JPH03120465A - Immunoassay device - Google Patents

Abstract

PURPOSE:To eliminate the need for the temp. control over the entire part of a part for measuring a fluorescent light quantity or to make measurement with high accuracy with only the rough temp. control by providing 1st and 2nd photodetectors having approximately the same temp. and photodetection sensitivity characteristics. CONSTITUTION:The light reflected by a parallel plane substrate 26 is received by the 1st photodetector 29 via a lens 28 and the quantity of exciting light is monitored. The monitored signal S1 is inputted to a power source part 30 of a light source and the impressed voltage is so controlled that always the specified light quantity is emitted from a low-ressure mercury lamp 21. Fluorescence is emitted from 4-methyl umbelliferone (4MU) when a slide 1 is irradiated with the exciting light. The fluorescence is detected by the 2nd photodetector 35. The signal S2 indicating the quantity of the fluorescence detected by the photodetector 35 is inputted to a signal processing section 31. The signal S1 is inputted as well to the signal processing section 31 where the signal S2 is standardized by the signal S1. Such measurement and computation are executed for 5 minutes at every 10 seconds and the quantity of 4MU is determined. The result thereof is collated with a prescribed calibration curve, by which the quantity of the antigens in blood is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an immunoassay device that uses antigen-antibody reactions to measure the amount of a predetermined antigen or antibody in a liquid to be measured, such as blood. It is.

(Prior Art) Various immunoassay methods for quantifying antigen-antibody reactions have been considered and put into practical use. These various immunoassay methods can be roughly classified into two types.

One of these is called the immunodiffusion method, in which insoluble complexes generated as a result of antigen-antibody reactions are precipitated within a gel, and the amount of this precipitated is measured. Turbidity measurement method (LASERNEPHELOME)
There is a method called TRY. The other method is called a labeled immunoassay, and by identifying either the antigen or the antibody in some way, it is possible to perform measurements with higher sensitivity than the sensitivity limit of the immunodiffusion method described above. Labels include radioisotopes, bacteriophages, enzymes, and fluorescent substances.

There are metals, etc.

Among the various measurement methods mentioned above, one of the labeled immunoassay methods using an enzyme as a label uses a support on which an antibody (antigen) that reacts with a predetermined antigen (antibody) is immobilized, and A liquid to be measured that is expected to contain the above-mentioned predetermined antigen is applied to the sample to cause an antigen-antibody reaction, and then the predetermined labeled antigen (antibody) is applied, and then A washing solution containing a substrate that is converted into a fluorescent substance by the catalytic action of the label is flowed over the support to flush out the labeled antigen (antibody) that has not reacted with the antibody (antigen), and at the same time The fluorescent substance is generated by the labeled predetermined antigen (antibody) reacting with the antibody (antigen), and the amount of the fluorescent substance is measured. A sequential reaction method is being considered that measures the presence or absence of antibodies (antibodies) and their amount.

In addition, the antibody immobilized on the support reacts with the antigen in the liquid to be measured, and then a labeled antibody that reacts with the antigen is spotted, and after the antigen and labeled antibody are reacted, a washing solution is used to remove the reaction. Known methods include the sandwich method in which labeled antibodies are flushed out, and the competitive method in which a labeled antigen or labeled antibody is added to a liquid to be measured and spotted on a support at the same time.

The above method has the advantage that it is much easier to handle than the conventional wet method due to the use of a support, and that it is possible to construct an apparatus that automatically sequentially measures a large number of liquids to be measured.

In an immunoassay device using the above principle, in order to measure the amount of the fluorescent substance generated, excitation light in a predetermined wavelength range is irradiated onto the support on which the fluorescent substance is generated to excite the fluorescent substance. This is carried out by photoelectrically measuring the amount of fluorescence emitted from the fluorescent substance.

(Problem to be Solved by the Invention) In measuring the amount of fluorescence as described above, conventionally, the amount of excitation light irradiated onto the support is monitored, and the amount of excitation light is used as the light source of the excitation light. Although it is possible to improve measurement accuracy by stabilizing the amount of excitation light through feedback and by taking this amount of excitation light into account during signal processing, it is still necessary to perform measurements with the necessary accuracy. For example, the entire fluorescent light amount measuring section is kept at ±1.0°C.
It is necessary to adjust the temperature with such precision that the device becomes large and complicated.

In view of the above circumstances, it is an object of the present invention to propose an immunoassay device that eliminates the need for temperature control of the entire fluorescent light FFI 71!1 constant section or only requires rough temperature control. .

(Means for Solving the Problems) The immunoassay device of the present invention applies a washing liquid containing a substrate that becomes a fluorescent substance by the action of the label to an antigen or antibody to which a label has been attached, and By generating a fluorescent substance, irradiating the fluorescent substance with excitation light, and measuring the amount of fluorescence emitted by the fluorescent substance, the amount of a given antigen or antibody in the liquid to be measured supported on the support can be determined. In the immunoassay device that measures
a light source that emits the excitation light; an irradiation optical system that guides the excitation light from the light source to the support; a first light receiver that monitors the amount of the excitation light; a second light receiver that measures the amount of fluorescence emitted from the fluorescent material; and a light receiving optical system that guides the fluorescence from the support to the second light receiver; a fluorescence light amount measurement unit comprising normalization means for normalizing the amount of the fluorescence obtained by the second light receiver with the amount of excitation light obtained by the first light receiver; The light receiver and the second light receiver are characterized in that they have substantially the same temperature and light-receiving sensitivity characteristics.

