JPH03120466A - Immunoassay device - Google Patents

Abstract

PURPOSE:To minimize the influence of 4-methyl unbellifer phosphate (MUP) and to improve measurement accuracy by irradiating a base with excitation light of a specific central wavelength. CONSTITUTION:A substrate and a fluorescent material are respectively the 4-methyl umbellifer phosphate (MUP) and 4-methyl umbelliferone (MU) and the base is irradiated with the excitation light having 370 to 375nm central wavelength and <=5nm DELTAlambda(1/10) when the wavelength band to have 1/10 intensity with respect to the central intensity of spectra is designated as DELTAlambda(1/10). The fluorescence emitted from the base is then photodetected via a filter having 460+ or -5nm central transmission wavelength and <=10nm DELTAlambda(1/10), by which the quantity of the prescribed antigens or antibodies in a liquid to be measured is measured. The measurement is made with the high accuracy in this way with substantially no influence of the fluorescence of the 4MUP or further the influence of Raman scattered light.

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 self-adjudication. 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 (antibody) is spotted on the sample to cause an antigen-antibody reaction, and further a labeled predetermined antigen (antibody) is spotted on the sample. ,
Furthermore, a washing solution containing a substrate that is converted into a fluorescent substance by the catalytic action of the label is then flowed over the support to flush out the labeled antigen (antibody) that has not reacted with the antibody (antigen). The above-mentioned fluorescent substance is generated by the labeled predetermined antigen (antibody) reacting with the above-mentioned 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 antigen (antibody) and its 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.

As one of the most suitable substrates and fluorescent substances, 4-methylumbellifer phosphoric acid (hereinafter referred to as r4
It is called MUPJ. ) and 4-methylumbelliferone (hereinafter referred to as r4MUJ) are known to exist.

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 determine the amount of the fluorescent substance produced, excitation light in a predetermined wavelength range is irradiated onto the support on which the fluorescent substance has been produced. This is done by exciting the fluorescent substance and photoelectrically measuring the amount of fluorescence emitted from the fluorescent substance.

(Issues to be Solved by the Invention) In measuring the amount of fluorescence light as described above, the wavelength λ-3 is emitted from a mercury lamp as excitation light for exciting 4MU.
It is considered to use an emission line of 85 nm to measure the fluorescence emitted from 4MU through an interference filter with a center transmission wavelength of λ-450nIl (CLI).
NICAL CHEMISTRY, Vol, 34. 9
．． 1988:172B-1732).

However, in addition to 4MU, 4MUP also exists on the support, and 4MUP itself has fluorescence, so with the combination of the excitation light λ-m385na and the light reception filter λ-450ns, the fluorescence of 4MU that is originally desired to be measured, There is a problem in that it is not possible to sufficiently separate the fluorescence of 4MUPs, which causes measurement errors.

In view of the above circumstances, it is an object of the present invention to provide an immunoassay device that reduces the influence of 4MUP as much as possible and improves measurement accuracy.

(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
When the substrate and the fluorescent substance are 4-methylumbellifer phosphate and 4-methylumbelliferone, respectively, and the wavelength band in which the intensity is 1/10 with respect to the center intensity of the spectrum is Δλ (1/10), The support has a center wavelength of 370 to 375ni and Δλ(1710)
is 5 nm or less, and the fluorescence emitted from the support is received through a filter whose central transmission wavelength is 460±5 na+ and Δλ (1/10) is 10 nm or less. It is characterized in that it is configured to measure the amount of the predetermined antigen or antibody in the liquid to be measured.

(Function) The immunoassay device of the present invention has a center wavelength of 37 cm on the support.
0 to 375 nm, Δλ (1/10) ≦ 5 nm excitation light, and the center transmission wavelength of the fluorescence emitted from the support was 460 ± 5 nap, Δλ (1/10) ≦ 10 nm.
Since the light is received through a filter of 4MU,
It is possible to perform n1 determination with good accuracy while suppressing the influence of P fluorescence.

(Example) Examples of the present invention will be described below with reference to the drawings.

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

FIG. 2(a) is a diagram schematically showing how antibody 2 is immobilized on slide 1, which is an example of a support, and blood containing antigen 3 is poured onto slide 1. It is. Depending on the amount of antigen 3 in the blood, as shown in FIG. 2(b), antibody 2a with which an antigen-antibody reaction has occurred and antibody 2b with which no reaction has occurred are generated, and the amount of antigen 3 in the blood increases. As the number increases, the number of antibodies 2a with which a reaction has occurred increases, and the number of antibodies 2b with which a reaction has not occurred decreases.

