JP2948935B2 - Antigen and antibody detection methods - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention particularly relates to a method for detecting a trace amount of an antigen or an antibody.

[0002]

2. Description of the Related Art An immunoassay for detecting an antigen or an antibody has attracted attention.

[0003] For example, detection and removal of allergens in foods and medicines are extremely important in diagnosis and prevention of allergy. Until now, allergens have been detected using RAST (Radioallergosorbenttes) using radioisotopes.
t) method, skin test (brick test, scratch test, intradermal test), histamine release test, etc. have been used. However, the RAST method has to use radioisotopes, and the skin test has a drawback that it involves risks such as anaphylactic shock. Therefore, development of a new allergen detection system is desired.

Therefore, the present inventors have reported that a fluorescent dye-labeled antibody extracted from a magnetic bacterium is immobilized and a trace amount of antigen is detected (ANALYTICAL CHEMISTRY, VOL. 6).
3, No. 3, FEBRUARY1, 1991 P268-P27
2).

In this method, a magnetic bacterium immobilized with a fluorescent dye-labeled antibody is allowed to react with an antigen in a liquid to cause aggregation based on the antigen-antibody reaction, and then the fluorescence intensity of the liquid dispersion after the aggregation and separation is measured. Then, the amount of decrease in the antibody is detected from the decrease in the fluorescence intensity, and the amount of the antigen is quantified.

[0006]

However, according to this method, as shown in the above-mentioned report P271, FIG.
It can only be quantified up to the amount of antibody amount reduced to about pg / ml,
Not suitable for trace quantification of antigen.

[0007] A main object of the present invention is to provide a method capable of accurately detecting an extremely small amount of an antigen or an antibody using ferromagnetic particles such as magnetic bacterial particles.

[0008]

This and other objects are achieved by the present invention which is defined below as (1) to (3).

(1) Immobilize fluorescently labeled antibodies or antigens on magnetic bacterial particles extracted from magnetic bacteria on ferromagnetic particles, and react them with the antigens or antibodies in a solution to perform antigen-antibody reactions. A method for detecting an antigen and an antibody, comprising the steps of magnetically separating and concentrating the resulting aggregate after the occurrence of the aggregation, and measuring the fluorescence concentration of the aggregate.

(2) The method for detecting an antigen or antibody according to (1), wherein the ferromagnetic particles have an organic thin film on a surface.

(3) The method for detecting an antigen or antibody according to the above (1) or (2), wherein the ferromagnetic particles are magnetic bacterial particles.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a specific configuration of the present invention will be described in detail.

A magnetic bacterium was discovered in the United States in the 1970's, and is called a magnetosome in which about 10 to 20 magnetite (Fe ₃ O ₄ ) single crystal microparticles having a particle size of about 50 to 100 nm are connected in the cell. Holds chain-like particles. The magnetic bacteria can detect the geomagnetism by holding the magnetosome and recognize the direction of the magnetic field lines. A magnetic bacterium is a microaerobic bacterium. By sensing geomagnetism, a magnetic bacterium can swim from an aerobic water surface to a microaerobic sediment surface layer along a magnetic field line. As indicated in the above-mentioned report, this was isolated as a single bacterium, and the magnetic bacterium that was able to be mass-cultured was a gram-negative spiral bacterium having a size of about 2 μm. Synthesize crystals.

The magnetic particles in the magnetic bacterium are hexagonal prisms, have a very uniform particle size and shape, have high purity, and have a magnetization of about 50 emu / g when the magnetization of bacterial cells containing the particles is converted per fine particle. It has been confirmed that the coercive force is 230 Oe and that it has a single magnetic domain structure.

Further, since the surface of the particles is covered with the organic thin film, the metal is hardly eluted and is stably present.
It has the property of being excellent in dispersibility in an aqueous solution. The organic thin film is a lipid bilayer having a thickness of about 4 nm and containing phosphatidylethanolamine as a main component. This organic thin film is preferably used without being removed.

The magnetic bacterial particles are usually primary particles alone, but may be 2 to 10 secondary particles.