(Function) In the immunoassay device of the present invention, since the first photoreceptor and the second photoreceptor have substantially the same temperature-light sensitivity characteristics, the temperature Drift can be suppressed to a very small level, and highly accurate measurements can be performed without adjusting the temperature of the entire fluorescence light amount measurement section, or by only making rough temperature adjustments.

(Example) FIG. 2 is an explanatory diagram for explaining the principle of immunoassay of the present invention.

FIG. 2(a) schematically shows that antibody 2 is immobilized on slide 1, which is an example of a support, and blood containing antigen 3 is spotted on slide 1. It is a diagram. Depending on the amount of antigen 3 in the blood, as shown in FIG. 2(b), antibody 2a with an antigen-antibody reaction and antibody 2b with no reaction occur, and the amount of antigen 3 in the blood increases. As the number increases, the number of antibodies 2a with which a reaction occurs increases, and the number of antibodies 2b with which a reaction does not occur decreases.

Next, as shown in FIG. 2(C), when the antigen 4 labeled 4a is spotted on the slide 1, a part of the antigen 4 is transferred to blood as shown in FIG. 2(d). The antigen 3 inside reacts with the antibody 2 that has not yet undergone an antigen-antibody reaction, and the rest remains unreacted.

Thereafter, when a fixed amount of washing solution containing the substrate 5 is poured over the slide for, for example, 30 seconds, first the unreacted antigen 4a is washed out, and then the labeled 4a is washed out, as shown in FIG. 2(e). The substrate 5 is transformed into a fluorescent substance 6 by the catalytic action. After flowing the cleaning solution onto the slide for 30 seconds, the slide is irradiated with excitation light for 5 minutes every 10 seconds, and the amount of fluorescence emitted from the fluorescent substance 6 is measured. is determined, and by comparing this with a predetermined standard curve, the amount of antigen 3 in the blood is determined.

FIG. 1 is a diagram showing the configuration of a fluorescence light amount measurement section that mainly measures the fluorescence light amount in an embodiment of the immunoassay device of the present invention to which the above measurement principle is applied.

The slide 1 is placed on the upper part of the fluorescence light amount measuring section 20 and is maintained at a constant temperature (for example, 37.5° C.) by a heater 41 built into the incubator 40 .

In this state, the syringe 11 of the spotting means 10 is used to
The blood 13 contained in the valve 15 is removed via the valve 14. A predetermined amount of liquid is applied onto the slide 1 via the nozzle 1B (see FIGS. 2(a) and 2(b)).

Next, a solution containing an enzyme-labeled antigen is spotted using a similar spotting means (not shown) in the same manner as the blood (see Figure 2).
(see e), (d)). Furthermore, after that, a fixed amount of a cleaning solution containing, for example, 4-methylumbellifer phosphate (hereinafter referred to as "4MUPJ") as a fluorescent substrate is poured onto the slide 1 for 30 seconds using a similar spotting means (not shown). After 30 seconds, the washing solution was stopped, and 4-methylumbelliferone (fluorescent material) (hereinafter referred to as r4MUJ) whose 4MUP was changed by the enzyme labeling was washed at 10 second intervals for 5 minutes. ) is measured.

The fluorescence light amount measuring section 20 uses a low-pressure mercury lamp 21 equipped with an elliptical mirror 21a as a light source. The light 22 emitted from the low pressure mercury lamp 21 passes through the slit 23° lens 2.
4. Interference filter 25. Parallel plane substrate 26. lens 27
Slide 1 is irradiated from below. The interference filter 25 filters light 22 emitted from the low-pressure mercury lamp 21.
It has a transmission wavelength band that passes only the emission line with wavelength λ-385na+, and 4MU on slide 1 has wavelength λ-385na+.
It is excited by 365na excitation light.

The light reflected by the parallel plane substrate 26 is received by the first light receiver 29 via the lens 2B, and the amount of excitation light is monitored. This monitored signal s1 is transmitted to the light source power supply section 3.
0, and the applied voltage is controlled so that a constant amount of light is always emitted from the low-pressure mercury lamp 20. The signal S1 is also input to the signal processing section 31.

When slide 1 is irradiated with the excitation light of λ-365n,
Fluorescence is emitted from 4MU. This fluorescence is caused by lens 32.
Interference filter 33. The second light receiver 3 via the lens 34
5 is photoelectrically detected. This interference filter 33 transmits light in a narrow wavelength band centered around λ-450 ns (for example, half width ±5 ng+).

