JP2728796B2 - Chemiluminescence measuring method and measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring chemiluminescence of a biological substance, a chemical substance and the like in a clinical test, a biochemical experiment, a physicochemical experiment and the like.

[0002]

2. Description of the Related Art In recent years, many methods have been put to practical use for quantifying trace substances such as antigens, antibodies, and hormones in blood by utilizing reactions performed in a living body such as an antigen-antibody reaction and a hormone-receptor reaction. I have. The most common of them are a radioimmunoassay (RIA) and an enzyme immunoassay (EIA) utilizing an antigen-antibody reaction. The radioimmunoassay has a measurement sensitivity of ng / ml to p.
It has advantages such as extremely high g / ml and is hardly affected by coexisting substances. On the other hand, the use of radioisotopes (radioisotopes, hereafter referred to as RI) requires costs for management facilities, waste disposal, measurement equipment, etc., and the short life of RI leads to short expiration of reagents. Have. Therefore, as a method for compensating for the drawbacks of RIA, EIA using an enzyme instead of RI was developed. EIA has spread rapidly due to the advantage of non-radioactive measurement, but since the detection method used to be mainly colorimetric,
The practical sensitivity was inferior to RIA. However, chemistry,
By using bioluminescence (hereinafter, both are referred to as chemiluminescence in the present specification), sensitivity equivalent to that of RIA can be obtained.
Recently it has been attracting attention. On the other hand, chemiluminescence can be measured not only by enzyme immunoassay but also by H ₂ O ₂ , ATP, and NADP.
It is also applied to the quantification of biological substances that directly act on luminescent substances.

As a method of measuring chemiluminescence, a method of taking a chemiluminescence with a polaroid photograph (for example, Japanese Patent Laid-Open No.
No. 8-38842) and a method using a luminometer which detects with a photomultiplier tube (for example, Japanese Patent Application Laid-Open No. 61-2093).
No. 40). The method using a polaroid photograph has the advantage that a plurality of light emitting sites can be measured at the same time without the need for a special device, but has the disadvantage that it takes a long time to take a picture and the measurement range is limited by photographic sensitivity. on the other hand,
The method using a luminometer directly converts luminescence into an electric signal, so it can be measured with high quantitativeness over a wide range. However, when a cell is used for measurement, measurement is performed for each sample (one luminescent site). For this purpose, a large cell moving device and a dispensing device are required. In addition, since the light emission intensity changes with time, quick measurement is required to accurately measure light emission sites that are simultaneously emitting light. Thus, conventionally, multiple
There has been no method and apparatus that can quickly and accurately measure a specimen having a light emitting site that emits light simultaneously .

[0004]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problem of the chemiluminescence measurement, and is directed to a sample having a plurality of simultaneously emitting light-emitting portions or a multi-sample simultaneously emitting light. And a method and apparatus for measuring the intensity of each chemiluminescence quickly and accurately.

[0005]

That is, the present invention provides a plurality of
Analyte or simultaneous onset having a light emitting part that simultaneously emission of
A method for measuring the emission intensities of the light emitting part of the multi-analyte being light, arranged in advance emitting site, along thereon
While moving the light measuring unit at a constant speed, the light emission intensity is continuously measured, and a continuous waveform graph of the light emission intensity is obtained, and the light emission intensity of each light emission portion is obtained with a peak value corresponding to each light emission portion of the waveform graph. And a method for measuring chemiluminescence.

Further, the present invention is an apparatus for measuring the emission intensities of the light emitting part of the multi-analyte that analyte or simultaneous emission having emitting <br/> light portion have multiple simultaneous emission, beforehand
The light-emitting portions are arranged , and a light-metering section that moves at a constant speed along the light-emitting portions and continuously measures the light-emitting intensity, obtains a continuous waveform graph of the light-emitting intensity, and a peak value corresponding to each light-emitting portion in the waveform graph. The present invention relates to a chemiluminescence measuring apparatus having a data analyzer for analyzing the light emission intensity of each of the light emission sites.

