JP2000199762A - Method and apparatus for biologically specific reaction measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method and apparatus for biologically specific reaction measurement capable of enhancing analytical sensitivity/measuring accuracy. SOLUTION: In a flow-through type biologically specific reaction measuring method and apparatus using a reaction solid phase, to which a ligand capable of capturing a substance to be measured is held, the reaction solid phase is moved on an absorbing material so as to measure a signal. According to this method and apparatus, the influence of the non-specific signal from the absorbing material can be excluded completely and, therefore, analytical sensitivity/ measuring accuracy can be enhanced markedly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD 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 the fields of clinical inspection, food inspection and the like.

[0002]

2. Description of the Related Art A biologically specific reaction measuring method for measuring a substance to be measured in 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 particularly known. No. 61-502214,
It is known as disclosed in JP-A-4-318462 and JP-A-6-50973. As a device used for such a measuring method, a porous material is generally used in which an absorbent is accommodated in a container having an upper opening and a ligand which specifically reacts with a substance to be measured is immobilized above the absorbent. A device provided with a matrix is used. In the measurement of a substance to be measured using such an apparatus, for example, when the substance to be measured is an antigen, a liquid to be measured (that is, an antigen) is added to the upper opening, and a ligand (that is, a ligand that specifically reacts with the substance to be measured) ,
Antibody) is passed through the immobilized porous matrix to capture the antigen. Next, by adding an antibody solution to which a label (for example, an enzyme) is bound,
The antigen is measured by binding the antibody to the antigen captured on the matrix and measuring a signal (for example, luminescence, radioactivity, etc.) based on the label. Each liquid that has passed through the porous matrix is absorbed by the absorbent.

[0003]

The substance to be measured is measured by 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 the method for measuring a biologically specific reaction described above, there is an effect that the substance to be measured can be easily measured. However, a signal based on the label (a non-specific signal) is also generated in the absorbing material in the container. Therefore, when measuring the signal of the reaction matrix, it is affected by the non-specific signal from the absorbing material, and there is a problem that the signal of the reaction matrix cannot be measured with high accuracy. As a means for solving such a problem, for example, a device equipped with a transmitted light reducing element between the absorbing material and the reaction matrix has been proposed for the purpose of reducing non-specific light signals transmitted from the absorbing material. (JP-A-8-506420). According to such an apparatus, transmitted light from the absorber can be reduced to some extent, but it has been difficult to completely eliminate the effect.

In order to solve such a problem, the present inventors have proposed a method for measuring a substance to be measured using a flow-through type biologically specific reaction measuring apparatus using a reaction matrix. As a result of examining a method that can eliminate the effects of non-specific signals, after reacting the label, the reaction matrix is moved, and a signal-generating substance solution (for example, Liquid) can prevent the generation of a non-specific signal in the absorbent, and can measure the signal of the reaction matrix alone. completed. The present invention has been made based on such findings, and the present invention provides a method and apparatus for measuring a flow-through type biological specific reaction of components in serum, plasma, and other body fluids usually performed in the field of clinical testing. In the above, there is provided a measuring method and a measuring apparatus which can eliminate the influence of a non-specific signal from an absorbing material and can remarkably increase the measuring accuracy.

[0005]

The gist of the present invention to solve the above-mentioned problems is to provide a flow-through type using a reaction solid phase (ie, a reaction matrix) holding a ligand capable of capturing a component to be measured. In the biological specific reaction measurement, the reaction matrix is moved on the absorbent,
After that, by adding the signal generating substance solution,
Prevents the generation of non-specific signals in the absorbent, measures the signal of only the reaction matrix, and the biological specific reaction measurement method and the reaction matrix is formed so as to be movable on the absorbent This is a biological specific reaction measuring device.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention has the above constitution, 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. For the reaction matrix, absorbent, container and the like, the above-mentioned documents can be referred to. In the present invention, after reacting the label, the reaction matrix is moved,
Thereafter, a signal generating substance liquid is added, and an example of an apparatus used for such measurement will be described with reference to the drawings.