A signal S2 representing the amount of fluorescence detected by the second light receiver 35 is input to the signal processing section 31. Signal processing section 81
As mentioned above, the signal S obtained by the first photoreceiver 29 is
l is also input, and the signal S2 is normalized with the signal S1. Such measurements and calculations are performed every 10 seconds for 5 minutes, and a graph representing the rate at which 4 MUs are generated is obtained. After the 5-minute measurement is completed, the amount of antigen in the blood 13 is determined based on the obtained graph. The value representing the determined amount of antigen is displayed on the display 3B.

Next, the temperature dependence of the fluorescence light amount measuring section will be explained.

The amount of light 22 emitted from the low-pressure mercury lamp 21! ,
The transmittance of the interference filter 2B is F1+the transmittance of the interference filter 33 is F2. It is assumed that the light receiving sensitivity of the first light receiver is Si+the light receiving sensitivity of the second light receiver is 82, and that these are functions of the temperature T.

At this time, the amount of excitation light is 1. , is 1,, = □-1 (T) ・Ft (T)
-(1) ko: Represented as a constant. In addition, fluorescent light m1 emitted from the slide. ．． is proportional to the excitation light mIo, and I am-ηsha1ex-yl・k6・1 (7)・Ft(T)・
...(2) Therefore, the value of the signal S2 detected by the second photoreceiver 35 is determined by the value of the signal S2 detected by the second photoreceiver 35, considering the transmittance of the interference filter 33 and the light receiving sensitivity of the second photoreceiver 35 in equation 21 above.
2'-to 1 ・I (T)・Ft (T)・Fz
(T)・St (T) ... (3)kl
: Represented as a constant.

Further, the signal Sl detected by the first light receiver 29 is 5l-kz 1 (T) ・Ft (T) φSl
(T)...(4) It is expressed as.

When the signal S2 detected by the second light receiver 35 is normalized by the signal S1 detected by the first light receiver 29 to obtain the normalized output S3, it becomes S3-52/5l St (T).

Here, the light receiving sensitivity Ss (T) of the first light receiver 29 and the light receiving sensitivity S2 (T) of the second light receiver 35 are in a proportional relationship, that is, even if there is temperature dependence, both change in the same way. In this case, Sz (T)/J (T) is a constant number, and the temperature dependence of the transmittance and transmission center wavelength of the interference filter is very small, and the fluorescence spectrum has a fairly wide wavelength width, so the interference filter 33 Even if the transmission center wavelength of the interference filter 83 changes somewhat, the amount of fluorescent light transmitted through the interference filter 83 hardly changes. Therefore, the final output S3 has very little temperature dependence. Therefore, the fluorescence light amount measuring section 20 hardly requires temperature adjustment.

(Effects of the Invention) As explained in detail above, the immunoassay device of the present invention has the following features:
Since the first photoreceiver that monitors the amount of excitation light and the second photoreceiver that measures the amount of fluorescence have approximately the same temperature-light sensitivity characteristics, even if the temperature drift of the photoreceiver is large, However, the temperature dependence of the entire fluorescence measurement section becomes extremely small, and temperature control of the entire fluorescence measurement section becomes almost unnecessary.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of the fluorescent light FF1FLL constant section that mainly measures the amount of fluorescent light in an embodiment of the immunoassay device of the present invention. FIG. 2 is the measurement principle of the immunoassay device of the present invention. It is an explanatory diagram for explaining. 1...Slide 3...Antigen 5...Substrate 1O...Spotting means 20...Fluorescence light amount measuring section 25, 33...Interference filter 35...Second light receiver 31... - Signal processing unit 40... Incubator 2... Antibody 4... Labeled antigen 6... Fluorescent substance 13... Blood 21... Low pressure mercury lamp 29... First light receiver 30...・Light source power supply section 36...Display (d) (e) (Subject to 6° correction) I401 Contents of 7° amendment in the "Detailed explanation of the invention" column of the specification dated January 2, 2019 (1) ) Specification section, page U, line 1, Patent Application No. 257,423 of 1999, r half-width ±5 nmJ is corrected to [half-width 5 naJ. 2゜Name of the invention Immunoassay device 3゜Relationship with the person making the amendment case

Claims (1)

[Scope of Claims] A cleaning solution containing a substrate that becomes a fluorescent substance by the action of the label is applied to an antigen or antibody to which a label has been attached, so that the antibody or antigen generates a fluorescent substance, and the fluorescent substance is excited. In an immunoassay device that measures the amount of a predetermined antigen or antibody in a test liquid supported on a support by irradiating light and measuring the amount of fluorescence emitted by the fluorescent substance, the excitation light is used. a light source that emits light; an irradiation optical system that guides the excitation light from the light source to the support; a first light receiver that monitors the amount of the excitation light; and a part of the excitation light that is transmitted to the first light receiver. a second light receiver that measures the amount of fluorescence emitted from the fluorescent substance; a light receiving optical system that guides the fluorescence from the support to the second light receiver; a fluorescence light amount measuring unit comprising a normalizing means for normalizing the amount of the fluorescence obtained by the light receiver with the amount of the excitation light obtained by the first light receiver, the first light receiver and An immunoassay device characterized in that the second light receivers have substantially the same temperature and light receiving sensitivity characteristics.