JPS63175768A - Method and instrument for immune analysis - Google Patents

Abstract

PURPOSE:To permit fluorescent immune analysis without making physical sepn. of a solid phase/liquid phase by passing a soln. after an antigen-antibody reaction to a flow cell by a pump, etc. CONSTITUTION:The soln. after the antigen-antibody reaction from a soln. tank 2 is fed into the flow cell 3 by the pump 1 and a beam of light is projected from a light source to the soln. after the antigen-antibody reaction passing the inside. The intensity of scattered light is changed by an immobilized antibody 6 when the beam of the light is projected to the soln. after the antigen- antibody reaction at the time when the soln. passes the inside of the flow cell 3. Then, the quantity of the fluorescent light in the immobilized antibody 6 changes according to the volumetric ratio of a fluorescent-labeled antigen and a non-labeled antigen 5 and, therefore, the intensity of said scattered light and the quantity of the fluorescent light can be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an immunoassay method and an immunoassay device for analyzing components contained in a living body by utilizing immune action.

[Conventional technology]

FIG. 4 is a diagram for explaining a conventional example of an immunoassay method, in which 21 shows a fluorescently labeled antigen, 22 shows an immobilized antibody, 23 shows beads, 24 shows an unlabeled antigen, and 25 shows an unknown sample.

In photoimmunoassay (immunoassay), when measuring a component (antigen) in an unknown sample, a method as shown in FIG. 4 is used.

First, as in a-1, a fixed amount of fluorescently labeled antigen 21 is reacted with a fixed amount of immobilized antibody 22 fixed on beads 23 or the like. Then, as shown in a-2, the solid phase (beads) is labeled with the fluorescently labeled antigen. After centrifuging this, the solid phase is suspended and the amount of fluorescence is measured (a-1).

Next, as shown in b-1, the fluorescent 1ffi antigen 21 and a known amount of unlabeled antigen 24 are reacted with the immobilized antibody 22 in the same manner as in the case of ta+, and then the labeled/unlabeled antigen 24 is reacted with the immobilized antibody 22 as shown in b-2.
Labeling is performed on the solid phase in proportion to the ratio of unlabeled antibody amounts, and this is treated in the same manner as +al to measure the amount of fluorescence.

Subsequently, in FC+, the amount of unlabeled antigen 24 is increased and the amount of fluorescence is measured in the same manner.

As described above, by measuring (al, (bl, (C)), a calibration curve in which the amount of fluorescence of the solid phase decreases as the amount of unlabeled antigen increases is obtained.

Therefore, by similarly measuring the amount of fluorescence of the unknown sample 25 as shown in (dl), the amount of antigen in the unknown sample 25 can be measured based on the calibration curve.

[Problem that the invention seeks to solve]

However, in the above-mentioned conventional immunoassay method, in order to separate the remaining free antibody after bouncing the antigen on the solid phase (immobilized antibody), steps such as centrifugation, removal of the supernatant, and further suspension of the solid phase are necessary. is necessary, and if you try to automate it, there is a problem that it will take a lot of time and effort. In addition to these methods of physically separating and measuring bound and free antigens, several methods have been considered that measure without separating them, but these also have various problems in sensitivity, applicability, etc. be.

The present invention solves the above-mentioned problems, and aims to provide an immunoassay method and an immunoassay device that can perform fluorescent immunoassay without physically separating a solid phase and a liquid phase. It is something.

[Means for solving problems]

To this end, the immunoassay method of the present invention circulates a solution containing a solid phase after antigen-antibody reaction, irradiates it with a light beam, and measures the scattered light and fluorescence, thereby determining the amount of fluorescence in each of the solid phase and liquid phase. The immunoanalyzer is characterized by obtaining data, and includes a flow cell that is irradiated with a light beam, a liquid feeding means that sequentially flows a solution containing a solid phase after antigen-antibody reaction, and detection of scattered light and fluorescence in the flow cell. detection means,
and a data processing means for analyzing the scattered light intensity and the fluorescence intensity to obtain respective amounts of fluorescence in the solid phase and the liquid phase.

[Effect]

In the immunoassay method and immunoassay device of the present invention, when a solution containing a solid phase after an antigen-antibody reaction is passed through and irradiated with a light beam, the intensity of the scattered light changes in the solid phase. I can recognize that. Furthermore, the amount of fluorescently labeled antigen can be detected based on the amount of fluorescence.

〔Example〕

Examples will be described below with reference to the drawings.

FIG. 1 shows one of the immunoassay method and immunoassay device according to the present invention.
FIG. 2, which is a diagram for explaining the embodiment, is a diagram showing an example of measurement of scattering intensity and fluorescence intensity in a flow cell.