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

Thereafter, when a washing solution (aqueous solution) containing 4MUPS is flowed over a complete slide for, for example, 30 seconds, first the unreacted antigen 4a is washed out, and then the labeled antigen 4a is washed out, as shown in FIG. 2(e). 4MUP due to the catalytic action of 4a
5 changes to 4MU6. 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 change in 4MU6 generated over time is determined by measuring the amount of fluorescence emitted from 4MU6, This determines the amount of antigen 3 in the blood.

FIG. 3 is a diagram showing the excitation spectrum and fluorescence spectrum of 4MUP and 4MU.

Graph 41 in the figure. Graph 42 represents the excitation spectrum and fluorescence spectrum of 4MU, respectively.

Also, graph 51. Graph 52 represents the excitation spectrum and fluorescence spectrum of 4MUPs, respectively.

As shown in this figure, 4MUP also has fluorescence, and when measuring the fluorescence of 4MUP, it is possible that the fluorescence of 4MUP is also measured at the same time, in which case a measurement error will be caused by the amount of contamination of the fluorescence of 4MUP.

Table 11 and Table 2 shown below show the experimentally determined 4MU and 4
This is a table showing the amount of fluorescence (relative value) when MUP is separated and excited independently with each excitation wavelength. It is something.

Table 1 However, when the excitation wavelength width is 5 nm, Table 2 As seen in Table 11 above, A/B, that is, 4MU
The ratio of the amount of fluorescence light of 4MU to the amount of fluorescence light of P becomes maximum when the center wavelength of the excitation light is 370 na in both cases where the excitation wavelength width is 5 nap and 3 nm.

It is also possible to reduce the influence of 4MU on the light receiving side. As seen in graph 52 of FIG. 3, the fluorescence spectrum of 4MUP extends to about 470 na+. Therefore, it is possible that light with a wavelength longer than 470no is received, but as the wavelength goes to the longer wavelength side, the intensity of the fluorescence spectrum of 4MU (height on the vertical axis) also decreases, and the amount of received light decreases. 460 due to a possible decrease in /N ratio.
It is best to measure at around na.

FIG. 1 is a diagram showing the configuration of a fluorescence light amount measurement section that mainly determines the fluorescence light amount by Δ or 1 in an embodiment of the immunoassay apparatus of the present invention.

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 taken out via the pulp 14. A predetermined amount is deposited on the slide 1 via the nozzle 10 (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).
e), see (d)). Furthermore, after that, a fixed amount of a washing solution containing 4-methylumbellifer phosphate (4MUP) as a fluorescent substrate is poured onto the slide 1 for 30 seconds by a similar spotting means (not shown) (Fig. 2(e)). reference). Stop the cleaning solution after 30 seconds, then 10 seconds.
4MUP with the above enzyme label for 5 minutes at second intervals.
The amount of 4-methylumbelliferone (4MU) that has changed is measured.

The fluorescence light amount measuring section 20 uses a lamp 21 equipped with an elliptical mirror 21a as a light source. As this lamp 21, for example, a Xe lamp or the like that emits light having a continuous spectrum is used. The light 22 emitted from the lamp 21 passes through the slit 23. Lens 24. Interference filter 25. Parallel plane substrate 26. Slide 1 from below via lens 27
irradiate. The interference filter 25 has a curve 61 (
(The vertical axis in FIG. 3 represents relative values), the center wavelength λ of the light 22 emitted from the lamp 21 is 370~
375 na and 11 to the center intensity of the spectrum.
It allows only the light whose wavelength band Δλ (1/10), which has an intensity of 10, is 5na or less to pass through.
The MU is excited by this excitation light.

The light reflected by the parallel plane substrate 2B is received by the first light receiver 29 via the lens 28, and the amount of excitation light is monitored. This monitored signal S1 is transmitted to the light source power supply unit 3.
0 and the applied voltage is controlled so that the lamp 20 always emits a constant amount of light. Moreover, the signal S1 is
It is also input to the signal processing section 31.

When slide 1 is illuminated with λ-370n excitation light,
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 has a transmission wavelength band shown by curve B2 shown in FIG. 1, and has a narrow wavelength band (Δλ(
1710)≦10no+) is transmitted.

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 31
As mentioned above, the signal S obtained by the first photoreceiver 29 is
1 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 amount of antigen determined is displayed on the display 36.

In the above embodiment, the slide 1 is irradiated with light that has passed through an interference filter with a center transmission wavelength λ-370 ns represented by the curve 61 in FIG. 3, and the fluorescence emitted from the slide 1 is Since the measurement is performed through an interference filter with a center transmission wavelength λ-48On- represented by the curve 62, the fluorescence of 4MUP is removed to an almost negligible extent, making it possible to accurately measure the amount of fluorescence of 4MU. can.