FIG. 1 is a view showing an example of an apparatus used in the measuring method of the present invention, wherein (A) is a schematic side view, (B) is a schematic plan view, and (C) is an X-ray of (B). X-ray cross-sectional schematic,
(D) is a schematic sectional view taken along line YY of (B). FIG. 2 is a schematic sectional view showing a use state of the device. FIG. 3 is a schematic cross-sectional view showing a use state of another example of the apparatus used for the measuring method of the present invention. The reactor shown in FIGS. 1 and 2 is a container 1 made of plastic or the like.
In this case, two absorbing materials 2-1 and 2-2 (for example, glass fiber or the like) are accommodated via a partition plate 3. A lid 4 is provided on the upper part of the container 1, and the lid 4 has an opening 5 at a position facing the absorbent 2-1.
Is fitted with a matrix holding member 7 to which a reaction matrix 6 is fixed. The lid 4 has a projection 9 formed 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 projection 9 and the groove 8. And can move on the container 1. That is, when the cover 4 is moved on the container 1, the reaction matrix 6 provided on the cover 4 can be moved from the absorbent 2-1 to the absorbent 2-2. I have.

[0008] The above-mentioned apparatus may be provided with a pretreatment filter 10 if necessary, and by providing the pretreatment filter 10, impurities in the sample can be removed. Clogging is prevented, and measurement accuracy and analysis sensitivity can be improved. Pretreatment filter 1
Numeral 0 is fixed to a filter holding member 11 made to fit 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, and includes a screen filter that captures impurities on the surface of the filter and a depth filter that captures impurities inside the filter. Is exemplified. As a preferred embodiment of the pretreatment filter 10, the pretreatment filter 10 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. Further, if necessary, other depth filters or screen filters having different retention particle diameters may be combined.

Referring to FIG. 2, an outline of an example of the method of the present invention for measuring a substance to be measured using the reaction apparatus having the above-described configuration, in which an antigen as a substance to be measured is measured will be described. First, the filter holding member 11 to which the pretreatment filter 10 is fixed is fitted into the opening 5 of the lid 4 (A
State). Thereafter, a sample liquid containing the 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 retaining (sensitizing) an antibody (monoclonal antibody or polyclonal antibody), and the antigen is captured by the reaction matrix 6 and passed through the sample solution. Is absorbed by the absorbing material 2-1. Next, the labeled antibody is supplied through the pretreatment filter 10, and the labeled antibody is bound to the antigen captured by the reaction matrix 6 (state B).
Thereafter, the filter holding member 11 is removed, and the opening 5
To supply a suitable washing solution to wash the reaction matrix 6. Next, the lid 4 is moved to move the washed reaction matrix 6 so as to come on the absorbent 2-2 (state C). Thereafter, a signal is generated by supplying a signal generating substance liquid from the opening 5,
The antigen (substance to be measured) can be measured (quantitatively or qualitatively) by measuring the signal of the reaction matrix 6 with a signal measuring device. 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 absorbing 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 significantly increased.

FIG. 3 is a schematic sectional view showing the use of another example of the apparatus used in the measuring method of the present invention. In this device, three absorbents (2-3, 2-
4, 2-5) are accommodated via the partition plate 3. 1 and 2 are denoted by the same reference numerals. The method of the present invention using the apparatus shown in FIG. 3 will be described with an example of measuring an antigen which is a substance to be measured. First, in the same manner as in FIG.
The filter holding member 11 to which is fixed is fitted into the opening 5 of the lid 4 (state A). Thereafter, a sample liquid containing the antigen to be measured is supplied to the pretreatment filter 10. The sample solution filtered by the pretreatment filter 10 is
After passing through the reaction matrix 6 holding (sensitizing) an antibody (monoclonal antibody or polyclonal antibody), the antigen is captured by the reaction matrix 6 and the sample liquid passed through 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 by the reaction matrix 6. After that, the filter holding member 11 is removed.
Next, by moving the lid 4, the reaction matrix 6 is moved so as to be on the absorber 2-4 (state B). In this state, an appropriate washing liquid is supplied from the opening 5 to wash the reaction matrix 6. The washing liquid that has passed through the reaction matrix 6 is absorbed by the absorbent 2-4. After the above-described washing, the lid 4 is further moved to move the washed reaction matrix 6 so as to come on the absorber 2-5 (state C). Thereafter, a signal is generated by supplying a signal generating substance liquid from the opening 5,
The antigen (substance to be measured) can be measured (quantitatively or qualitatively) by measuring the signal of the reaction matrix 6 with a signal measuring device. 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 used as the absorbent 2-
3 and the intermediary of the absorber 2-4 that has absorbed only the washing liquid, it is possible to substantially completely prevent signal generation in the absorber 2-5 that has absorbed the signal generating substance liquid. it can. 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 above 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; generation of optical signals based on labels by signal generating substances (chemiluminescent substrates, fluorescent substance substances, chromogenic substances, etc.) and optical measurement methods thereof are in this field. It is well known. In the above-described measurement method and apparatus, the filtration step using the pretreatment filter 10 may be omitted depending on the properties of the sample. In the above example, the absorbing material is separated into a plurality of absorbing materials (2-1 and 2-2 and 2-3, 2-4 and 2-5) by the partition plate 3. Depending on the filling amount and the amount of filling, it can be carried out without providing the partition plate 3. Further, after the reaction is completed, by removing the lid 4 from the container 1, the reaction matrix 6 in which the signal has been generated can be preserved. Further, in order to facilitate the movement of the lid 4, the lid 4
An uneven portion may be provided on the surface to make it easier to grasp the lid 4. As described above, the present invention can be implemented with appropriate modifications.

