JP2553360B2 - Immunoassay method and immunoassay reagent used therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to an immunoassay method and an immunoassay reagent used therefor, and more specifically, to a specific antigen or antibody present in a sample with high sensitivity by a simple operation. The present invention relates to a measurable immunoassay method and an immunoassay reagent used therefor.

[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, enzymes or enzyme-related substances have been used to carry out a labeled immunoassay which is superior in sensitivity. As a labeling substance, radioisotopes have conventionally been widely used from the point of sensitivity, but radioisotope reagents have a half-life, are unstable, and have problems with radioactivity and the use of expensive facilities. Measuring methods using fluorescent substances or enzymes as labels have come to attract more and more attention in conjunction with studies on improving their sensitivity.

For immunoassays using fluorescent substances or enzymes as labeling substances,
At present, a method using a heterogeneous system that requires a step of separating those bound by an antigen-antibody reaction from those bound by an antigen-antibody reaction, and a method using a homogeneous system that does not require such a separation step There is a way to use the system.

As an immunoassay using a heterogeneous system, there are a two-antibody method and a solid-phase method which are also used in a radioisotope-labeled immunoassay, and these methods separate an unreacted substance and a reacted substance. Is required,
This separation step is complicated to perform, and thus has a defect that it is difficult to speed up the determination. On the other hand, an immunoassay using a homogeneous system has been proposed for the purpose of simplifying and speeding up quantification by eliminating the need for a separation step. However, in practice, the sensitivity is low and the measurement range is low. The fact is that it has not been put to practical use for quantification of antigens or antibodies due to its small size.

As a method for overcoming such a problem, in recent years, a liposome having a marker-encapsulated ester phospholipid and cholesterol as a constituent component is prepared and sensitized with an antigen or an antibody, and the sensitized liposome is used as a sample. The antigen or antibody covalently bound to the liposome is allowed to react with the antibody or antigen in the sample to form an antigen-antibody complex, and the complex is specifically destroyed to measure a marker flowing out from the liposome. On the other hand, a sensitized liposome similar to the above is allowed to coexist with various known amounts of antigens or antibodies, and a standard calibration curve is prepared in advance by measuring the efflux marker in the same manner as above, and the above-mentioned measurement result for the sample is the above-mentioned standard. A method for quantifying an antibody or an antigen by being matched with a calibration curve was provided. According to this immunoassay method, the above-mentioned problems can be solved and the test substance can be easily quantified in a homogeneous system in a short time.

[Problems to be solved by the invention]

However, in the immunoassay method using a liposome composed of this ester-type phospholipid and cholesterol, retention stability of the labeling substance encapsulated in the liposome inside the liposome is insufficient, and the labeling substance is not stored during liposome storage. There was a problem of leakage, which affected the quantitative value of the test substance.

[Means for solving problems]

In view of such circumstances, the present inventor has conducted various studies to solve the above-mentioned problems, and as a result, by incorporating an amide-type phospholipid into the phospholipid that is a constituent component of the liposome, the retention of the labeling substance in the liposome Improved stability,
The inventors have found that the measurement sensitivity of the test substance is high, and have completed the present invention.

That is, the present invention has the following general formula (I) (Wherein each of R ¹ and R ² represents an alkyl group having 11 to 17 carbon atoms) labeled with an amide type phospholipid in an amount of 5% by weight or more and a liposome containing cholesterol as an essential component From the liposome reagent, a liposome reagent, in which a substance is encapsulated and an antigen or antibody that specifically reacts with the test substance is immobilized on the outside through a cross-linking agent, is allowed to react with the test substance in the presence of complement. The present invention provides an immunoassay method characterized by quantifying a test substance by measuring a liberated labeling substance, and an immunoassay reagent containing the liposome reagent.

The liposome used in the present invention contains phospholipids including amide-type phospholipids and cholesterol as essential components. The amide type phospholipid is represented by the general formula (I).

Examples of the phospholipid other than the above-mentioned amide type phospholipid include ester type phospholipids that are usually used in the preparation of liposomes, such as phosphatidycholine, phosphatidylethanolamine, and phosphatidic acid.

The amide type phospholipid is preferably incorporated in the phospholipid constituting the liposome in an amount of 5% by weight or more.

The amount of cholesterol used is preferably 0.5 to 2 times the total amount of phospholipids in order to obtain stable liposomes.

The labeling substance encapsulated in the liposome must be hydrophilic and can be quantified when eluted outside the liposome. Such a substance, for example, does not show fluorescence due to self-quenching at a high concentration, but has 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 decomposed by the action of oxidase to cause enzyme consumption or hydrogen peroxide production; relatively large ionic compounds such as tetrapentyl ammonium; nicotinamide adenine Coenzymes such as dinucleotide (NAD); radical compounds such as methylpiorogen are preferable. These compounds are appropriately selected in consideration of factors such as the detection method, sensitivity and liposome stability.