FIG. 4 is a diagram showing the Raman scattered light spectrum of water. The numerical value written at the top of each graph represents the wavelength of light irradiated onto water, and each graph represents the spectrum of Raman scattered light emitted from water when irradiated with light of that wavelength.

Raman scattering is the process of converting monochromatic light into a substance (here, cleaning liquid (water)).
) is a phenomenon in which scattered light with a slightly different wavelength from the incident light is mixed into the scattered light.

The spectrum of Raman scattered light generated by the cleaning solution (water) when slide 1 is irradiated with excitation light with a center wavelength of 370 nm is shown in graph 71 in FIG.
3 (see FIG. 1) is centered around 480 na+ as shown by the curve 62 in FIG. It has no effect on the

In the above example, the center wavelength of the excitation light is λ-370ns.
The center wavelength of fluorescence reception is λ-480 nm.

Furthermore, when a continuous spectrum light source and an interference filter are used, the transmission spectrum width of the interference filter has a center wavelength of λ-385n, which is the center wavelength of the bright line spectrum of the mercury lamp.
limited by m. In this case, consider the case where the center wavelength of the transmission spectrum of the interference filter is λ-370 nm. In other words, if there is no overlap between the transmission spectrum of the interference filter and the emission line spectrum of the mercury lamp, 4MU is lower than when the emission line spectrum of the mercury lamp is used for excitation.
The influence of P can be reduced. The emission line spectrum width of a mercury lamp is Δλ(1/10)−2 to 3 nm, and in order to eliminate overlap with this, the transmission spectrum width of the interference filter must be Δλ(1/10)≦5na+. It is enough.

Furthermore, the transmission spectrum width of the interference filter on the fluorescence receiving side is limited by the Raman spectrum of the cleaning liquid. In this case, consider the case where the center wavelength of the excitation light is λ-375na. According to FIG. 4, when the center wavelength of the excitation light is 375 nm, the Raman spectrum has a tail up to around 445 nm. Therefore, if the center wavelength of fluorescence reception is the shortest λ-445ni, the transmission spectrum width of the interference filter for fluorescence reception is Δλ (1/10)≦10
nm is sufficient.

Furthermore, although the above embodiments were explained using a sequential reaction method, the present invention is also applicable to the Sand-Deutsch method, competitive method, and the like.

(Effects of the Invention) As explained in detail above, the immunoassay device of the present invention has the following features:
The support has a center wavelength of 370 to 375 nm, Δλ (1/1
0) Excitation light of ≦5na was irradiated, and the center transmission wavelength of the fluorescence emitted from the support was 460±5nap.

Since the light is received through a filter with Δλ(1/1G)≦1Ona+, it is possible to perform highly accurate measurement with almost no influence of fluorescence from 4MUPs and almost no influence of Raman scattered light.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of a fluorescence light amount measuring section that mainly measures the fluorescence light amount in an embodiment of the immunoassay device of the present invention. FIG. 2 is a diagram showing the measurement principle of the immunoassay device of the present invention. Figure 3 is a diagram showing the excitation spectrum and fluorescence spectrum of 4MUP and 4MU, and Figure 4 is a diagram showing the Raman scattered light spectrum of water. 1... Slide 2... Antibody 3... Antigen 4... Labeled antigen 5... 4MUP (
4 methyl umbellifer phosphate) 6...4 MU (4 methyl umbelliferone) 10...
13...Blood 20...Fluorescence light amount measuring section 21...
・Low pressure mercury lamp 25, 33...Interference filter 29
...First light receiver 35...Second light receiver 30
...Light source power supply section 31...Signal processing section 3B.
...Display 40...Incubator (d) (e) (f) 6゜ Subject of amendment January 2, 1999 "Detailed description of the invention" column of the specification Mr. Bunki Furuta, Commissioner of the Japan Patent Office 7゜Date of amendment (1) Page 17, line 13 of the specification, "λ-445nm J" is corrected to "λ-455nm J." 2゜Name of the invention Immunoassay si is 3゜Relationship with the case of the person making the amendment

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 substrate and the The fluorescent substances are 4-methylumbellifer phosphate and 4-methylumbelliferone, respectively, and when the wavelength band in which the intensity is 1/10 of the center intensity of the spectrum is Δλ (1/10), The center wavelength is 370 to 375 nm and Δλ (1/10)
is 5 nm or less, and the fluorescence emitted from the support is received through a filter whose central transmission wavelength is 460±5 nm and Δλ (1/10) is 10 nm or less. An immunoassay device characterized in that it is configured to measure the amount of the predetermined antigen or antibody in the liquid to be measured.