JPS61128168A - Immunological analysis - Google Patents

Abstract

PURPOSE:To rapidly perform immunological analysis with high accuracy, by reacting a specimen solution with an insoluble carrier particle allowed to support an antibody or antigen by using a magnetizable particle or marked particle and applying a magnetic field to the reaction mixture to separate the magnetized particle or flocculated particle before detecting a marker. CONSTITUTION:An antibody or antigen is respectively supported by a particle (L- particle) prepared by marking an insoluble carrier, obtained by emulsion polymerization of styrene, by a fluorescent dye and an insoluble carrier particle (M-particle), same as mentioned above, containing a magnetic powder made magnetizable through the application of the magnetic field of magnetite. A specimen containing an antigen or antebody to be measured is added to an equi-amount mixture of both carrier particles and, after reaction, a magnetic field is applied to the reaction mixture. When the antigen to be measured is set to Ag, M-particle/Ag, M-particle/Ag/L-particle and Ag/L-particle are generated and the mutual agglutinated substance of M-particles and the agglutinated substance of M-particles and L-particles can be easily separated. The market of each separated agglutinated substance is measured. Because the fluorescence of the marker increases in proportion to the concn. of the antigen in the specimen, immunological analysis is enabled with high accuracy within a short time by using a preliminarily obtained calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to an immunoassay method using insoluble carrier particles.

(Conventional technology) The present invention relates to a method for measuring antigen-antibody reactions.

Conventionally, antibodies or antigens supported on insoluble carrier particles such as latex are reacted with antigens or antibodies or a mixture thereof in a liquid medium, and as the reaction progresses, the transmittance of the anti-mixture decreases (i.e., the absorbance decreases). A method is known in which the rate of the antigen-antibody reaction is measured based on the increase in the number of antigens and antibodies, and the concentration of the antigen or antibody in the subject is determined from the rate (Japanese Patent Publication No. 7, JR7Jr).

According to this method, the concentration of antigen or antibody can be determined rapidly and with high accuracy.

(Problem to be solved by the invention) However, in this method, in terms of sensitivity, the number n y
Reliable measurement values can only be obtained up to about /d, and items that require even more sensitive measurements such as growth hormone y, insulin, and digoxin are not necessarily fully satisfied.

Therefore, the present inventors conducted various studies to further improve the sensitivity in the measurement of antigen-antibody reactions as described above, and as a result, they arrived at the present invention.

(Means for Solving the Problems) The gist of the present invention is to support an antibody or antigen on an insoluble carrier particle, and react the supported antibody or antigen with the antigen or antibody or a mixture thereof in a liquid medium. In the method of measuring the degree of aggregation of the reaction mixture as the reaction progresses, the reaction mixture is caused to react using magnetizable particles and label particles as the carrier particles, and then a magnetic field is applied to the reaction mixture, so that the reaction mixture is magnetized. Magnetized particles or aggregated particles containing magnetized particles are separated from the reaction mixture, and labeled particles in the aggregated particles are detected by the label to determine the degree of aggregation of the reaction mixture. ! [This is an immunoassay method characterized by measuring as an increase in the degree of

The present invention will be explained in detail below.

First, the present invention involves supporting an antibody or an antigen on an insoluble carrier particle as described above, reacting the supported antibody or antigen with the antigen or antibody, or a mixture thereof in a liquid medium, and causing aggregation of the reaction mixture. Applied in the method of measuring degree.

The insoluble carrier particles are substantially insoluble in the liquid medium used in the measurement and have a size of about 10μ or less, preferably O,OS~! , fine particles of organic polymeric substances having an average particle size of 0μ, such as polystyrene, styrene-
Organic polymer latex obtained by emulsion polymerization such as butadiene copolymer, or inorganic oxides such as silica, silica-alumina, and alumina are used.

In the method of the present invention, such insoluble carrier particles (for example, latex particles) are made to carry (sensitize) an antibody or antigen that can react with the antigen and/or antibody in the analyte to be measured.

In this case, the antibody or antigen may be physically or chemically adsorbed onto the carrier. Antibodies are made up of proteins, while antigens are made up of proteins, polypeptides, antibodies, and polypeptides. It comes from various things such as S species, Shiga, pollen, etc. Many methods have already been proposed for carrying such antibodies or antigens on insoluble carrier particles. When an insoluble carrier carries an incomplete antigen such as a hapten, it is advantageous to chemically modify the carrier, for example with a coupling agent, and chemically adsorb the antigen.

A suspension is used to sensitize the above antibody or antigen and the concentration of insoluble carrier particles (e.g., latex particles) is usually 0.00/wt% or more, preferably about 0.0/-/wt%. .

The sensitized carrier is reacted with the antigen or antibody 6 or a mixture thereof in a liquid medium under substantially constant conditions.

In the method of the present invention, two types of insoluble carrier particles are used: magnetizable particles and labeled particles.

Magnetizable particles (hereinafter referred to as 0 particles) refer to particles that are not magnetized during a reaction but become magnetized by applying a magnetic field and can be easily separated from a liquid medium, such as magnetite, ferrite, etc. used for. for example,
In this case, latex particles containing magnetite, ferrite, etc. are used for sensitization and reaction.

