JPH10318929A - Method for measuring emission reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently measure an emission amount, by adding an emission stopper to a container completed to be measured, stopping an emission and then measuring the emission of a next container. SOLUTION: A detector 5 is moved over a sample well on a microplate 6. A constant amount of an emission initiator is guided into the sample well by a tube 4 from a container 1 storing the emission initiator, whereby an emission reaction is brought about. An emission amount for a predetermined time is measured. After the measurement, a constant amount of an emission stopper is introduced by the tube 4 into the sample well from a container 2 storing the emission stopper, whereby the emission is stopped. A next sample well is measured afterwards. The tube 4 may be provided with a pump 3 for sending the constant of the emission initiator or emission stopper to the sample well. In this method, a crosstalk between adjacent samples is eliminated and light condensation efficiency is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for eliminating crosstalk in a system for detecting a component in a sample using a luminescence reaction.

[0002]

2. Description of the Related Art An immunoassay utilizing an antigen-antibody reaction has been widely adopted as a method for measuring a physiologically active substance present in a living body in a measured amount. In particular, radioimmunoassay (RIA) has been rapidly developed because of its high sensitivity, high specificity, and simple operation. However, regarding the handling of radioactive materials used for labels, there are various restrictions such as equipment and facilities, and disposal problems after use,
Various non-radioactive immunoassays using a non-radioactive substance such as an enzyme, a fluorescent dye or a chemiluminescent substance for labeling instead of a radioactive substance have been developed and put into practical use. Among them, the chemiluminescence immunoassay has been particularly noted as having a detection sensitivity comparable to that of RIA.

At present, various improvements have been made to improve the reproducibility by full automation and to efficiently detect even weak light emission, and to enhance and stabilize the light emission reaction for a long time. Luminescent reagents have also been developed and are commercially available.

[0004]

By the way, in order to analyze a plurality of samples efficiently, a plurality of containers accommodating each measurement sample are prepared, and after the reaction, the luminescence amount of each container is measured with a time difference. Measurement methods are widely used. Generally, in the measurement by the conventional method, a 96-well microplate having a shape in which reaction vessels are arranged in 8 rows × 12 rows is used, and individual reaction vessels are used in a fully automatic measuring device.

[0005] In order to improve the performance of luminescence determination, it is necessary to construct a measurement system capable of detecting slight luminescence. For this purpose, various devices have been devised for the reaction vessel.

For example, as a method for efficiently detecting weak light with the 96-well microplate described above, there is a method of changing from a conventional black plate to a white plate.
This allows the light that has been absorbed in the well to be reflected and reaches the detector, so that weak light emission can be measured efficiently. However, since the light is easily transmitted in the white well, when the sample in the next well is measured with the sample in the next well emitting strong light,
The amount of light emission may be added (crosstalk) due to the influence of light leakage, which causes a problem in the accuracy of light emission measurement. This is particularly problematic in a light emitting system having a long light emitting life. To solve this, it is necessary to take measures such as thickening the walls of the wells, covering the sides of the wells with a light-blocking material such as a light-blocking paint or a metal material, and devising a structure. Merchandise is also commercially available, but none are perfect, so black plates are still often used.

[0007] Further, even when individual reaction vessels are used, the same problem occurs depending on the material and structure of the vessels. In addition to the above-mentioned measures, it is necessary to separate the sample to be measured from other samples and to shield light. A possible measure is to design around the detector. However, in this case, the design takes time,
It is expected that development will be difficult.

The present invention has been made in view of the above circumstances, and provides a simple method for eliminating crosstalk, thereby enabling the use of a reaction vessel capable of efficiently measuring the amount of luminescence, and the detection sensitivity. The purpose is to improve.

[0009]

According to the present invention, there is provided a method for measuring a luminescence reaction in which a target component in a measurement sample is sequentially detected by a luminescence reaction using a reaction container which individually accommodates a plurality of measurement samples. The method is characterized in that the luminescence is stopped by adding a luminescence terminator to the container after the measurement, and then the luminescence of the next container is measured.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention is particularly useful in a chemiluminescent immunoassay. Chemiluminescence reactions used for detection include luminol or isoluminol derivative-hydrogen peroxide, acridinium ester derivative-hydrogen peroxide, NADH-electron carrier-isoluminol-microperoxidase (m-POD), lucigenin-reduction And adamantyl dioxetane derivatives.

[0011] In addition, the reaction system is not particularly limited as long as the reaction system can arbitrarily stop the luminescence reaction.

[0012] The luminescence terminator can be appropriately selected according to the system in which the reaction is carried out. For example, for a substance that emits light in an alkaline state such as lucigenin, the light emission can be stopped almost instantaneously by adding an acid such as sulfuric acid.

To carry out the present invention in a chemiluminescence immunoassay, first, an antibody or an antigen directly labeled with a chemiluminescent compound is used, and an antigen-antibody reaction is performed by a competition method or a sandwich method. Next, B / F separation is performed, and the chemiluminescent compound remaining on the solid phase is detected by the respective chemiluminescent reactions exemplified above. In addition, an enzyme can be used as a label. In this case, a substrate is added to the enzyme remaining on the solid phase to cause chemiluminescence. Enzymes used as labels include horseradish peroxidase, glucose oxidase, β-D-galactosidase, dehydrogenase, alkaline phosphatase, invertase and the like.

