JPH01244371A - Method for measuring specific coupling reaction - Google Patents

Abstract

PURPOSE:To determine the quantity of a material to be measured in a short time, by applying a one-way magnetic field into reaction liquid which incorporates fine grains comprising magnetic bodies, and releasing the application. CONSTITUTION:Fine grains comprising magnetic bodies wherein material that is coupled with a material to be measured is fixed and a sample is mixed. Said mixed liquid 3 is contained in a container 2. Light is projected to the container 2 from a light source 1. Then, the light is scattered with the composite material comprising the aggregated fine grains. The scattered light is detected with a photodetector 4. When a one-way magnetic field is applied into the liquid 3 from a device 5, the fine grains comprising the magnetic bodies are aligned in the direction of the magnetic field. The intensity of the scattered light becomes the maximum value. When the device 5 is turned OFF, the speed of the dispersion of the aggregated grains becomes slow since the average grain size is large. When the time constant of the detected output of the photodetector 4 is obtained, the concentration of the material to be measured can be determined.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for measuring specific binding reactions using a magnetic field.

[Conventional technology]

Japanese Patent Application Laid-Open No. 62118255 is a conventional technique for measuring a compound produced by an immune reaction using a magnetic field. This involves applying a periodically displacing magnetic field to a reaction solution containing fine particles made of magnetic material, causing the magnetic fine particles to rotate in synchronization with the magnetic field, and detecting antigens or antibodies from the scattered light of the periodically displacing aggregated particles. It is a method of qualitative or quantitative analysis.

[Problem to be solved by the invention]

In the prior art, it was necessary to periodically displace the magnetic field. Furthermore, since the i-field is displaced and the measurement is performed after the required reaction time has elapsed, there is a problem that the measurement time becomes long.

The present invention has been made in view of these problems, and it is an object of the present invention to provide a method that shortens the time required for measurement with a simple configuration.

[Means and actions to solve the problem]

FIG. 1 is a conceptual diagram of this invention. Light emitted from a light source 1 is projected onto a container 2. The container 2 contains a mixed solution (reaction solution) 3 of a sample and fine particles made of a magnetic material on which a substance that binds to the substance to be measured is immobilized. Scattered light from a composite composed of fine particles (magnetic force) or fine particles aggregated via the substance to be measured in the container 2 is detected by a photodetector 4 installed at an arbitrary angle. The magnetic field generator 5 applies a unidirectional magnetic field to the reaction liquid 3. The inventor has confirmed that when a magnetic field is applied, both aggregated and non-aggregated particles made of a magnetic material align in the direction of the magnetic field. The scattered light intensity reaches its maximum value in this state.

When the magnetic field generator 5 is turned off and the application of the magnetic field is removed, the aligned fine particles made of magnetic material are dispersed again.

At this time, since the aggregated particles have a large average particle size, the speed of dispersion becomes slow.

Therefore, by canceling the application of the magnetic field to the reaction liquid 3 and determining the time constant of the detection output of the photodetector 4, the concentration of the substance to be measured can be determined. Furthermore, the concentration of the substance to be measured can be determined from the ratio of the intensity of scattered light during application of the magnetic field to the intensity of scattered light after the application of the magnetic field is removed.

The composite, which is the source of part or all of the scattered light from particles in the reaction solution, is formed by the combination of a substance fixed on fine particles made of a magnetic substance and a substance to be measured.

The substance immobilized on the magnetic particles is a component involved in the ligand-receptor binding reaction and the antigen-antibody reaction, such as antigenic substances and antibodies, immunoglobulins and antibodies of foreign animals against this immunoglobulin, It is one component of a binding pair such as a cell membrane protein and an antibody, or biotin and avidin, and the other component is the substance to be measured.

In addition, this composite basically consists of magnetic particles (referred to as magnetic particles) on which a substance that binds to the substance to be measured is immobilized,
Although it is composed of a substance to be measured, it also includes composites and aggregated particles that are composed of other substances bound to this composite.

〔Example〕

FIG. 2 is a diagram showing an embodiment of an apparatus for carrying out the method of the present invention. Coherent light or incoherent light can be used as the light source 10, and in this embodiment, a He-Ne gas laser (wavelength: 632.8 nm) is used. A light beam 11 emitted from a light source 10 passes through an ND filter 12 to adjust the amount of light, and then is focused near the center of a container 14 by a condenser lens 13. The light beam from the condenser lens 13 passes through a polarizing plate 15 made of, for example, a Glan-Thompson prism, and is projected onto a container 14 as linearly polarized light. The container 14 contains spherical fine particles made of a magnetic material with a specific binding substance immobilized on the surface, such as Ni particles having a particle size of 0.1 μm to 0.05 μm and having immunocloprin immobilized on the surface.
, Co, or an alloy thereof, containing a buffer solution in which a ferromagnetic material with no spontaneous magnetization is dispersed, and then a sample containing an antigen or an antibody is added to prepare the antigen-antibody reaction solution 1.
Accommodates 6. The light scattered by the particles in the container 14 is made incident on a collimator 17 having a pair of pinholes,
The light is made incident on a photodetector 19 through a polarizing plate 18 arranged so that only the polarized light component perpendicular to the polarization plane of the polarizing plate 15 passes. The photodetector 19 is a silicon photodiode,
Any element that converts an optical signal into an electrical signal, such as an avalanche photodiode or a photomultiplier tube, can be used. Helmholtz coils 20 and 21 are arranged to sandwich the container 14 and generate a magnetic field perpendicular to the laser beam.
Place. The output signal from photodetector 19 is sent to current-voltage converter 22 . The signal is supplied to a data processing device 25 via an amplifier 23 and a low-pass filter 24.