In addition, as label particles (hereinafter referred to as 0 particles), particles containing fluorescent dyes, radioisotope (R engineering) 11
Examples include particles containing particles such as silver, pigments that can be easily eluted into a solvent by adding an organic solvent, and enzymes such as peroxidase.

For example, when using a fluorescent dye, 1. Insoluble carrier particles, for example, may be those in which a fluorescent dye is mixed with the reaction solution during latex polymerization and the dye is injected into the latex particles. It may be dyed.

Examples of fluorescent dyes include rhodamine dyes (eg, rhodamine-4G, tetramethylrhodamine), acridine dyes (eg, acridine orange), cyanine dyes, rare earths, eosin, fluorescein, coumarin derivatives, stilbene derivatives, and the like.

The ratio of the magnetizable particles to the labeled particles is not particularly limited, but a ratio of about 1, etc. is usually used.

For antigen-antibody reactions, -antibody method and double antibody method can be applied. - In the case of the antibody method, (1) When 0 particles are sensitized with the same antibody and added together with a buffer into a solution containing an antigen, the antigen-antibody reaction causes aggregation of the 2 particles, and the 0 particles are separated from each other. Simultaneous reactions cause agglomeration of ■-■, ■-■, ■-■, or a combination of these. By applying a magnetic field to the reaction mixture after incubation for a certain period of time,
Agglomerated particles, including little or no particles, can be easily separated from the reaction mixture.

In the case of the two-antibody method, what? , by using two types of antibodies that react with each antigen (denoted as O), by sensitizing 0 particles and 0 particles with different antibodies and allowing them to react with the antigen, ■-0. ■−〇−■, 〇−■ particles are obtained. By applying a magnetic field to the reaction solution that has been removed from the tube for a certain period of time, it is possible to easily separate 2 particles or 0 particles and aggregates of 0 particles.

In these cases, if you collect 0 particles or aggregates of 0 particles and 0 particles at the bottom of the reaction container, remove the supernatant, and measure the labeled substance in the remaining particles, it will be proportional to the antigen concentration in the reaction solution. A labeled substance is observed.

To measure a labeled substance, for example, in the case of a fluorescent dye, it is collected at the bottom of a reaction vessel that does not absorb excitation light and fluorescence, and then the excitation light is irradiated from the bottom of the vessel and the fluorescent light that comes out is quantified for 11 seconds. If you use a system that

There is no need to remove the supernatant. After removing the supernatant, an organic solvent such as alcohol is added to elute the dye from the particles, resulting in a highly sensitive measurement with a high degree of FF.

In addition, for example, as a measurement method using a flow cell using latex containing a fluorescent dye as a label particle, a#ht- is applied from the lateral direction of the flow cell, and 0 particles or agglomerates containing 0 particles in the reaction solution flowing through the flow cell. By applying sufficiently focused excitation light to the areas where particles are attracted and leaked in the horizontal direction due to magnetic force, it is possible to increase the number of fluorescent particles that are attracted and leaked due to aggregation with zero particles. It can be associated with an increase in antigenicity.

Measurements are usually carried out after a certain period of incubation after the start of the reaction.

By the above method, the degree of agglutination of the reaction mixture is measured as an increase in label intensity after a certain period of time, and a calibration curve of antigen or antibody prepared in advance with samples with known concentrations is used to determine the degree of agglutination of the antibody or antibody in the sample. The concentration of antigen can be measured.

(Effects of the Invention) According to the method of the present invention, an antigen-antibody reaction using latex can be performed with high precision without being affected by coexisting substances in the reaction solution.
Highly sensitive antigen or antibody quantification is possible.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example / S-based caries (manufactured by Rhone-Poulenc) with an average particle diameter of / μm and latex of Dov ChemiCe-1 fi/, /am were dyed with tetramethylrhodamine (dye/mmol per latex/kl). Use) Sensitize each fluorescent latex with an anti-API' antibody and add the buffer and these two types of latex (equal volumes) to neifetoprotein (AFF) solutions at various concentrations. O
7% by weight).

Fell for cell culture after a certain period of time (inner diameter 7111!, depth/
A fixed amount of the particles is transferred to an ), a magnet is placed below, and the aggregated particles including ■- particles and 0 particles are collected at the bottom of the felt. After that, the supernatant was removed, and the remaining fluorescent particles and zero particles were treated with an argon ion laser! The intensity of fluorescence emitted from the fluorescent particles was measured by applying excitation light of lμ, jnm. FIG. 1 shows the number of photons counted per second plotted against the AFP concentration (the line represents the measurement).

Using this calibration curve, the antigen concentration in a sample with unknown AFF concentration can be measured with high sensitivity.

[Brief explanation of drawings]

FIG. 1 shows the first line for AFP obtained in Example 1. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney, Yukuyo - and 1 other person Figure 1 AFP concentration range L)

Claims (1)

[Claims] (1) An antibody or antigen is supported on insoluble carrier particles, and the supported antibody or antigen is reacted with the antigen or antibody or a mixture thereof in a liquid medium, and the degree of aggregation of the reaction mixture is determined as the reaction progresses. In this method, a reaction is performed using magnetizable particles and labeled particles as the carrier particles, and then a magnetic field is applied to the reaction mixture to separate magnetized particles or aggregated particles containing the magnetized particles from the reaction mixture. and detecting labeled particles in the aggregated particles using the label, thereby measuring the degree of aggregation of the reaction mixture as an increase in label intensity.