The measuring method of the present invention can be applied to any method for measuring a substance to be measured based on a biologically specific reaction. Examples of such a method include an antigen-antibody reaction and a nucleic acid hybridization method. Hybridization, sugar chain-lectin reaction,
Examples include a reaction with an enzyme-substrate or an inhibitor, a physiologically active substance-receptor reaction, a biotin-avidin reaction, and the like. Therefore, as a substance to be measured by the method and the device of the present invention, for example, antigen, antibody, DNA, cDNA, RNA,
Examples include saccharides, enzymes and their inhibitors, physiologically active substances and their receptors, biotinylated substances, avidinated substances and the like.

[0013]

According to the method and apparatus of the present invention, the effect of non-specific signals from the absorbing material can be completely eliminated, so that there is an effect that the analytical sensitivity and the measurement accuracy can be significantly increased.

[0014]

Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to the examples. Example 1 Preparation of Reaction Apparatus As shown in FIG. 1, a sensitized membrane obtained by sensitizing an anti-HBs antigen monoclonal antibody on a nitrocellulose membrane having a pore size of 0.45 μm (reaction matrix 6)
Was held in the reaction matrix holding member 7, and a reaction device provided with the absorbing materials 2-1 and 2-2 below the holding member. Measurement of HBs antigen Immunoassay for HBs antigen was performed using the above apparatus. That is, 100 μl of a PBS buffer solution was added to the opening 5 of the device, and then 200 μl of a 4-fold diluted sample was added.
Three minutes later, 100 µl of a peroxidase-labeled anti-HBs antigen monoclonal antibody solution was added. After washing twice by adding 200 μl of a washing solution containing a PBS buffer as a main component, 100 μl of a chemiluminescent substrate solution was added, and the luminescence signal was measured by a luminometer to perform an immunoassay. 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 a 1% BSA-PBS alone containing no HBs antigen were used. Performed using a negative control sample (NC). In the immunoassay, a method according to the present invention in which a labeled antibody solution is added, washing is performed, the reaction matrix 6 on the absorber 2-1 is moved to the absorber 2-2, and a chemiluminescent substance is added. (Slide type),
The method of adding a labeled antibody solution and a chemiluminescent substance on the same absorbent (fixed type) was performed, and the luminescence count was measured.
Table 1 shows the results. As shown in Table 1, when the signals of the positive control (PC) and the negative control (NC) obtained in the slide type were compared, the ratio was clearly higher in the slide type and higher in the measurement in the slide type. 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 (B).
3 is a schematic sectional view taken along the line YY of FIG.

FIG. 2 is a schematic sectional view showing a use state of the apparatus of FIG. 1;

FIG. 3 is a schematic sectional view showing a use state of another example of the device 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 5 Opening 6 Reaction matrix 7 Reaction matrix holding member 8 Groove 9 Projection member 10 Pretreatment filter 11 Filter holding member

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Takuya Odawara 1-2-1 Muroya, Nishi-ku, Kobe International Reagent Co., Ltd. R & D Center (72) Inventor Yoshihiro Saito 1-2-1-2 Muroya, Nishi-ku, Kobe City (72) Inventor Tetsuo Ueno 1-2-1 Muroya, Nishi-ku, Kobe International Reagents Company R & D Center

Claims (2)

[Claims] In a flow-through type biological specific reaction measuring method using a reaction solid phase holding a ligand capable of capturing an analyte, a signal is measured by moving the reaction solid phase on an absorbent. A method for measuring a biologically specific reaction, characterized in that: 2. In a flow-through type biologically specific reaction apparatus using a reaction solid phase holding a ligand capable of capturing an analyte, the reaction solid phase is formed so as to be movable on an absorbent material. A biological specific reaction measuring device characterized by the above-mentioned.