liposome-sensitized antibody (IgG fraction) obtained from goat was used, even if an antigen-antibody complex was formed on the liposome surface, guinea pig complement was not activated and complement activation was observed. No associated marker release from the liposomal membrane is seen.

As a measurement method that improves on these points, there is the Sangertian method (Japanese Patent Laid-Open No. 138465/1985), but this method also requires the antibodies to immunize two types of animals, and has the drawback that the procedure is complicated. I got it.

[Means for solving problems]

In this situation, the present inventor has conducted diligent research, and as a result, if two or more kinds of monoclonal antibodies recognizing different epitopes are immobilized on the liposome, the antigen in the sample can be detected with high sensitivity by a simple operation. They have found that they can be quantified and have completed the present invention.

That is, the present invention provides an immunoassay reagent characterized in that two or more kinds of monoclonal antibodies recognizing different epitopes are immobilized on a liposome encapsulating a labeling substance via a crosslinking agent. .

The liposome in the immunoassay reagent of the present invention has a broad meaning including an erythrocyte ghost membrane. The liposome may be any liposome as long as it has been conventionally used, but a liposome composed of phospholipid or glycolipid and cholesterol is preferable.

For example, when synthesizing liposomes from phospholipids and cholesterol, stable liposomes are easily obtained when the ratio of these is around 1: 1. The fatty acid residue in the phospholipid preferably has 12 to 18 carbon atoms, and more preferably has an even number.

The labeling substance encapsulated in the liposome must be a substance that is hydrophilic and can be quantified when eluted outside the liposome. As such a substance, for example, at a high concentration, no fluorescence is shown due to self-quenching, but at a low concentration (10 ⁻³ M
The fluorescent compound such as carboxylfluorescein that emits extremely strong fluorescence in the following); a luminescent compound such as luminol and luciferin that emits light by an oxidation reaction outside the liposome; a specific absorption band in the visible region or ultraviolet region. Absorbing compounds (water-soluble dyes, etc.) possessed; sugars such as glucose and sucrose that are cleaved by the action of an oxidase to cause oxygen consumption or hydrogen peroxide production; relatively large ionic compounds such as tetrapentylammonium; nicotinamide adenine Coenzymes such as dinucleotide (NAD); radical compounds such as methylpiorogen are preferable. However, enzymes are not used as labeling substances in the present invention. These compounds are appropriately selected in consideration of factors such as the detection method, sensitivity and liposome stability.

Monoclonal antibodies immobilized on liposomes are obtained by, for example, immunizing the human CRP antigen BALB / c mouse, and fusing its splenocytes with the mouse myeloma cell line NS-1.
It can be obtained by selectively collecting and recognizing different epotopes from the monoclonal antibodies against the CRP antigen.

The immunoassay reagent of the present invention is produced, for example, by the following method. First, a lipid and a cross-linking agent are reacted in a solvent to introduce into the lipid molecule a functional group capable of binding to the antibody immobilized on the liposome. Then, the obtained functional lipid, cholesterol and other lipids if necessary are placed in a flask, a solvent is added and reacted, and then the solvent is distilled off and dried by suction. 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 suspension of sensitized liposomes.

On the other hand, two or more different antibodies to be immobilized on the liposome and a cross-linking agent are reacted in a buffer to introduce a cross-linking group, and then, if necessary, a reagent that reduces the cross-linking group (for example, dithiothrey). (DTT) to obtain a modified antibody. When the lipid is treated with the activator in the above step, this step is unnecessary.

Finally, the immunosensitized reagent of the present invention is obtained by reacting the sensitized liposome with the modified antibody in a buffer solution. Such a reagent is usually obtained as a microcapsule containing a labeling substance and carrying two or more different antibodies immobilized on the surface.

Examples of the crosslinking agent in the above production method include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (p-maleimidophenyl) butyrate (SMPB), N-succinimidyl 4- ( p-maleimidophenyl) acetate (SMP
A), N-succinimidyl 4- (p-maleimidophenyl) propionate (SMPP), N- (γ-maleimidobutyryloxy) succinimide (GMBS) and N- (ε.
-Maleimidocaproyloxy) succinimide (EMC
S).

Antigens that can be quantified using the immunoassay reagent of the present invention include, for example, tumor markers (CRP, AFP, BFP, CEA and POA).
Etc.) Immunoglobulins (IgA, IgE, IgG and IgM etc.), hormones (insulin, T ₃ and T ₄ etc.) and drugs.

Quantification of the antigen in the specimen sample using the reagent for immunoassay of the present invention is performed, for example, as follows. First, the reagent, a sample containing an antigen, and complement are mixed in an appropriate buffer (eg, gelatin-veronal buffer) to cause a binding reaction between the antigen-antibody and complement. Then, the labeling substance is released from the inside of the liposome in proportion to the reaction amount. Then, an analysis method according to the labeling substance (for example,
When the labeling substance is a fluorescent substance, it can be quantified by a fluorescent analysis method, and the amount of the antibody in the sample can be measured by, for example, a calibration curve prepared in advance.

The complement used in the quantitative operation is not particularly limited, but guinea pig serum is usually used. However, serum of rabbit, mouse, human and the like may be used.

〔Example〕

 Next, reference examples and examples will be described.