The antigen or antibody immobilized on the outside of the liposome is an antibody against the test substance when the test substance is an antigen, and is the antigen when the test substance is an antibody.
The antibody used here is not limited to polyclonal or monoclonal as long as it has specific reactivity to the antigen.

A liposome reagent (hereinafter, simply referred to as a liposome reagent) having a labeling substance encapsulated inside and an antigen or antibody that specifically reacts with a test substance immobilized on the outside through a cross-linking agent used in the present invention is, for example, It is manufactured as follows. First, react the phospholipid and the crosslinking agent in a solvent,
A functional group capable of binding to the antigen or antibody immobilized on the liposome is introduced into the phospholipid molecule. Then, the obtained functional phospholipid, 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, a 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 functional liposomes.

On the other hand, an antigen or antibody to be immobilized on the liposome is reacted with a cross-linking agent in a buffer to introduce a cross-linking group, and then, if necessary, a reagent that reduces the cross-linking group (for example, dithiothreitol; Further reaction with DTT) yields a modified antigen or modified antibody. This step is unnecessary when the lipid is treated with the activator in the above step.

Finally, the liposome reagent is obtained by reacting the functionalized liposome with the modified antibody in a buffer.

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).

The immunoassay method of the present invention is performed as follows using the above liposome reagent. First, a liposome reagent, a sample containing a test substance, and complement are mixed in an appropriate buffer solution. Then, the antigen or antibody in the liposome reagent reacts with the test substance, the complement is activated by the antigen-antibody reaction, and the labeling substance is released from the inside of the liposome in proportion to the amount of the antigen-antibody reaction. Then, quantification is performed by an analysis method corresponding to the labeling substance (for example, if the labeling substance is a fluorescent substance, a fluorescent analysis method), and, for example, by a calibration curve prepared in advance, the amount of the test substance in the sample Can be measured.

Before carrying out the analysis of the present invention, a sample is prepared in advance with phospholipids selected from phosphatidic acid, dicetyl phosphate, phosphatidyl serine, phosphatidyl glycerol, cardiolipin and lipid A, cholesterol and a crosslinking agent as constituent components. It is preferable to remove the complement component contained in the sample by treating with a liposome.

The complement used in this method is not particularly limited, but serum of guinea pig, rabbit, mouse, human and the like, particularly guinea pig serum is preferable.

Examples of test substances that can be quantified by the method of the present invention include tumor markers (CRP, AFP, BFP, CEA, POA, etc.), immunoglobulins (IgA, IgE, IgG, IgM, etc.), hormones (insulin, T ₃ and T _{3 4} etc.) and drugs.

In carrying out the method of the present invention, it is convenient to prepare a liposome reagent on which an antigen or antibody corresponding to the test substance is immobilized, or the liposome reagent and complement as an immunoassay reagent in advance.

〔The invention's effect〕

According to the present invention, since the amide-type phospholipid is contained as a constituent lipid of the liposome, when the liposome membrane is formed, the liposome membrane is stabilized by hydrogen bond,
As a result, the retention stability of the labeling substance in the liposome was improved. Therefore, the method of the present invention improves the spontaneous leakage of the labeling substance, which has been conventionally observed during storage of the liposome reagent, and as a result, improves the accuracy of measurement, and is extremely useful in immunodiagnosis.

〔Example〕

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

Example 1 (Encapsulation of labeling substance in liposome) Dimyristoylphosphatidylcholine (DMPC), cholesterol (Chol), dithiopyridylated dipalmitoylphosphatidylethanolamine (DTP-DPPE) and
Each chloroform solution of (1,2-dimyristoylamide-1,2-deoxyphosphatidylcholine CDDPC) was added to a 10 ml pear-shaped flask so as to have the composition shown in Table 1.

From these, chloroform as a solvent 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 in a dicator for 1 hour. 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 and add 0.15M sodium chloride.
0.05M Tris-HCl buffer with 0.02% sodium azide pH
7.4 (TBS) was added, the mixture was centrifuged at 12,000 × g for 15 minutes, and the supernatant was removed with an aspirator. After repeating this operation 3 times, the pellet was added with 500 μ of 0.15 M sodium chloride solution.
Suspend in 01M HEPES buffer (pH7.4) and use 6 types of 1-
Methoxy-5-methylphenazinium sulphate-encapsulated liposomes were prepared.

Example 2 (Preparation of goat IgG-sensitized liposome reagent) 0.01M HEPES buffer containing 0.15M sodium chloride (pH
7 μ in 2.0 ml of goat IgG (2.0 mg / ml) equilibrated with 7.4)
Of 30 mM N-hydroxysuccinimidyl-3- (2-pyridyldithio) propionate in ethanol was added and reacted at room temperature for 30 minutes. Then, using a Sephadex G-25 column equilibrated with 0.1 M acetate buffer (pH 4.5) containing 0.15 M sodium chloride, the dithiopyridylated goat IgG was recovered and the buffer was exchanged at the same time. Recovered dithiopyridylated goat IgG solution 2.0
15 ml of dithiothreitol was added to ml, and the mixture was reacted at room temperature for 20 minutes. Then 0.01M containing 0.15M sodium chloride
Sephadex G equilibrated with HEPES buffer (pH 7.4)
The thiolated goat IgG (1.5 mg / ml) was separated using a -25 column.