First, the measuring method of the present invention will be described in detail. In the present invention, examples of the sample having a plurality of light- emitting sites emitting at the same time include various ones. Mast immunosystems (manufactured by US Mast Immunosystems, Inc. Measurement of the light emission of each thread 14 of the measurement container (FIG. 4). This is an allergy diagnostic agent and is disclosed in Japanese Patent Application Laid-Open No. 58-50163.
No. 7, Japanese Patent Application Laid-Open No. 60-89553, Japanese Patent Application Laid-Open No.
The details are described in JP-A-82165 and JP-A-62-22770. As shown in FIG. 4, various kinds of allergy-specific IgE antibody quantification reagents are contained in a plastic container into which serum or a luminescence reagent is injected. Each thread that binds each allergen (cause of allergy) is arranged. When the serum of an allergic patient is injected into this container, the specific antibodies in the serum bind to the allergen. In this method, the complex is reacted with an enzyme-labeled secondary antibody (IgE), and then chemiluminescence is induced by a combination of luminol and hydrogen peroxide, and the amount of antibody in the serum is determined by measuring the amount of luminescence. Heretofore, the measurement of the light emission amount has been conventionally performed by exposure to a polaroid film and measurement of the transmittance voltage of the obtained film, which required a long time for exposure and low measurement accuracy.

[0008] In addition, the method of the present invention is particularly effective in the Southern blotting method in which a DNA fragment or RNA cleaved with a restriction enzyme is electrophoresed, transferred to a membrane such as cellulose, and a complementary sequence is examined with a DNA probe. In some cases, chemiluminescence is used as a Northern blotting method, or a western blotting method in which a protein is electrophoresed, the protein is transferred to a membrane such as cellulose, and an antigen is searched for with a specific antibody. All of these can be obtained by moving one-dimensionally as described later, but measurement can also be performed by moving two-dimensionally described later.

In the method of the present invention, the above-mentioned sample is set in a measuring device and the luminescence intensity is measured. The set sample needs to be completely shielded from external light at the time of measurement. The measuring unit of the measuring device is moved at a constant speed in a constant direction over the light emitting portion of the set sample, and the light emitting intensity is continuously measured. Thereby, as shown in FIG.
The emission intensity graph becomes a waveform graph with a fixed period,
Waveforms can be clearly distinguished from each other,
The waveform graph can be easily identified and identified. Here, moving the photometric unit at a constant speed and continuously measuring the light emission intensity means that the light intensity is stopped at a pitch of about 0.05 mm to 0.5 mm in order to capture the data of the light emission intensity as a digital signal, and the data is stopped. This includes a case where the taking and moving are continuously repeated, that is, a case where the object appears to have a constant speed as a result. The method of the present invention also includes a case where the measurement unit is fixed and the sample is moved at a constant speed. This is because there is no difference in the relative operation of the measurement unit and the sample. In addition, in order to remove the effects of noise in the electrical signal, smoothing processing is performed using a moving average method that averages the measured values of several signals before and after the signal, a weighted moving average method submitted by Zarbic Golay, etc. You can also

In the method of the present invention, in the case of a specimen having a plurality of luminescent sites on a straight line (such as cellulose in the above-mentioned mast immunosystems or blotting method),
The measurement is performed by moving the photometric unit once along the linear direction in which the light emitting portion exists, continuously measuring the light emission intensity, obtaining a waveform graph, and obtaining a peak value corresponding to each light emitting portion of the obtained waveform graph. The light emission intensity of the light emitting portion can be obtained (one-dimensional movement ). This means that a plurality of specimens can be arranged on a straight line, for example, the luminescence intensity in a well of a microtiter plate can be similarly measured. In these cases, if the plurality of light emitting sites are arranged in a straight line or a plurality of specimens arranged in a straight line, and a plurality of the light emitting sites are further arranged in parallel, the movement is measured in a direction perpendicular to the scanning direction. It is also possible to measure all at once by moving the part or sample once and repeating it (two-dimensional movement) .

[0011]

The luminescence reactions useful for the measurement method of the present invention include luminescence reactions based on chemiluminescence, so-called chemiluminescence, and bioluminescence, so-called bioluminescence. As an analysis method, a chemiluminescent immunoassay in which an antigen or antibody is labeled with a chemiluminescent substance to perform an antigen-antibody reaction to detect luminescence, or an antigen or antibody reaction in which the antigen or antibody is labeled with an enzyme such as peroxidase to perform chemiluminescence. Chemiluminescent enzyme immunoassay for detection, bioluminescent enzyme immunoassay for labeling an antigen or antibody with a bioluminescence-related enzyme and performing an antigen-antibody reaction to detect luminescence, labeling the antigen or antibody with a bioluminescence coenzyme Various immunoassays such as a homogeneous bioluminescent cofactor immunoassay for detecting an inactivation of coenzyme by bioluminescence by performing an antigen-antibody reaction, and DNA or RN having a base sequence complementary to the target DNA or RNA.
A is labeled with a chemiluminescent substance or enzyme and the target DNA or RN
A that forms a hybrid with A and detects this by emission D
There is an NA probe method and the like.

