JPS6080767A - Immunoanalysis method - Google Patents

Abstract

PURPOSE:To analyze sample from a phosphoric acid compd. which flows out of a microcapsule sealed internally with the phosphoric acid compd. by mixing the microcapsule and a sample contg. an antibody or antigen. CONSTITUTION:A microcapsule which has a microcapsule film conjugating an antigen or antibody and soluble with complement activity and is sealed internally with a phosphoric acid compd. is used. A reagent having the microcapsule sealed internally with the phosphoric acid compd. and a test sample, for example, blood sample, are mixed, by which antigen antibody reaction is induced and the film is broken down by the film dissolving effect of the complement activated by the antigen antibody reaction. The phosphoric acid compd. sealed in plural kinds of the microcapsules is liberated in the system. The phosphoric acid compd. flowing out of the microcapsules is detected and therefore the analysis of the antigen and antibody in the test sample is accomplished in short time.

Description

[Detailed description of the invention] [Technical field of invention] This invention belongs to the technical field of immunoassay methods.

[Technical background of the invention and its problems]

Conventional immunoassay methods use, for example, an antigen-antibody reaction between a radioisotope-labeled antibody (or antigen) and an antigen (or antibody) in a sample collected from a living body to identify a specific antigen (or antibody) in a sample. ) and radioimmunoassay method for quantitative analysis of enzyme-labeled antibodies (or antigens) and antigens (or antibodies) in samples collected from living organisms.
Enzyme immunoassay methods are used to quantitatively analyze a specific antigen (or antibody) in a sample by obtaining an antigen-antibody complex through an antigen-antibody reaction with a sample, and then using an enzyme reaction with a labeled enzyme on the antigen-antibody complex. be.

However, the previous geoimmunoassay method is limited.

Since radioactive substances are used, the disadvantage is that the equipment is time-consuming, and both radioimmunoassay and enzyme immunoassay methods require several hours to several tens of hours to reach sufficient detection sensitivity. The disadvantage is that it takes a long time for analysis.

In order to solve the above drawbacks, sheep red blood cells, liposomes, etc. are made into microcapsules, a marker component is encapsulated in these microcapsules, and the marker component that flows out from the microcapsules destroyed by complement activity is quantified. Techniques that allow immunoassays are possible. In order to realize this method, the marker component must be suitable for the quantitative analysis method and must exist stably within the microcapsule.

Conventionally, substances with large molecular weights such as trimethylphenylammonium ions, enzymes, and albumin are considered suitable as such marker components.

However, although such a substance having a large molecular weight can be stably present in microcapsules, it has the disadvantage that the encapsulation efficiency when encapsulating it in microcapsules is extremely low. Furthermore, when a substance with a large molecular weight reaches a high a degree, the solution becomes viscous, making it difficult and complicated to handle.

On the other hand, there are reports of encapsulating glucose, a low molecular weight compound, in liposomes as microcapsules and subjecting it to analysis (J, A, Haxbyetal et al.
,, Proc, N-A, S,, A1.2083
(1977) Liposome encapsulation has poor stability, and furthermore, when serum is used as a sample, it is affected by glucose present in blood.

Therefore, regarding immunoassay methods using microcapsules that can amplify and measure immune reactions, we are investigating marker components that have good encapsulation efficiency, are easy and simple to handle, and can stably exist within microcapsules. was requested.

[Purpose of the invention]

The present invention has been made based on the above-mentioned circumstances, and provides an immunoassay method using microcapsules encapsulating a low molecular weight compound that is easy and convenient to handle and can stably exist within the microcapsules. The purpose is to provide.

[Summary of the invention]

Summary of the present invention for achieving the purpose of MMe (!) Microcapsules having a microcapsule membrane that binds an antigen or an antibody and can be dissolved by complement activity, and a monononic acid compound encapsulated therein. and a sample containing an antibody or an antigen, the microcapsule membrane is dissolved, and the sample is analyzed by analyzing the monononic acid compound that flows out.