Methods for extracting magnetic bacterial particles from magnetic bacteria include physical pressure crushing using a French press, alkali boiling, enzymatic treatment, and ultrasonic crushing. Magnetic bacterial particles extracted by any of the methods are also used. Its surface is covered with an organic thin film. When an enzyme such as lysozyme or protease is used, it can be extracted in the form of magnetosomes retained in the cells, and when ultrasonic treatment is used, a product in which each is dispersed can be obtained. Therefore, it is desired to use an extraction method more suitable for the purpose of use. However, when obtaining a large amount of magnetic bacterial particles, it can be said that ultrasonic crushing is suitable at present. Although this organic thin film can be removed by chemical treatment,
It is preferable to leave it for immobilization of antigens and antibodies described below.

The ferromagnetic particles used in the present invention may preferably be magnetic fine particles coated with an organic thin film of a phospholipid layer having a binding functional group.

Here, examples of the binding functional group include an amino group, a carboxyl group, a hydroxyl group and the like, and an amino group and a carboxyl group are generally used. Examples of such a phospholipid having a binding functional group include phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidyl-N-methylethanolamine, phosphatidylcholine, phosphatidylglycerol, sphingomyelin, and phosphatidylthreonine.

As the ferromagnetic fine particles, for example, F
_{e 3 0 4, γ-Fe} 2 O 3, Co-γ-Fe 2 O 3,
(NiCuZn) O.Fe ₂ O ₃ , (CuZn) OF
e ₂ O ₃ , (Mn · Zn) O · Fe ₂ O ₃ , (NiZ
_{n) O · Fe 2 O 3} , SrO · 6Fe 2 O 3, BaO ·
_{6Fe 2 O 3, Fe 3 0} 4 was covered with SiO _2, (particle size of about 200A) [Enzyme Microb. Technol. , Vol.2, p.2~
10 (1980)], composite fine particles of various polymer materials (nylon, polyacrylamide protein, etc.) and ferrite, magnetic metal fine particles, and the like.

Such particles are produced, for example, by forming a ribosome comprising a lipid bilayer of a phospholipid having a binding functional group in an aqueous medium, and synthesizing ferrite in the ribosome by a coprecipitation method. Is done.

[0022] Fluorescently labeled antibodies and antigens are immobilized on such ferromagnetic particles as magnetic bacterial particles. Usable antibodies and antigens include all those used in immunoassays. There is no restriction on the fluorescent substance, and there is no restriction on the immobilization on the antibody or antigen. The amount of the fluorescent substance fixed to the antibody or the like is generally about 1 to 10 molecules per molecule of the antibody or the like.

In order to immobilize a fluorescently labeled antibody or the like on magnetic bacterial particles, various polyfunctional compounds such as glutaraldehyde or various coupling agents are preferably used by using an organic thin film such as magnetic bacterial particles. What is necessary is to use it. The amount of the fluorescently labeled antibody or the like immobilized on magnetic bacterial particles or the like is generally about 1 to 10 molecules per particle.

In the present invention, such magnetic bacterial particles or the like on which such fluorescently labeled antibodies or antigens are immobilized are preferably ultrasonically dispersed in a buffer. Then, for example, an antigen-antibody reaction between the fluorescently labeled antibody-immobilized magnetic bacterial microparticles and the allergen is performed. At the time of the reaction, it is preferable to apply a magnetic field from the outside to promote aggregation of the magnetic bacterial particles and the like. Reaction time is 1-6
It is about 0 minutes.

After completion of the reaction, unreacted fine particles are magnetically separated from magnetic bacteria particles and the like agglomerated by the antigen-antibody reaction. In order to separate the aggregated particles, utilizing the high magnetic field sensitivity of the aggregated particles, the aggregated particles in the liquid after the reaction are decanted while being collected by a magnet, or the liquid is caused to flow in and out. However, only the aggregated particles may be captured by the magnet. Generally, the aggregated particles are approximately 10 aggregates such as magnetic bacterial particles that are primary or secondary particles.

The separated agglomerated ferromagnetic particles are dispersed in a buffer containing gelatin, if necessary, and the fluorescence intensity is measured. At this time, 50 pg / ml or more, for example, 50 to 100
A trace amount of antigen of 0 pg / ml can be determined.

[0027]

【Example】

Now, the present invention will be described in further detail with reference to Examples.

Example The following operation was performed according to the above-mentioned report (ANALYTICAL CHEMISTRY).

