JPH1073597A - Method for detecting or measuring immunological active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform highly sensitive detection quickly by applying the minute voltage of AC or DC on reagent, wherein antigen or antibody is immunized on the carrier of a specified particle diameter. SOLUTION: Immunological active substance is selected from the material, wherein latex or the like is used as a carrier by immunological aggregation reaction, and e.g. infection marker such as CRP, solidifying-line melting-system marker such as D-dimer, tumor marker such as AFP and the like are used. When latex or the like is used as the carrier particles, it is recommendable that the average particle diameter is about 0.01-1μm. As the minute voltage, the AC voltage is recommendable. The voltage in the range, wherein a reaction system does not cause electrolysis, is used. The detecting method is as follows. The specimen is added to the solution, wherein buffer is the main component and processed at the specified temperature and time. Then, after the reagent, wherein antigen is sensitized to the antibody, is added, the suspension of the reagent is added into the solution, wherein the buffer is the main component. Processing is performed at the specified temperature and time. After the specimen is added and the minute voltage is applied, the change in turbidity is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting or measuring an immunologically reactive substance by an immunological agglutination reaction using carrier particles. More specifically, the present invention relates to a method for rapidly or simply detecting and measuring an immunologically reactive substance with high sensitivity by applying a small voltage derived from alternating current and / or direct current to the reaction system.

[0002]

2. Description of the Related Art An immunologically reactive substance forms an insoluble aggregate by an immunological agglutination reaction, and by detecting this, it is possible to detect or measure the immunologically reactive substance. . As a method for detecting or measuring an immunologically reactive substance, for example, an enzyme immunoassay and a radioimmunoassay have been conventionally used. These methods have high sensitivity and high accuracy. However, because reagents are unstable due to the use of enzymes and radiation, and there are restrictions on storage and storage, detailed considerations, techniques, and special equipment are required for measurement. Was sought.
In addition, since these methods require time for measurement, it is difficult to deal with them in an emergency test, and high-sensitivity and quick methods have been actively studied.

[0003] Since 1970, an optical analysis method (turbidimetry or counting method) has been developed as a method for measuring immunological agglutination using a carrier such as latex or liposome. These immunological agglutination reactions are generally started by stirring with a stirring blade or the like, and are performed at a temperature of 37 ° C to 45 ° C. At this time, the time required for the measurement (reaction) is 5 to 20 minutes. This is quicker than enzyme immunoassay or radioimmunoassay, but in terms of measurement sensitivity and specificity,
Although it is being improved step by step, it is inferior to the above method and its application range is limited.

There is known a method for measuring an immunological agglutination reaction quickly and with high sensitivity by applying a DC pulse or an AC voltage to the reaction system. For example, Biosensors by Tamiya et al., 3 (3), pp. 139-146 (198)
8) In a cell equipped with electrodes, a reagent in which human immunoglobulin G was bound to 1.0 μm latex particles and immunoglobulin G were mixed, and the peak value was 200 V (electric field intensity 200
V / mm) and a DC pulse voltage of
It is stated that a cohesion of 0% was obtained. Japanese Patent Application Laid-Open No. 7-83928 proposes a method in which an alternating voltage is applied to the reaction system so that the electric field strength becomes 5 to 50 V / mm in the presence of a salt of 10 mM or more. In each case,
By applying these fluctuating voltages, the carrier particles are formed into pearl chains, thereby promoting the formation of aggregates.

[0005]

However, it is difficult to form a pearl chain with a latex reagent (particle size less than 1 μm) generally used for immunological agglutination, and high sensitivity cannot be expected. The above-mentioned patent publication also describes that the preferable average diameter of the carrier particles is 0.5 to 10 μm. The present inventors have found that the 0.1 to 0.1 used in latex turbidimetry, which is generally evaluated as being highly sensitive, rapid and simple.
The present inventors have found a method for detecting or measuring an immunologically reactive substance with higher sensitivity and higher sensitivity than the conventional method using a reagent consisting of 5 μm carrier particles, and have reached the present invention.