These luminescence reactions are performed for each container, and the amount of luminescence is measured.
After measuring the light emission from the container for a predetermined time, the reaction stop agent is added to the container to stop the light emission, and then moved to the next container, a light emission initiator is added, and the light emission amount is measured for a predetermined time. By doing so, the light emission itself of the adjacent well disappears, so that light does not leak and the light emission amount can be measured without crosstalk.

To carry out the measuring method of the present invention, for example, FIG.
An apparatus as shown in FIG. The detector 5 is moved over the sample well to be measured on the microplate 6 (or the microplate 6 is moved below the detector 5), and the tube 1 is transferred from the container 1 containing the luminescence initiator.
A certain amount of a luminescence initiator is introduced into the sample wells to start the luminescence reaction, and the luminescence amount for a predetermined time is measured. After the measurement, a certain amount of the issuance terminator is introduced into the sample well from the container 2 containing the issuance terminator through the tube 4, and the light emission is stopped. After that, the process proceeds to the measurement of the next sample well. The tube 4 may include a pump 3 for introducing a certain amount of a luminescence initiator or a luminescence terminator into the well. Also,
As shown in FIG. 2, a lens 8, a photomultiplier tube 7, a well lid 9, and a reagent such as a luminescence initiator and an emission stop agent are introduced into the well of the detector 5, as shown in FIG. And a reagent dispensing port 10 for performing the same.

Next, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples. .

Lucigenin (bis-N-methylacridinium nitrate) emits light under alkaline conditions, and its emission is significantly enhanced by the presence of a suitable oxidizing agent and reducing agent.
As a measurement sample, a strong luminescence sample in which luminescence of lucigenin was enhanced by cortisol was used, and it was examined how much the luminescence leaked to an adjacent well (which should not emit light). We decided to check the effect of disappearing.

Specifically, the operation was performed as follows.

Example White plate with crosstalk (LUMINO, Labsystems)
PLATE FB, EM) and black plate without crosstalk (La
bsystems FLUOROPLATE FB, EB) was used. In the center of five consecutive wells, a strong luminescent sample (50 μl of 1.0 × 10 ⁻⁴ M cortisol + 8.0 × 10 ⁻⁴ % lucigenin 50)
μl), and in the other four wells, a non-luminescent sample (100 μl of water) was added.
(LUMSOKAN, ystems).

First, measurements were made on white and black plates without addition of H ₂ SO ₄ used as a luminescence initiator. Move the detector to the first well and add 1N KO
H was dispensed in an amount of 50 μl to make it alkaline, and light emission was started, and the total amount of light emission for 5 seconds from 20 seconds after dispensing was determined. After that, the detector was moved to the next well, and the second to fifth wells were similarly measured sequentially.

Next, in the white and black plates, H
It was measured for the case of ₂ SO ₄ addition. The detector was moved to the first well, 50 μl of 1N KOH was dispensed to make it alkaline, and luminescence was started, and the total amount of luminescence for 5 seconds from 20 seconds after dispensing was determined. Next, 1N H ₂ SO ₄ was added to 50
The solution was neutralized by dispensing μl to stop luminescence. After that, the detector was moved to the next well, and the second to fifth wells were similarly measured sequentially.

Since the third intense luminescent sample is measured, crosstalk should be detected fourth.
The first, second and fifth samples were measured to determine the blank level.

Results The results are shown below. Table 1 shows the average of three tests.

[0024]

[Table 1]

When FLUOROPLATE (black plate) is used, no crosstalk is observed for the fourth sample regardless of the presence or absence of the luminescence terminator (H ₂ SO ₄ ).

It can be seen that when LUMINOPLATE (white plate) is used, the amount of light emission is higher than that of FLUOEOPLATE, and the effect of collecting light is higher. However, without the quencher, the luminescence of the fourth sample was 2.88, and 1, 2,
The light emission amount is clearly higher than the light emission amount of the fifth non-emission sample, which indicates that there is crosstalk.

Here, a measurement was carried out to stop the light emission of the intensely luminescent sample by adding a luminescence terminator. The luminescence of the fourth sample was 1.06, and the luminescence of the other non-luminescent sample was The value has not changed.

As described above, it was confirmed that the addition of the luminescence terminator enables the measurement of the luminescence amount without crosstalk using a white plate having a high light-collecting effect.

[0029]

According to the present invention, since the crosstalk between adjacent samples can be eliminated, it is possible to employ a reaction vessel having good light-collecting efficiency. This makes it possible to increase sensitivity while maintaining accuracy.

[Brief description of the drawings]

FIG. 1 shows an example of a measuring apparatus used for the measuring method of the present invention.

FIG. 2 shows a partial cross-sectional view of a measuring device used in the measuring method of the present invention.

[Explanation of symbols]

 1: luminescence initiator 2: luminescence stop agent 3: pump 4: tube 5: detector 6: well of microplate 7: photomultiplier tube 8: lens 9: well lid 10: reagent dispensing port 11: sample 12: micro Plate wall / bottom

Claims (1)

[Claims] 1. A method for sequentially detecting a target component in each measurement sample by a luminescence reaction using a reaction container that individually stores a plurality of measurement samples, wherein a luminescence terminator is added to the container after the measurement. A luminescence reaction measuring method, wherein the luminescence of the next container is measured after the luminescence is stopped.