[Embodiments of the invention]

In the living body, substances taken in from the outside world are broken down and reconstituted as necessary components of the living body, but during the metabolic process, various compounds often exist in a phosphorylated state. It is well known that Such metabolic reactions often take place within cells. The membrane surrounding cells is mainly composed of a lipid bilayer consisting of phospholipids, cholesterol, and proteins, and this lipid bilayer acts as a barrier. cannot freely pass through. Some phosphoric acid compounds even accumulate at high aa levels in cells. Such phosphoric acid compounds include, for example, 2 in human red blood cells.
6-bisphosphoglycerin powder and the like are famous.

Microcapsules used in immunoassays are red blood cells, liposomes, etc., and their membranes are linker-lipid bilayers.

Therefore, we expected that a phosphoric acid compound, even a low-molecular-weight one, would be able to stably exist within microcapsules, and after conducting various studies, we arrived at the present invention.

The immunoassay method according to the present invention uses microcapsules having a microcapsule membrane that binds to antigens or antibodies and can be dissolved by complement activity, and encapsulating a phosphoric acid compound therein.

The antigen or antibody can be bound to the microcapsule membrane using known methods such as the chromium chloride method, the glutaraldehyde method,
This can be achieved by applying enzyme immobilization methods such as the bivalent crosslinking reagent method and the method using condensing agents.

As the microcapsules, red blood cells of animals such as sheep can be suitably used. In addition, even red blood cells of other animals or animal cells other than red blood cells have antibodies, (or antigens,) bound to their cell membranes, and contain chemically inert and separable substances within their cells. If possible, it can be used as a microcapsule in this invention. Furthermore, rehomme can also be used as an artificial membrane.

The complement includes guinea pig serum.

The phosphoric acid compound to be encapsulated in microcapsules is
Any material may be used as long as it can stably hold the microcapsule membrane within the microcapsule without loading it; for example, alkyl phosphate, glucose-6
-+)yllJ NADH.

adenosine-6-phosphate (AT)'), adenosine-2-phosphate (ADP), adenosine-1-phosphate (
AMP), choline phosphate, galactose-1-phosphoric acid,
The choice of which of these various phosphoric acid compounds to use, including xanthosine-1-phosphate and phosphoenolpyruvate, depends on the detection method used in the analysis process and the quality of the presence of similar substances in the test sample. However, it can be determined as appropriate.

Encapsulation of a phosphoric acid compound into microcapsules can be carried out by a known method. When the microcapsules are sheep red blood cells, the analogy is that of Dee D'Orazio (D, Do
Phosphoric acid compounds can be encapsulated in microcapsules by the method of Razio et al. (D, L)o
razio etal, , Anal, (JCm,
, 49°2083 (1977) It. Furthermore, when the microcapsules are liposomes, CM, which can be encapsulated by the method of Haga et al.
etal, , Bi.

chem, 118, 286 (1981):).

In the method of the present invention, an antigen-antibody reaction is induced by mixing a reagent having a microcapsule containing a phosphoric acid compound with a test sample, such as a blood sample, and the complement membrane activated by the antigen-antibody reaction is The lytic action destroys the membrane, thereby liberating the phosphoric acid compound encapsulated in the microcapsules of 'tX e into the system.

Detection of the phosphoric acid compound liberated within the system can be carried out by various methods, but a method involving an enzymatic reaction is preferred from the viewpoint of both specificity and sensitivity. When using an enzymatic reaction, it is desirable to coexist the enzyme used in a reagent containing microcapsules in advance. Therefore, the enzyme used must not harm the microcapsules. In addition, the enzymatic reaction depends on temperature and p
Since the pH is highly dependent on pH, the reaction conditions for enzymatic reactions should be set at pH 5 to 9. Preferably, the temperature is set at 10-50°C. The enzyme reaction can be quantified, for example, by optical or electrical quantitative methods such as absorbance measurement, fluorescence photometry, and luminescence photometry.

Next, enzymatic reactions and quantitative methods for various phosphoric acid compounds will be briefly illustrated.

(1) Phosphatase thread When an alkyl phosphate ester is used as a marker component, it is decomposed by an alkyl phosphate phosphatase into an alkyl alcohol and an inorganic phosphoric acid, resulting in 461J-free phosphoric acid, such as molybdenum blue. The alkyl phosphate ester is quantified by measuring the absorbance using a method or the like.