[0006]

That is, the present invention relates to a method for detecting or measuring an immunologically reactive substance by immunological agglutination using a reagent obtained by sensitizing a carrier to an antigen or an antibody. And applying a minute voltage derived from alternating current and / or direct current to the reaction system. In the present invention, the immunological agglutination is an antigen-antibody reaction,
This method utilizes the reaction with a specific receptor, etc., and includes both qualitative and quantitative methods. The immunologically reactive substance in the present invention can be selected from substances that can be measured by the above-described immunological agglutination reaction by an agglutination method using latex, liposome, or the like as a carrier. For example, CR
P, HBs antigen, HBs antibody, HCV antibody, HIV antibody, infectious disease marker such as TP antibody, coagulation / fibrinolytic marker such as D-dimer, protein C, protein S, ATIII, tumor such as AFP, CEA, CA19-9 Markers, hormones such as TSH, prolactin, and insulin,
Tissue components such as β2m, myoglobin and myosin, DNA
And the like. The detectable concentration is generally about 0.05 ng / ml to 50 μg / ml, preferably 0.1 ng / ml.
ng / ml to 10 μg / ml. As the antigen or carrier to be sensitized, an antigen or antibody capable of immunologically recognizing the above immunologically reactive substance is used.

[0007] The carrier particles of the present invention include latex,
Liposomes, colloidal gold and the like can be mentioned, but latex particles are preferred. As the latex particles, those generally used in an immunological agglutination method can be used.
The average diameter of the carrier particles is 0.01 in the case of latex particles.
１1 μm is preferred. More preferably 0.05-0.5μ
m. Most preferably, it is 0.1 to 0.3 μm. When the average diameter exceeds 1 μm, Brownian motion of the particles and the like hardly occur, which is not preferable. The sensitization of the latex particles with the antibody or antigen can be carried out, for example, by adsorbing or binding by a conventionally well-known method.

[0008] The minute voltage of the present invention is derived from AC and / or DC, and is more preferably an AC voltage.
DC voltage readily induces electrolysis, thus narrowing the range of indicated conditions. Further, a voltage in a range where the reaction system does not substantially cause electrolysis, for example, the electric conductivity of the reaction system is 17.
At 4 mS / cm (equivalent to physiological saline), the electric field strength is 7.
AC voltage (frequency 10 KHz) of 8 V / mm or less. The application time of the AC voltage is preferably as long as possible within a range in which the reaction system does not substantially cause electrolysis, and is preferably several tens seconds to several minutes. More preferably 30 seconds to 2
Min application. However, the cause of the electrolysis depends on factors such as the electrical conductivity and applied voltage of the reaction system reagent, the material of the electrode, the area, and the application time. .

The alternating current frequency of the present invention does not greatly affect the speed of immunological agglutination within the range studied, but is preferably a frequency of 1 KHz to 1 MHz. The waveform of the AC voltage in the present invention may be any of a continuous wave and a pulse wave, and may have an arbitrary shape, but is preferably a sine wave, a square wave, or a rectangular wave.

In the present invention, a typical apparatus for applying a minute voltage to a reagent and a typical measuring apparatus for detecting or measuring an immunologically reactive substance are described, for example, in FIG. 1 (or FIG. 5).
As shown in the figure, a power supply device that can apply any voltage such as a DC pulse voltage, an AC voltage, and a DC voltage, a cell with electrodes in which electrodes are attached to a cell for a spectrophotometer, and a voltage / frequency / pulse width monitor. It consists of an oscilloscope, a spectrophotometer for measuring turbidity, and the like. As the power supply device, an arbitrary pulse generator, for example, a commercially available function generator or cell stimulator can be used. As a spectrophotometer cell, a commercially available glass cell, plastic cell, or the like can be used, and as an electrode material, platinum, nickel, or the like can be used.