Reference Example 1 (Production of Monoclonal Antibody) BALB / c mice were immunized with human CRP antigen purified from human CRP positive sera by a known method, and their splenocytes were fused with mouse myeloma cell line NS-1 to give humans. A monoclonal antibody against the CRP antigen was obtained. Of the antibodies thus obtained, there were five types of monoclonal antibodies that could be used in the present invention. As a result of examining the epitope recognition site in the CRP antigen by EIA by the competition method, they were divided into at least two groups recognizing different epitopes. About these,
Further, the activity of complement activation was examined and the following results were obtained.

Example 1 (Preparation of liposome encapsulating marker) DPPC (5 mM, 100 μ): Chol (10 mM, 50 μ): DTP-DPPE
(1 mM, 10 μ) = 1: 1: 0.02 (molar ratio) (DTP-DPPE is dipalmitoylphosphatidylethanolamine into which dithiopyridyl is introduced by an amide bond), and the above proportions were set in a 10 ml flask. When a lipid is loaded and the solvent chloroform is stripped off by an evaporator, a film-like substance is deposited on the inner surface of the flask. After vacuum-drying this film for 1-2 hours, 0.1N NaOH solution of carboxyfluorescein used as a marker
Add 100μ and stir vigorously on a vortex mixer for 5-10 minutes.

Then, excess carboxyfluorescein was added to 10,000 × G
When it is removed by centrifugation with, a liposome (MLV type) in which carboxyfluorescein is encapsulated as a marker is obtained.

This liposome contains 0.01M HEPES buffer (0.15M NaCl, p
H7, 5) Suspend in 500μ and store.

Example 2 (Antibody sensitization of liposome) (a) Mouse monoclonal antibody No. 1 obtained in Reference Example 1
And No. 2 are preliminarily dialyzed with 0.01 M HEPES buffer to obtain 0.5 ml of antibody No. 1 (2.0 mg / ml) and antibody No. 2 (2.0 mg / m 2
l) To a mixture of 0.5 ml, an ethanol solution of N-hydroxysuccinimidyl-3- (2-pyridyldithio) propionate (SPDP) was added to a concentration of 0.1 mM, and the mixture was reacted at room temperature for 30 minutes with occasional stirring. . Then, excess SPDP and antibody are separated using a Sephadex G-25 column, in which case 0.1M acetate buffer (0.15M NaCl, pH 4.5) is used.
The buffer solution is exchanged and the wavelength is 280 nm by the spectrophotometer.
Confirm elution of antibody with (fraction volume is 1 ml each).

Solid solution of dithiothreitol (DTT) was added to the dithiopyridylated antibody solution at 50 mM, and the solution was added at room temperature for 30 minutes.
React for minutes.

After that, the reaction solution is treated with a Sephadex G-25 column to separate excess DTT and antibody. At this time,
Perform buffer exchange with 0.01 M HEPES buffer (pH 7.5) and confirm elution of the antibody with a spectrophotometer at a wavelength of 280 nm (fraction volume is 1 ml each).

Then, the resulting thiolated antibody solution (1mg / ml) 500μ
Was added to 500 μ of the liposome suspension obtained in Example 1, gently stirred for 15 to 20 hours, and centrifuged to detect two monoclonal antibodies, which are the reagents for immunoassay of the present invention. The prepared antibody-sensitized liposome is obtained.

The antibody-sensitized liposomes thus obtained were 1 ml of gelatin-
Suspend in veronal buffer and store at 4 ° C.

The dilution (Quantification of CRP) Example 3, gelatin - veronal buffer (GVB ^-) to 0.1m
Buffer solution (GVB ²⁺ ) containing M MgCl ₂ and 0.03 mM CaCl ₂
It was used. For the measurement, the antibody-sensitized liposome stock solution prepared in Example 2 was used after being diluted 100-fold with GVB ²⁺ . or,
The guinea pig complement was similarly diluted with GVB ²⁺ to 1 to 5 units before use. The measurement was performed as follows. Dispense 25μ of sample (CRP antigen) into a 96-well microplate,
Then, 5 μl of 100 times diluted liposome suspension and 25 μl of complement (3 units) are dispensed in this order. After reacting at 37 ° C for 1 hour, 100 µl of veronal buffer containing 10 mM EDTA is added to stop the complement reaction. Since the amount of antigen in the sample is proportional to the amount of carboxyfluorescein released from the liposome, excitation is performed at 490 nm, and the fluorescence emission amount at 530 nm is measured. The fluorescent intensity was expressed as a marker lease% based on the fluorescent amount obtained by breaking the liposome with a surfactant as 100%. The calibration curve thus obtained is as shown in FIG. The calibration curve (x-x) when using the reagent of the present invention is the calibration curve (●-when one kind of mouse monoclonal antibody-sensitized liposome is used.
The sensitivity was extremely high compared to ().

[Brief description of drawings]

FIG. 1 is a calibration curve when CRP antigen was measured using the reagent of the present invention.

Claims (2)

[Claims] 1. A reagent for immunoassay, wherein two or more kinds of monoclonal antibodies recognizing different epitopes are immobilized on a liposome encapsulating a labeling substance via a crosslinking agent. 2. The reagent for immunoassay according to claim 1, wherein the monoclonal antibody belongs to the IgG and IgM classes and can activate guinea pig complement.