The chemiluminescent substance used in the present invention includes luminol (5-amino-) which emits light by reacting with hydrogen peroxide.
2,3-dihydro-1,4-phthalazinedione) and isoluminol and its derivatives, lucigenin (10,10 ′)
-Dimethyl-9,9 'biacridium dinitrate), acridium salts and esters thereof,
-Dioxetane, indole derivatives such as lophine (2,4,5-triphenylimidazole) and 3-methylindole which emit light by oxygen in the presence of a strong base, tetrakis (dimethylamino) ethylene and sidimethyl which emit light when exposed to oxygen or air There is a Schiff base that emits light in the presence of t-butyl in a formamide solution. Among them, a system using peroxidase in the presence of luminol or isoluminol and hydrogen peroxide is most often used. There is also a method in which a fluorescent substance such as perelin, 8-anilinonaphthalene-1-sulfonic acid, or dansyl amino acid coexists with an oxalic acid derivative such as an oxalic acid diester to excite the fluorescent substance to emit light.

The bioluminescent substance used in the present invention includes luciferases such as fireflies and luminescent bacteria, and various photoproteins such as aequorin obtained from Oan jellyfish. These luminescent substances can be used together with a component for amplifying luminescence, that is, an enhancer. These enhancers include phenol derivatives such as 6-hydroxybenzothiazole and iodophenol.

Next, a chemiluminescence measuring apparatus of the present invention for carrying out the chemiluminescence measuring method of the present invention will be described in detail.
The measuring device of the present invention arranges the luminescent sites in advance, and
A light meter that moves at a constant speed along the top and continuously measures light emission intensity, obtains a continuous waveform graph of light emission intensity, analyzes peak values corresponding to each light emission portion of the waveform graph, and analyzes each light emission portion. And a data analysis unit for setting the light emission intensity.

One example of the photometric unit is shown in FIG. 1 (perspective view) and FIG. 2 (side sectional view). In FIG. 1, 105 dedicated reaction vessels from Mast Immunosystems are fixed on a fixed base 11 and placed on a plate holder 12 as samples. As shown in FIG. 1, the photometric unit 1 includes lenses 2 and 3, slits 4 and 5 elongated in a direction perpendicular to the moving direction, a photomultiplier tube 8, and an amplifier 7. The photometric unit 1 moves at a constant speed in the X direction shown in the figure along the guide rod 9 and can continuously measure the emission intensity. Also,
The plate holder 12 can move in the Y direction shown in the drawing. As shown in the cross-sectional view of FIG. 2, the emitted light enters from the lower surface of the photometric unit, is reflected via the reflection mirror 6 toward the photomultiplier tube 8, and is converted into an electric signal.

The light intensity measured by the photometric unit 1 and converted into an electric signal is obtained by a data analysis unit by obtaining a continuous waveform graph of the light emission intensity and analyzing the peak value corresponding to each light emitting portion of the waveform graph. It is converted into the luminous intensity of each luminous site. A microprocessor is used as the data analysis unit. FIG. 3 shows a schematic diagram of an example of the measuring device of the present invention. Light from the sample enters the photomultiplier tube via a reflection mirror, is taken into a microprocessor via an amplifier and an A / D converter, is processed, and is output to a printer. On the other hand, the microprocessor also controls the drive motor of the photometer and the plate holder. In particular, the photometric unit fetches data of a predetermined number of samplings, and then minutely feeds the photometric unit to the next position.

According to the method and apparatus for measuring chemiluminescence of the present invention as described above, for example, when the sample is a multi-sample in a well of a microplate, the influence of adhesion of a luminescent substance, non-uniformity of sedimentation, etc. And when mast immunosystems is used as the sample, highly accurate data can be obtained regardless of the variation in the interval between the yarns in the plastic container. In addition, the measurement is completed in a short time because the photometry unit moves at a constant speed, as compared with the case where the photometry unit stops at the light emitting portion once and performs the measurement.

[0019]

Examples of the present invention will be described below. In the present example, the photometric unit shown in FIG. 1 was used, and the chemiluminescence measuring device shown in FIG. 3 was used. The sample to be measured was prepared by reacting a sample serum and enzyme-labeled antibody in accordance with the reaction vessel of the allergy diagnostic agent Mast Immunosystems (Mast Immunosystems, USA, imported from Hitachi Chemical Co., Ltd.) (FIG. 4, instruction manual for the product). After reaction, filled with luminol solution for chemiluminescence)
Was used as shown in FIG. 1 so that five samples were measured at one time. The actual movement and measurement of the photometric unit is 0.1
By repeating the measurement of the luminescence intensity each time the sample was moved at an interval of 88 mm and stopped, the luminescence intensity at about 1,000 points was continuously measured for one reaction vessel. As shown in FIG. 1, when the light intensity was measured by moving the photometric unit along the plastic container (X direction), a waveform graph as shown in FIG. 5 was obtained. The scanning time per line was 20 seconds. This is numerically analyzed with a microprocessor,
Each peak value was assigned to the emission amount of each allergen, and the results shown in Table 1 were obtained.