As a method for detecting or measuring the presence of an immunologically reactive substance, a sample is added to a buffer-based solution, treated at room temperature to 37 ° C. for 0.5 to 3 minutes, and then treated with a carrier. After adding a suspension of a reagent sensitized with an antigen or an antibody to the solution, or adding the suspension of the above reagent to a solution containing a buffer as a main component, and treating at room temperature to 37 ° C. for 0.5 to 3 minutes. And
Then, after adding a specimen, while applying a minute voltage or after applying a minute voltage, the method is carried out by, for example, a method of calculating a turbidity change amount using the above-described measuring device. In the latter method of adding a sample to a solution containing a reagent, a change in turbidity can be measured after applying the sample while applying a minute voltage.

The reagent used in the present invention is usually used by dispersing it in an aqueous medium. The salt concentration in the aqueous medium containing the reagent is not particularly limited, but is preferably 0 to 0.
100 mS / cm. More preferably, 2 to 60 mS
/ Cm. Examples of the salt include potassium chloride, sodium chloride, sodium nitrate, ammonium nitrate and the like, and potassium chloride and sodium chloride are particularly preferable. As a composition of the aqueous medium containing the reagent, a composition well known to those skilled in the art can be used. For example, proteins such as albumin, surfactants such as polyethylene glycol, Tween and Triton, chlorides such as choline chloride, amino acids such as arginine and asparagine, and preservatives such as sodium azide may be added. It is preferable that the electric conductivity of the reaction system is small. If the electric conductivity is low, the intensity of the electric field that can be applied to the reaction system can be increased, so that the effect of promoting the immunological agglutination reaction can be easily obtained.

[0013]

EXAMPLES The present invention will be described in detail below with reference to examples and comparative examples, but these do not limit the present invention.

Example 1 AC voltage characteristics / applied voltage electric field strength (1) Preparation of AFP latex reagent 5 mg of anti-AFP antibody was dissolved in 9.5 ml of glycine buffer (hereinafter abbreviated as GBS), and 0.5 ml of latex was dissolved. (Particle size: 0.22 μm, solid content: 10% suspension) was added, and the mixture was stirred at room temperature for 2 hours, and the sensitized latex suspension was centrifuged to remove the supernatant. The precipitate was suspended in 33 ml of 0.2% BSA-GBS to prepare an AFP latex reagent.

(2) AC Voltage Applying Apparatus Using the experimental apparatus shown in FIG. 1, an oscilloscope was used to set the condition of the potential applied between the electrodes and apply an AC voltage.
(Distance between electrodes: 3 mm) (3) Measurement method Phosphate buffer (15 mM phosphoric acid, 0.4 mM) containing 0.2% bovine serum albumin in a glass cell with electrodes attached
% Polyethylene glycol 6000, 2% sodium chloride, 0.1% sodium azide, below 0.2% BSA
-PBS) 600 μl, AFP latex reagent 3
Dispense 00 μl, and apply an alternating current (AC) voltage (0 V to ± 7.5
V, frequency 10 KHz, sine wave) for 2 minutes. Thirty seconds after the start of the application, 60 μl of the sample was added, and the time course of turbidity change was immediately measured using a spectrophotometer (Hitachi U-3200), and the turbidity difference (ΔOD572) for 5 minutes was measured.
nm). The AFP standard was diluted with 0.2% BSA-GBS to obtain an AFP concentration of 0 to 2.5 ng /.
A sample of ml was prepared and used for measurement. Electric field strength of applied voltage is 0V, ± 1V, ± 3.5V, ± 4.5V, ± 7.5V
And the sample of each concentration was measured.

(4) Results The results are shown in Table 1 and FIG. As is clear from FIG. 2, according to the present invention, compared to the conventional method (applied voltage 0 V),
This shows that it is possible to detect or measure an immunologically reactive substance with high sensitivity by promoting an immunological agglutination reaction. Also, it can be seen that increasing the electric field strength of the applied voltage can further promote the immunological agglutination reaction.