(2) Dehydrogenase thread For example, when glucose-6 monophosphate is used as a marker component, glucose
Sulf-6-phosphate and NAD (oxidized nicotinamide adenine dinucleotide) are decomposed into 6-phosphogluconic acid in the presence of glucose-6-phosphate dehydrogenase, and the resulting NADH (oxidized nicotinamide adenine dinucleotide) is decomposed into 6-phosphogluconic acid. Glucose-6-phosphate is quantified by measuring the absorbance of dinucleotide) at 340 nm.

(3) When bacterial luciferase-based nicotinamide adenine dinucleotide (NADH) is used as a marker component, NA is determined by measuring the light emitted by reacting according to the first and second linear reaction formulas. DH quantification 7, NADH+FM
N (flavin mononucleotide) + kl oxide reductase NAD + FMNH2... (1 bacterial lucinoerase 1i'MNH2 + decanal → -02 FMN + decanoic acid + H2U + 11 L...
・, (2) (4) When firefly luciferase-based adenosine-3-phosphate (ATP) is used as a marker component, ATP is quantified by measuring the light emitted by reacting according to the following sixth reaction formula. do.

Firefly luciferase ATP + luciferin + 0 □ --- 1 → qiP + pyrophosphate oxidized luciferin + bL ・・・・・・・・・
・(3)(5) Oxidase system choline phosphate, calactosine-1-phosphate, xanthosine-1-phosphate, adenosine-1-phosphate (AMP),
When using phosphoenolpyruvate etc. as a marker component,
The marker component is quantified by quantifying the amount of oxygen consumed or the amount of hydrogen peroxide produced using the following fourth to eighth reaction equations.

Bequinaldehyde D-galactohexodialdose (5)
Uric acid Allantoin Adenosine 5'carboxylic acid (7)
(Mitsuhashi et al. 1 Japanese Biochemical Society lecture abstract (81)) Said 4th
In the ~8th reaction equation, the amount of oxygen consumed can be measured, for example, with an oxygen electrode.

The amount of hydrogen peroxide produced can be determined by, for example, ① measuring absorbance using a combination of peroxidase and a coloring agent such as 4-aminoantipyrine; ② ac measuring with a Bolaro type electrode; jJ, a method of directly quantifying hydrogen hydride, (2) a method of measuring 43Q nm light emitted in the presence of luminol and iron ions, etc.

Although the method of the present invention has been explained above, it goes without saying that the method of the present invention is not limited to the above explanation and can be implemented with appropriate modifications within the scope of the gist of the present invention. .

Since the method of this invention uses a phosphoric acid compound as a marker component, it can be applied to a wide variety of detection systems depending on the purpose such as measurement sensitivity.

When a nucleic acid-based compound is used as the phosphoric acid compound, the liquid containing the nucleic acid-based phosphoric acid compound that has flowed out from the microcapsules can be quantified by directly measuring absorbance or fluorescence without going through an enzymatic reaction. be able to.

[Effect of emitting]

According to this invention, (1) it is possible to stably retain a low molecular weight phosphoric acid compound within the microcapsule;

■It is possible to encapsulate the Helinoseki compound in the capsule more easily and efficiently than with conventional methods.

(2) Since the phosphoric acid compound released from the microcapsules destroyed by the complement activity associated with the antigen-antibody reaction is detected, the antigen-antibody in the test sample can be analyzed in a short time.

- Detection of the phosphoric acid compound leaked from the microcapsules can be carried out by various methods, particularly by employing microscopic detection methods such as fluorescence measurement and luminescence measurement.

Φ has excellent effects such as.

Claims (2)

[Claims] (1) By mixing a sample containing the antibody or antigen with a microcapsule that binds an antigen or antibody and has a microcapsule membrane that can be dissolved by complement activity and encapsulates a phosphoric acid compound inside. , an immunoanalytical method characterized in that a sample is analyzed by dissolving a microcapsule membrane and analyzing the phosphoric acid compounds that flow out. (2) The immunoassay method according to claim 1, wherein the phosphoric acid compound flowing out is analyzed by an enzymatic reaction.