JP4067208B2 - Biological specific reaction measuring method and apparatus - Google Patents

Description

【図面の簡単な説明】
【図１】本発明に係る装置の一例を示す概略図である。図中、（Ａ）は側面概略図、（Ｂ）は平面概略図、（Ｃ）は（Ｂ）のＸ−Ｘ線断面概略図、（Ｄ）は（Ｂ）のＹ−Ｙ線断面概略図である。
【図２】図１の装置の使用状態を示す断面概略図である。
【図３】本発明に係る装置の他の例の使用状態を示す断面概略図である。
【符号の説明】
１ コンテナー
２−１〜２−５ 吸収材
３ 仕切り板
４ 蓋体
５ 開口部
６ 反応マトリックス
７ 反応マトリックス保持部材
８ 溝
９ 突起部材
10 前処理フィルター
11 フィルター保持部材[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and an apparatus for measuring a substance in a sample by a biological specific reaction. Such a measuring method and apparatus are used especially in fields such as clinical examinations and food inspections.
[0002]
[Prior art]
A biological specific reaction measuring method for measuring a substance to be measured by a flow-through type using a reaction solid phase holding a ligand capable of specifically capturing the substance to be measured, for example, a porous matrix, is disclosed in JP-A-61-502214. No. 4, JP-A-4-318462, JP-A-6-50973, and the like. As an apparatus used for such a measurement method, a porous material in which an absorbent material is usually contained in a container having an upper opening and a ligand that specifically reacts with a substance to be measured is immobilized above the absorbent material. A device provided with a matrix is used. For example, when the substance to be measured is an antigen, a measurement substance (that is, an antigen) solution is added to the upper opening, and the ligand (that is, specifically reacts with the substance to be measured) is measured. , Antibody) is passed through the immobilized porous matrix to capture the antigen. Next, an antibody solution to which a label (for example, an enzyme) is bound is added to the upper opening so that the antibody binds to the antigen captured on the matrix, and a signal based on the label (for example, luminescence, radiation) For example, the antigen is measured. Each liquid that has passed through the porous matrix is absorbed by the absorbent material.
[0003]
[Problems to be solved by the invention]
A biological specific reaction measurement method for measuring a substance to be measured in a flow-through type using a reaction solid phase (hereinafter referred to as a reaction matrix for convenience) holding a ligand capable of specifically capturing the substance to be measured. According to this, it is possible to easily measure the substance to be measured. However, a signal based on the label (non-specific signal) is also generated in the absorbent material in the container. For this reason, when measuring the signal of the reaction matrix, there is a problem that the signal of the reaction matrix cannot be measured with high accuracy due to the influence of a non-specific signal from the absorbent material. As a means for solving such a problem, for example, a device equipped with a transmitted light reducing element between the absorbent and the reaction matrix is proposed for the purpose of reducing non-specific light signals transmitted from the absorbent. (JP-A-8-506420). According to such an apparatus, the transmitted light from the absorber can be reduced to some extent, but it has been difficult to completely eliminate the influence.
[0004]
In order to solve such a problem, the present inventors, in a method for measuring a substance to be measured using a flow-through type biological specific reaction measuring device using a reaction matrix, As a result of examining methods that can eliminate the influence of the signal, after reacting the labeling substance, the reaction matrix is moved, and a signal generating substance solution (for example, a substrate solution for enzyme labeling) on the absorbent that does not absorb the labeling substance By adding, it is possible to prevent the generation of a non-specific signal in the absorbent material, and it is possible to measure the signal of the reaction matrix alone, and thus the present invention has been completed by finding that more sensitive measurement can be performed.
The present invention has been made on the basis of such knowledge, and the present invention relates to a flow-through type biological specific reaction measuring method and apparatus for components in serum, plasma and other body fluids, which are usually performed in the field of clinical examination. Provides a measurement method and a measurement apparatus that can eliminate the influence of non-specific signals from the absorbent material and can significantly increase the measurement accuracy.