[0017]

[Table 1]

Example 2 AC voltage characteristics / frequency (1) Preparation of AFP latex reagent An AFP latex reagent was prepared in the same manner as in Example 1. (2) AC voltage application device The measurement was performed using the same device as in Example 1. (3) Measurement method An alternating voltage (sine wave) having a frequency of 10 Hz to 100 KHz was applied so that the electric field strength became ± 6 V, and the turbidity change with respect to the frequency of each sample was measured in the same manner as in Example 1. (4) Results The results are shown in Table 2 and FIG. From the results shown in FIG. 3, it can be seen that the immunological agglutination reaction can be uniformly promoted when the AC frequency is between 1 and 100 KHz.

[0019]

[Table 2]

Example 3 AC voltage characteristics / application time (1) Preparation of AFP latex reagent In the same manner as in Example 1, an AFP latex reagent was prepared. (2) AC voltage application device The measurement was performed using the same device as in Example 1. (3) Measurement method An alternating voltage (sine wave) having an electric field strength of ± 7.5 V and a frequency of 10 KHz was applied for 0 to 4 minutes, and the turbidity change with respect to the application time of each sample was measured in the same manner as in Example 1. (4) Results The results are shown in Table 3 and FIG. From the results shown in FIG. 4, it can be seen that the longer the application time of the AC voltage, the more the immunological agglutination reaction can be promoted.

[0021]

[Table 3]

Example 4 (1) Preparation of AFP Latex Reagent AFP latex reagent was prepared in the same manner as in Example 1. (2) DC voltage applying device Using the experimental device of FIG. 5, a DC voltage was applied by setting the condition of the potential applied between the electrodes using an oscilloscope.
(3 mm between electrodes) (3) Measurement method 0.2% BSA-PB was placed in a glass cell with electrodes attached.
S600 μl of SFP and 300 μl of AFP latex reagent were dispensed, and a DC voltage of 1 V was applied for 50 seconds. Ten seconds after the start of the application, 60 μl of the sample was added, and the turbidity was measured immediately using a spectrophotometer (Hitachi U-3200). After stirring for 5 minutes, the turbidity was measured and the turbidity difference was measured for 5 minutes. (ΔOD
572 nm). In addition, 0.2% BSA-GB
Using S, dilute the AFP standard to give an AFP concentration between 0 and 1.
A sample of 25 ng / ml was prepared and used for measurement. (4) Results The results are shown in Table 4 and FIG. From the results shown in FIG. 6, according to the method of the present invention, the immunological agglutination reaction is promoted as compared with the conventional method (applied voltage of 0 V), and the immunologically reactive substance can be detected or measured with high sensitivity. It can be seen that it is.

[0023]

[Table 4]

[0024]

According to the present invention, it is possible to detect or measure an immunologically reactive substance in a shorter time and with higher sensitivity than the conventional measurement method.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an AC voltage application experimental device used in an example of the present invention.

FIG. 2 is a graph showing the relationship between human AFP concentration, application of an AC voltage, and turbidity change.

FIG. 3 is a graph showing a relationship between a human AFP concentration, a frequency of an AC voltage, and a turbidity change.

FIG. 4 is a graph showing the relationship among human AFP concentration, AC voltage application time, and turbidity change.

FIG. 5 is a schematic diagram of a DC voltage application experimental device used in an example of the present invention.

FIG. 6 is a graph showing a relationship between a human AFP concentration and a change in turbidity due to application of a DC voltage.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshinori Yoshimura 1-1-13-7 Sengen, Tsukuba, Ibaraki Prefecture Eagle 1201 (72) Inventor Masao Karube 1-3-16 Higashiarima, Miyamae-ku, Kawasaki-shi, Kanagawa

Claims (3)

[Claims] 1. A method for detecting or measuring an immunologically reactive substance by an immunological agglutination reaction using a reagent sensitized to an antigen or an antibody on a carrier, wherein an alternating current and / or direct current is applied to the reaction system. Applying a minute voltage derived from the method. 2. The method according to claim 1, wherein the minute voltage is a voltage at which the reaction system does not substantially cause electrolysis. 3. The method according to claim 1, wherein the carrier has an average particle size of 0.01 to 1 μm.