In FIG. 1, 1 is a pump, 2 is a solution tank, 3 is a flow cell, 4 is a fluorescently labeled antigen, 5 is an unlabeled antigen, and 6 is an immobilized antibody. The solution tank 2 contains the solution after performing the antigen-antibody reaction as shown in FIG. It is. In the flow cell 3, the solution after the antigen-antibody reaction is fed from the solution tank 2 by the pump 1, and a light beam is irradiated from the light source to the solution after the antigen-antibody reaction flowing inside. When the solution after the antigen-antibody reaction flows through the flow cell 3 in this way, when it is irradiated with a light beam, the intensity of the scattered light changes due to the immobilized antibody 6, and the fluorescently labeled antigen 4 and the unlabeled antigen 5 are separated. The amount of fluorescence in the immobilized antibody 6 changes depending on the capacity ratio. In the present invention, the intensity and amount of fluorescence of this scattered light are measured.

Next, measurement of scattered light and fluorescence in the flow cell 3 will be explained. Now, when we measure the intensity of the scattered light when a narrow beam of about 10 μm is irradiated from the light source, we find that the second
As shown in Figure fa), it increases when a solid phase of several μm to several tens of μm flows into a certain part of the beam in the flow cell 3, and is weak when only a liquid phase flows. Furthermore, when fluorescence is measured, it changes depending on the amount of fluorescently labeled antigen, as shown in Q) 1 in Figure 2.

Therefore, we set a threshold level for the scattering intensity,
When the total amount of fluorescence when the scattering intensity is below this threshold level, that is, the amount of fluorescence of free fluorescently labeled antigen, is measured, the amount of unlabeled antigen can be determined from the calibration curve obtained from Figure 4 (al or C1). Can be done.

FIG. 3 is a diagram showing an example of the configuration of a measurement system for the amount of scattered light and fluorescence. In the measurement system shown in FIG. 3, a forward scattered light detector 19 is provided in front of a flow cell 17 through a lens 18 to measure the intensity of the scattered light, and a detector 19 is provided in front of a flow cell 17 through a lens 14, a half mirror 12, and a filter 11.
3 to measure the amount of fluorescence. Moreover, in this example, a plurality of detectors 13 are prepared to detect fluorescence of different wavelengths.

In this way, antigens labeled with fluorescent substances of different wavelengths and antibodies against them are immobilized on the same solid phase or separately, and the immobilized antibodies and the labeled antigen are reacted with the same sample and then measured. , multiple items can be measured with one sample.

Note that the present invention can be modified in various ways and is not limited to the above embodiments.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, in fluorescence immunoassay using immobilized antibodies or antigens using the immobilized competitive method, after the antigen-antibody reaction, the solution is caused to flow through the flow cell using a pump or the like. The solid phase can be detected by measuring scattered light, and the amount of unlabeled antibody can be determined by measuring the amount of fluorescence in the solid phase or liquid phase.

Therefore, fluorescence immunoanalysis can be performed without physically separating solid phase/liquid phase.

[Brief explanation of the drawing]

FIG. 1 shows one of the immunoassay method and immunoassay device according to the present invention.
Figure 2 is a diagram illustrating an example of measuring scattered light and fluorescence intensity in a flow cell; Figure 3 is a diagram illustrating a configuration example of a measurement system for scattered light and fluorescence amount; Figure 4 is a diagram for explaining an example. FIG. 2 is a diagram for explaining a conventional example of an immunoassay method. l...pump, 2...solution tank, 3...flow cell, 4...fluorescent labeled antigen, 5...unlabeled antigen, 6...
・Immobilized antibody. Applicant JEOL Co., Ltd. Agent Patent Attorney Ryukichi Abe (2 others) Figure 1 P Figure 2 (cL) 11 River C Figure 2 (1)) Figure 3 Edge 2'3 (S( Figure 4

Claims (2)

[Claims] (1) Flowing a solution containing a solid phase after antigen-antibody reaction,
An immunoassay method characterized by obtaining data on the amount of fluorescence in a solid phase and a liquid phase by irradiating a light beam and measuring scattered light and fluorescence. (2) A flow cell that is irradiated with a light beam, a liquid feeding means that sequentially flows the solution containing the solid phase after the antigen-antibody reaction, a detection means that detects the intensity of scattered light and fluorescence in the flow cell, and the scattered light intensity and fluorescence An immunoassay device characterized in that it is equipped with a data processing means for analyzing intensity and obtaining respective amounts of fluorescence in a solid phase and a liquid phase.