[0005]
[Means for Solving the Problems]
The gist of the present invention made to solve the above problems is that in a flow-through type biological specific reaction measurement using a reaction solid phase (that is, a reaction matrix) holding a ligand capable of capturing a component to be measured. Biology consisting of measuring the signal of the reaction matrix only by preventing the generation of non-specific signal in the absorbent material by moving the reaction matrix on the absorbent material and then adding the signal generator solution. A biological specific reaction measuring device comprising a specific reaction measuring method and a reaction matrix formed so as to be movable on an absorbent material.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present invention has the above-described configuration, and a flow-through type biological specific reaction measuring method and apparatus, which are the basic principle of the measuring method and apparatus of the present invention, are known as described above, and include a reaction matrix, an absorbent, You can refer to the documents listed above for containers.
The present invention is characterized in that after reacting a labeled substance, the reaction matrix is moved, and then a signal generating substance solution is added, and an example of an apparatus used for such measurement will be described with reference to the drawings. .
[0007]
FIG. 1 is a diagram showing an example of an apparatus used in the measurement method of the present invention, where (A) is a schematic side view, (B) is a schematic plan view, and (C) is a cross-sectional view taken along line XX of (B). Schematic, (D) is a schematic cross-sectional view along line YY of (B). FIG. 2 is a schematic cross-sectional view showing the usage state of the apparatus. Further, FIG. 3 is a schematic cross-sectional view showing a use state of another example of the apparatus used in the measurement method of the present invention.
In the reactor shown in FIGS. 1 and 2, two absorbent materials 2-1 and 2-2 (for example, glass fiber) are accommodated in a container 1 made of plastic or the like via a partition plate 3. A lid 4 is provided on the top of the container 1, and the lid 4 has an opening 5 at a position facing the absorbent 2-1, and a matrix in which a reaction matrix 6 is fixed to the opening 5. A holding member 7 is fitted. The lid 4 has a protruding member 9 formed so as to fit into a groove 8 provided on the side surface of the container 1, and the lid 4 is coupled to the container 1 by the protruding member 9 and the groove 8. And move on the container 1. That is, when the lid 4 is moved on the container 1, the reaction matrix 6 provided on the lid 4 can be moved from the absorbent 2-1 to the absorbent 2-2. Yes.
[0008]
The above-mentioned apparatus may be provided with a pretreatment filter 10 as necessary. Since the pretreatment filter 10 can remove impurities in the sample, the reaction matrix 6 is prevented from being clogged. As a result, measurement accuracy and analysis sensitivity can be improved. The pretreatment filter 10 is fixed to a filter holding member 11 that is produced so as to fit in the opening 5 of the lid 4. The pretreatment filter 10 is not particularly limited as long as it can remove impurities in the sample, but there are a screen filter that captures impurities on the surface of the filter and a depth filter that captures impurities inside the filter. Illustrated. As a preferable aspect of the pretreatment filter 10, it is composed of two or more depth filters having different retention particle diameters, and more preferably, the retention particle diameter is 0.2 to 0.4 μm in order from the side corresponding to the reaction matrix 6. It consists of a depth filter and a depth filter of 1 μm or more. Furthermore, if necessary, other depth filters or screen filters having different retention particle diameters may be combined.
[0009]
An outline of an example of measuring an antigen, which is a substance to be measured, as an example of the method of the present invention for measuring a substance to be measured using a reaction apparatus having such a configuration will be described based on FIG. First, the filter holding member 11 to which the pretreatment filter 10 is fixed is fitted into the opening 5 of the lid 4 (state A). Thereafter, a sample solution containing an antigen to be measured is supplied to the pretreatment filter 10. The sample solution filtered by the pretreatment filter 10 passes through the reaction matrix 6 holding (sensitized) the antibody (monoclonal antibody or polyclonal antibody), and the antigen is captured and passed through the reaction matrix 6. Is absorbed by the absorbent 2-1. Next, the labeled antibody is supplied through the pretreatment filter 10, and the labeled antibody is bound to the antigen captured in the reaction matrix 6 (state B). Thereafter, the filter holding member 11 is removed, and an appropriate cleaning solution is supplied from the opening 5 to clean the reaction matrix 6.
Next, the washed reaction matrix 6 is moved so as to come on the absorbent material 2-2 by moving the lid 4 (state C). Thereafter, a signal is generated by supplying a signal generating substance liquid from the opening 5 and the antigen (substance to be measured) is measured (quantitative or qualitative) by measuring the signal of the reaction matrix 6 with a signal measuring device. it can. The signal generating substance liquid that has passed through the reaction matrix 6 is absorbed by the absorbent 2-2.