First, the magnetic bacterium Aquaspirillum magnetotac
ticum Strain AMB-1 was cultured until the early stationary phase. Extraction of the magnetic bacterial particles from the cells was performed by sonication. In addition, extraction of magnetic bacterial particles from crushed bacterial cells was performed using Sm-
This was performed using a Co magnet.

As the fluorescently labeled antibody, mouse immunoglobulin E (IgE) on which fluorescein isocyanate (FITC) was immobilized was used. Since the magnetic bacterial particles are covered with a lipid membrane, the antibodies were immobilized by glutaraldehyde treatment.

More specifically, the extracted magnetic bacterial particles were dispersed in an ultrasonic washer (Tocho UC 0310100W),
% Glutaraldehyde solution for 1 hour at room temperature. After washing with phosphate buffered saline (PBS pH 7.4), the cells were washed with FITC-labeled mouse IgE antibody for 12 hours.
Incubation was performed at ℃ for immobilization. After removing the unreacted antibody by washing, it was dispersed in PBS and stored at 4 ° C.

Separately, a model allergen, dinitrophenylated bovine serum albumin (DNP-BS
A) was converted to a gelatin-veronal buffer (GVB pH 8.3).
(Gelatin 0.1%) to a concentration of 0 to 1000 ng / ml. This diluted standard sample was prepared for each measurement.

50 μg of the antibody-immobilized magnetic bacterial particles,
0 μl of the sample was mixed and incubated at 37 ° C. for 15 minutes. In the agglutination reaction based on the antigen-antibody reaction, Sm-C
o A magnetic field was applied by a magnet to shorten the reaction time.

After the antigen-antibody reaction, the magnetic bacteria particles that had undergone the agglutination reaction and the unreacted particles were magnetically separated and concentrated, and the amount of the particles that had undergone the agglutination reaction was measured using the fluorescence intensity as an index. That is, decantation is performed in a state where the aggregated particles are captured by the magnet, and the separated and concentrated particles are improved using a flow microcell (12 μl), and a magnetic field is applied to the outside of the cell to suppress the sedimentation of the particles. ,
The fluorescence intensity was measured. Aggregated particles are 1% gelatin
GVB including Thermomixer (Thermonics Model T
M-105) and the fluorescence intensity was measured with a fluorescence spectrophotometer (Hitachi F-1200). The excitation light is 490
nm and fluorescence were detected at 520 nm, and the measurement was performed using a 10 × 10 mm quartz cell. The fluorescence intensity was evaluated at a value 15 minutes after the value was stabilized.

Table 1 shows the fluorescence intensity at a DNP-BSA concentration of 0 to 500 pg / ml. In this case, incubation was performed without adding the antigen, and the relative intensity was calculated with the background value measured as 100. DNP-BSA concentration 5
From 0 pg / ml, an increase in relative fluorescence intensity was observed, and detection with higher sensitivity was possible.

[0037]

[Table 1]

It should be noted that quantification could be performed with high accuracy even at 500 pg / ml to 1000 mg / ml.

Further, FITC-labeled mouse IgE antibody-immobilized magnetic bacterial particles, mouse IgG, and bovine serum albumin (BSA) were mixed, and reacted at 37 ° C. for 15 minutes, and the fluorescence intensity was measured. The fluorescence intensity increased only when incubated with DNP-BSA, and did not decrease with other proteins. Also,
When DNP-BSA is mixed with other proteins,
The increase was seen only when NP-BSA was included. From this, selective measurement of the antigen based on the antigen-antibody reaction was possible.

[0040]

According to the present invention, an antigen or an antibody can be quantified with high sensitivity and accuracy to an extremely small amount.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) G01N 33/543 581 G01N 33/543 541 G01N 33/543 575

Claims (3)

(57) [Claims] 1. An antibody or antigen which has been fluorescently labeled is immobilized on ferromagnetic particles, and the antibody or antigen is reacted with the antigen or antibody in a solution to cause aggregation based on an antigen-antibody reaction. A method for detecting an antigen and an antibody, wherein the method comprises the steps of: isolating and concentrating the mixture; and measuring the fluorescence concentration of the aggregate. 2. The method according to claim 1, wherein the ferromagnetic particles have an organic thin film on a surface. 3. The method for detecting an antigen and an antibody according to claim 1, wherein the ferromagnetic particles are magnetic bacterial particles.