FIG. 3 is a graph showing the attenuation of orthogonal polarization components of scattered light by particles. The apparatus shown in Fig. 2 was used for the measurement. Polystyrene L/7 Latex Ecstaball LMP266 (manufactured by Rhone-Poulenc) with an average particle diameter of 0.87 μm and a Fe'o' content of 14% (W/W) was used as the magnetic fine particles. 150 μl of LMP266 0.1 wt% suspension was placed in container 14, and 0-10% sodium chloride solution 15
μl was added to cause aggregation by salting out. 0% is the reference. After adding the sodium chloride solution, the Helmholtz coils 20 and 21 were energized for 5 minutes to apply a unidirectional magnetic field of 50 G. The change in the output signal of the photodetector 19 after the application of the magnetic field was removed was measured. 4 +m5e
c'''C magnetic field was turned off. The higher the concentration of NaCl and the greater the degree of aggregation, the slower the rate of decrease of the output signal.

FIG. 4 is a graph showing the relationship between the sodium chloride concentration and the time constant of the output signal.

Based on the measurement data shown in FIG. 3, for each concentration, a time constant, which is the time taken to decrease to 1/e from the value at the time of magnetic field release, was determined.

FIG. 5 is a graph showing the relationship between the sodium chloride concentration and the ratio of output signal current.

Based on the measurement data in Figure 3, the output signal current value for each concentration and when the magnetic field is released (4m5ec)! . And the output current value at 10m5ec after a certain period of time! , the ratio R-(R
-11/I. ) was sought.

From FIGS. 4 and 5, there is clearly a correlation between the sodium chloride concentration, the time constant of the output signal current, and the ratio of the output signal current. Therefore, by determining the time constant of the output signal current or the ratio of the output signal currents, the concentration of sodium chloride can be determined from the time constant of the output signal currents or the ratio of the output signal currents.

In this example, colloidal particles undergo salting out depending on the concentration of sodium chloride, and the higher the concentration of sodium chloride, the larger the aggregates of colloidal particles grow, so the experiment was conducted using sodium chloride. The correlation between the time constant of the output signal current and the ratio between the output signal current and the output signal current was clarified. Therefore, it can be applied to reactions in which aggregates of magnetic particles are formed depending on the amount added.

In the embodiment described above, polarized light vi15°1 is provided before and after the container 14.
8 is arranged so that the photodetector receives only the orthogonal component of the polarized light incident on the reaction solution, thereby cutting out the single scattered light and transmitted light of non-agglomerated particles floating in the reaction solution. The concentration can be measured accurately from

In the above embodiment, since the magnetic field is applied immediately after adding the sample to the magnetic particle suspension, the reaction can be promoted by aligning the magnetic particles in the direction of the magnetic field.

According to the embodiment described above, the measurement can be carried out in a short period of time before and after the application of the magnetic field is removed.

In FIG. 5, the output signal measurement time required to determine the output signal current ratio is 6IllSeC.

〔Effect of the invention〕

According to the present invention, the amount of a substance to be measured can be determined in a short time by a simple operation of applying a magnetic field and releasing the application.

[Brief explanation of the drawing]

FIG. 1 is a conceptual diagram of the present invention, FIG. 2 is a diagram showing an example of an apparatus for carrying out the method of the present invention, FIG. 3 is a graph showing attenuation of orthogonal polarization components of light scattered by particles, and FIG. The figure is a graph showing the relationship between the sodium chloride concentration and the time constant of the output signal current, and FIG. 5 is a graph showing the relationship between the sodium chloride concentration and the ratio of the output signal current. 1, 10- light source 2.14-... container 3.
16...--Reaction liquid 4.11-... Photodetector 5
-------- Magnetic field generator 12 -------
---・NO filters 15, 18 ------Polarizing plate 20.21 ---- Helmholck coil 22.
--- Current-voltage converter 23 --- Amplifier 24 --- Low-pass filter 25 --- Data processing device

Claims (1)

[Claims] 1. Light from a light source is applied to a reaction solution containing a compound produced by a reaction between a substance to be measured and fine particles made of a magnetic material on which a substance that binds to the substance to be measured is immobilized. project the
In the method of detecting scattered light from particles in a reaction solution with a photodetector, a magnetic field is applied in one direction into the reaction solution, and then the application of this magnetic field is released, and the number of points in the detection output of the photodetector is measured. A method for measuring a specific binding reaction, characterized in that the amount of a substance to be measured is determined based on the number of time points. 2. Projecting light from a light source into a reaction solution containing a compound produced by a reaction between a substance to be measured and fine particles made of a magnetic material on which a substance that binds to the substance to be measured is immobilized;
In a method of detecting scattered light from particles in a reaction solution using a photodetector, a magnetic field is applied in one direction into the reaction solution, and during this time the intensity of the scattered light while the magnetic field is being applied is detected by the photodetector, and the intensity of the scattered light is detected by the photodetector. The application of the magnetic field is canceled, the scattered light intensity after the magnetic field is removed is detected by the photodetector, and the ratio of the scattered light intensity during the application of the magnetic field to the scattered light intensity after the magnetic field is removed is determined, and the ratio of this scattered light intensity is calculated. A method for measuring a specific binding reaction, characterized in that the amount of a substance to be measured is determined based on.