The thiolated goat IgG (1.0 mg / m
l) 500 μ was added to each of the 6 types of liposome suspensions 500 μ prepared in Example 1, reacted for 20 hours in a cold place (4-10 ° C.), and then centrifuged at 12,000 × g for 15 minutes, The supernatant was removed with an aspirator. After repeating this operation three times, the pellet was mixed with 1 ml of TBS (GTB containing 0.1% gelatin).
S ^- were suspended in), was prepared goat IgG sensitized liposome reagents.

Example 3 (Study of retention stability of labeling substance) Six kinds of goat IgG-sensitized liposome reagents prepared in Example 2 were diluted 200 times with TBS to prepare a liposome suspension 25 thereof.
μ was mixed with 10% Triton X-100 solution or 25 μT of TBS, and the mixture was heated at 37 ° C. for 20 minutes. Next, 1% TBS containing 1.5 mM nitrotetrazolium blue (NTB) and 6 mM NADH was added.
After adding 25μ and heating at 37 ° C for 10 minutes, 50μ of 2N hydrochloric acid and 2% Triton X-100 was added to stop the reaction, and the wavelength was 570nm.
The absorbance was measured with. As a result, as shown in Fig. 1, S /
The N ratio (absorbance obtained when TBS 25 μm was added / 10% Triton X-100 25 μm) was improved in proportion to the addition ratio (molar ratio) of DDPC to the liposomes.

Further, instead of TBS 25 μ, guinea pig serum (complement source) or 10% Triton X-100 solution was mixed with the liposome suspension, and the same experiment as above was performed. as a result,
As shown in FIG. 2, leakage of non-specific labeling substance from liposomes caused by addition of complement was also improved.

Example 4 (Storage stability of liposome reagent) The goat IgG-sensitized liposome reagent prepared in Example 2 was
It was stored as a suspension at 4-6 ° C. Then, during storage in the same manner as in Example 3, the color development due to leaked 1-methoxy-5-methylphenazinium sulphate was measured, and S / N
The ratio was calculated. As a result, as shown in Table 2, improvement in storage stability was observed in liposomes containing a phospholipid having an amide bond, as compared with liposomes.

Example 5 (Measurement of anti-goat IgG antibody titer) 6 kinds of goat IgG-sensitized liposome reagents prepared in Example 2
The anti-goat IgG antibody titer was measured using the same type. 6 types
The liposome suspension was 0.5 mM magnesium chloride and 0.1 mM.
GTBS containing 5 mM calcium chloride^-(GTBS ) Diluted 200 times
did. 25 μl of these liposome diluents, guinea pig complement
(2CH₅₀) 25μ, and anti-goat IgG antibody (rabbit IgG
Picture) GTBS A 2-fold dilution series than a 1000-fold dilution with
25 μm of the assembled sample was mixed and reacted at 37 ° C. for 20 minutes.
Next, GTBS containing 1.5 mM NTB and 6 mM NADH^-125μ
After reacting for 10 minutes, 2N hydrochloric acid, 2% Triton X-10
Add 50μ of 0 to stop the reaction and increase the absorbance at a wavelength of 570nm.
It was measured. As a result, as shown in FIG. 3, anti-goat IgG antibody
Absorbance also increased in proportion to the amount.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between the molar ratio of DDPC (phospholipid having an amide bond) in the liposome component of the present invention and the S / N ratio in Example 3 of the liposome. FIG. 2 shows the complement values of the added complement and liposomes in Example 3.
It is a drawing showing the relationship with the S / N ratio. FIG. 3 is a drawing showing the relationship between the antibody dilution ratio and the absorbance when using liposomes in Example 5.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuaki Yoshikawa 2-11-1, Sugamo, Toshima-ku, Tokyo Within Nissui Pharmaceutical Co., Ltd. (56) Reference JP-A-61-225190 (JP, A)

Claims (2)

(57) [Claims] 1. The following general formula (I): (Wherein each of R ¹ and R ² represents an alkyl group having 11 to 17 carbon atoms) labeled with an amide type phospholipid in an amount of 5% by weight or more and a liposome containing cholesterol as an essential component From the liposome reagent, a liposome reagent, in which a substance is encapsulated and an antigen or antibody that specifically reacts with the test substance is immobilized on the outside through a cross-linking agent, is allowed to react with the test substance in the presence of complement. An immunoassay method characterized by quantifying a test substance by measuring a released labeling substance. 2. The following general formula (I) (Wherein each of R ¹ and R ² represents an alkyl group having 11 to 17 carbon atoms) labeled with an amide type phospholipid in an amount of 5% by weight or more and a liposome containing cholesterol as an essential component An immunoassay reagent containing a liposome reagent in which a substance is encapsulated and an antigen or antibody that specifically reacts with a test substance is immobilized on the outside via a cross-linking agent.