[0020]

[Table 1]

The yarns in the plastic containers used in the examples are arranged at intervals of about 2.8 mm. When the variation in the intervals was closely examined, the maximum was about ± 0.3 mm. Thus, even if there is an error in the dimensional accuracy, the present invention obtains the light emission amount by calculation from the peak value, so that highly accurate data can be obtained.

[0022]

According to the method and apparatus for measuring chemiluminescence of the present invention, the intensity of each chemiluminescence of a sample having a plurality of simultaneously emitting luminescent sites or a plurality of samples simultaneously emitting light can be measured quickly and accurately. Can be measured.

[Brief description of the drawings]

FIG. 1 is a perspective view showing a main part of an example of a chemiluminescence measuring apparatus of the present invention.

FIG. 2 is a side sectional view showing a main part of one example of the chemiluminescence measuring apparatus of the present invention.

FIG. 3 is a schematic view showing one example of a chemiluminescence measuring apparatus of the present invention.

FIG. 4 shows a plurality of useful chemiluminescence measurement methods of the present invention.
It is a perspective view which shows the sample (reaction container of Mast Immunosystems) which has the light emission part which emits simultaneously .

FIG. 5 is a waveform graph of emission intensity measured in the example of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Photometry part 2 ... Lens 3 ... Lens 4 ... Slit 5 ... Slit 6 ... Reflection mirror 7 ... Amplifier 8 ... Photomultiplier tube 9 ... Guide rod 10 ... Dedicated reaction vessel 11 ... Fixed table 12 ... Movable plate holder 13 ... Plate guide 14 ... thread

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yutaka Kimura 3517 Higashiishikawa Katsuta-shi Ibaraki Pref.Corona Electric Co., Ltd. (72) Inventor Tsutomu 3517 Higashiishikawa Katsuta-shi Ibaraki Pref.Corona Electric Co. (56) References JP-A-2-268256 (JP, A) JP-A-2-28772 (JP, A)

Claims (5)

(57) [Claims] 1. A method for measuring the luminous intensity of each luminous site of a sample having a plurality of luminous sites emitting at the same time or a plurality of analytes emitting at the same time , comprising:
While arranging , moving the photometric unit at a constant speed along it , continuously measuring the light emission intensity, obtaining a continuous waveform graph of the light emission intensity, and having a peak value corresponding to each light emitting portion of the waveform graph. A method for measuring chemiluminescence, wherein the luminescence intensity of each luminescence site is used. 2. It has a plurality of light-emitting portions which emit light simultaneously.
Of each luminescent site of a sample or multiple samples that emit light simultaneously
This is a method for measuring light intensity, in which light emitting sites are arranged in a straight line in advance , and the photometric unit is moved at a constant speed along the light emitting sites.
As well as continuously measuring the light emission intensity,
Degree continuous waveform graph, and each light emitting part of the waveform graph
With the peak value corresponding to the position,
A method for measuring chemiluminescence. 3. An apparatus for measuring the luminous intensity of each luminous part of a sample having a plurality of luminous parts emitting at the same time or of multiple specimens emitting at the same time , wherein the luminous part is determined in advance.
Array , move along it at a constant speed, and continuously measure the light emission intensity, obtain a light measurement unit, obtain a continuous waveform graph of the light emission intensity, and analyze the peak value corresponding to each light emitting part of the waveform graph. A chemiluminescence measuring device comprising a data analysis unit for setting the luminescence intensity of each luminescence site. 4. It has a plurality of light emitting parts which emit light simultaneously.
Of each luminescent site of a sample or multiple samples that emit light simultaneously
This is a device for measuring light intensity.
Arrange on a straight line, move at a constant speed along it and emit light
Metering unit that continuously measures the intensity, continuous wave of emission intensity
A shape graph is obtained, and the peaks corresponding to each light emitting portion of the waveform graph are obtained.
Data solution by analyzing the peak value and obtaining the luminous intensity of each luminous site
A chemiluminescence measuring device comprising a analyzer. 5. A photometric portion, the lens, the moving direction and the direction perpendicular to the long slit, chemiluminescence measuring device according to claim 3 or 4 characterized by having a photomultiplier tube and amplifier.