In this method, since the labeled antibody is not absorbed by the absorbent material 2-2, no signal is generated even if the signal generating substance is absorbed. Therefore, the signal of the reaction matrix 6 can be measured without being affected by the non-specific signal, and the measurement accuracy can be remarkably improved.
[0010]
FIG. 3 is a schematic cross-sectional view showing a use state of another example of an apparatus used in the measurement method of the present invention. In this apparatus, three absorbents (2-3, 2-4, 2-5) are accommodated in the container 1 via the partition plate 3. In addition, the same code | symbol is attached | subjected to the same member as the apparatus of FIG.
The method of the present invention using the apparatus shown in FIG. 3 will be described with an example of measuring an antigen to be measured.
First, similarly to FIG. 2A, the filter holding member 11 to which the pretreatment filter 10 is fixed is fitted into the opening 5 of the lid 4 (state A). Thereafter, a sample solution containing an antigen to be measured is supplied to the pretreatment filter 10. The sample solution filtered by the pretreatment filter 10 passes through the reaction matrix 6 holding (sensitized) the antibody (monoclonal antibody or polyclonal antibody), and the antigen is captured and passed through the reaction matrix 6. Is absorbed by the absorbent 2-3. Next, the labeled antibody is supplied through the pretreatment filter 10, and the labeled antibody is bound to the antigen captured in the reaction matrix 6. Thereafter, the filter holding member 11 is removed.
Next, the lid 4 is moved to move the reaction matrix 6 so as to be on the absorbent 2-4 (state B). In this state, an appropriate cleaning solution is supplied from the opening 5 to clean the reaction matrix 6. The cleaning liquid that has passed through the reaction matrix 6 is absorbed by the absorbent 2-4.
After the washing, the lid 4 is further moved to move the washed reaction matrix 6 so as to come on the absorbent 2-5 (state C). Thereafter, a signal is generated by supplying a signal generating substance liquid from the opening 5 and the antigen (substance to be measured) is measured (quantitative or qualitative) by measuring the signal of the reaction matrix 6 with a signal measuring device. it can. The signal generating substance liquid that has passed through the reaction matrix 6 is absorbed by the absorbent 2-5.
In this method, the labeled antibody is absorbed by the absorbing material 2-3, and the absorbing material 2-4 that absorbs only the washing solution is present in the middle. Therefore, the absorbing material 2- absorbing the signal generating substance solution is present. The signal generation at 5 can be substantially completely prevented. Therefore, the signal of the reaction matrix 6 can be measured without being affected by the non-specific signal, and the measurement accuracy can be further improved.
[0011]
Examples of the signal measurement include chemiluminescence measurement, fluorescence measurement, and colorimetric measurement. The measurement may be either instrumental measurement or visual measurement depending on the measurement method. In addition, enzymes, fluorescent substances, etc. can be used as labels; optical signals based on labels generated by signal generating substances (chemiluminescent substrates, fluorescent source substances, chromogenic substances, etc.) and their optical measurement methods, etc. Is well known.
In the measurement method and apparatus described above, the filtration step in the pretreatment filter 10 can be omitted depending on the properties of the sample. Moreover, in said example, although the absorber is isolate | separated into the some absorber (2-1 and 2-2 and 2-3, 2-4 and 2-5) by the partition plate 3, the kind of absorber Depending on the filling amount, the partition plate 3 may be provided without being provided. Further, the reaction matrix 6 in which a signal is generated can be stored by removing the lid 4 from the container 1 after the reaction is completed. In order to facilitate the movement of the lid body 4, an uneven portion may be provided on the surface of the lid body 4 so that the lid body 4 can be easily grasped. Thus, the present invention can be implemented with appropriate modifications.
[0012]
The measurement method of the present invention can be applied to any method as long as it measures a substance to be measured based on a biological specific reaction. Examples of such a method include antigen-antibody reaction, nucleic acid hybridization, sugar Examples thereof include chain-lectin reaction, enzyme-substrate or inhibitor reaction, bioactive substance-receptor reaction, biotin-avidin reaction, and the like. Accordingly, the substances to be measured by the method and apparatus of the present invention include, for example, antigens, antibodies, DNA, cDNA, RNA, saccharides, enzymes and inhibitors thereof, physiologically active substances and receptors thereof, biotinylated substances, and avidin. An example is a chemical substance.
[0013]
【The invention's effect】
According to the method and apparatus of the present invention, it is possible to completely eliminate the influence of a non-specific signal from the absorbent material, so that the analysis sensitivity and measurement accuracy can be remarkably improved.
[0014]
【Example】
Hereinafter, the present invention will be described with specific examples, but the present invention is not limited to the examples.
Example 1
(1) Production of reaction apparatus As shown in Fig. 1, a sensitized membrane (reaction matrix 6) in which an anti-HBs antigen monoclonal antibody was sensitized on a nitrocellulose membrane having a pore diameter of 0.45 µm was reacted. A reaction apparatus was prepared which was held by the matrix holding member 7 and provided with absorbent materials 2-1 and 2-2 below.
(2) Measurement of HBs antigen Immunoassay of HBs antigen was performed using the above apparatus. Specifically, 100 μl of PBS buffer solution was added to the opening 5 of the device, 200 μl of a sample prepared by 4-fold dilution was added, and 100 μl of peroxidase-labeled anti-HBs antigen monoclonal antibody solution was added 3 minutes later. After washing by adding 200 μl of a washing solution containing PBS buffer as a main component twice, 100 μl of a chemiluminescence substrate solution was added, and the luminescence signal was measured with a luminometer, and immunoassay was performed. In the above measurement, as a sample, a positive control sample (PC) prepared in 1% BSA-PBS so that the HBs antigen was 5 IU / ml, and only 1% BSA-PBS containing no HBs antigen were used. A negative control sample (NC) was used.
In the immunoassay, after adding a labeled antibody solution and performing washing, the reaction matrix 6 on the absorbent material 2-1 is moved onto the absorbent material 2-2, and the chemiluminescent substance is added. (Slide type) and a method of adding a labeled antibody solution and a chemiluminescent substance (fixed type) on the same absorbent material, and the luminescence count was measured. The results are shown in Table 1. As shown in Table 1, when the signals of the positive control (PC) and negative control (NC) obtained by the slide type are compared, the ratio of the slide type is clearly larger and the measurement by the slide type is higher. It turned out to be sensitivity.
[0015]
[Table 1]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of an apparatus according to the present invention. In the figure, (A) is a schematic side view, (B) is a schematic plan view, (C) is a schematic cross sectional view taken along line XX of (B), and (D) is a schematic cross sectional view taken along line YY of (B). It is.
FIG. 2 is a schematic cross-sectional view showing a use state of the apparatus of FIG.
FIG. 3 is a schematic cross-sectional view showing a use state of another example of the apparatus according to the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Container 2-1 to 2-5 Absorbent material 3 Partition plate 4 Lid body 5 Opening part 6 Reaction matrix 7 Reaction matrix holding member 8 Groove 9 Projection member
10 Pre-processing filter
11 Filter holding member

Claims (5)

Using biological specificity reactor a flow-through type reaction solid phase is retained ligand capable of capturing a substance to be measured is movably formed on the absorber, measure the signal generated in the reaction solid phase organisms In a specific biological reaction measurement method, a sample solution is supplied to the reaction solid phase, and the reaction solid phase to which the sample solution is supplied is labeled and is based on a substance to be measured and a biological specific reaction. and supplying a liquid substance capable of binding Te, organism, wherein the step Before moving the reaction solid phase on absorbent, and measuring the signal generated on the basis of the label, further comprising a Specific reaction measurement method. 2. The biological specific reaction measurement method according to claim 1, wherein in the measurement step, signal measurement is performed by instrumental measurement. The biological specific reaction measurement method according to claim 1, further comprising a step of supplying a signal generating substance solution to the reaction solid phase between the transfer step and the measurement step. 4. The biological specific reaction measurement method according to claim 3, wherein the label is an enzyme, and the signal generating substance solution is a chemiluminescent substrate solution. A biological specific reaction measurement which is used in the biological specific reaction measurement method according to any one of claims 1 to 4, wherein the reaction solid phase is formed so as to be movable on an absorbent material. apparatus.

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