JPH1073595A - Method for detecting or measuring presence of immunological active substance and reagent used for method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve the highly sensitive detection quickly by using the reagent, which detects immunological active substance, as the carrier having the specified particle diameter, on which a minute voltage is applied beforehand. SOLUTION: Immunological active substance is selected from the substance, which uses latex and the like as a carrier based on immunological aggregation reaction as a carrier. For example, infectious disease marker such as CRP and tumor marker such as D-dimer and the like are used. As the carrier particled, e.g. altex and the like are used. It is recommndable that the average particle diameter of the barrier particles is about 0.01-1μm. As the minute voltage, DC, DC pulses or AC are used. The voltage in the range, wherein the water-based medium containing reagent dose not cause electrolysis, is used. When the pretreating reagent, wherein the minute voltage is applied on the reagent comprising the carrier which is acted by antigen or antibody, is used, it is recommendable that the reagent is used as quickly as possible after the pretreating, desirably immediately after the treatment. The device for applying the minute voltage comprises, e.g. a container with an electrode, an oscilloscope and the like, which can apply the arbitrary voltage such as a DC voltage.

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 detecting or measuring an immunological reactant quickly and simply and with high sensitivity by using a reagent to which a minute voltage is applied in advance.

[0002]

2. Description of the Related Art Since an immunologically reactive substance forms an insoluble aggregate by an immunological agglutination reaction, it is possible to detect or measure the presence of the immunologically reactive substance by detecting this. It is. 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, reagents are unstable due to the use of enzymes and radiation, and there are restrictions on storage and preservation, so 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 (turbidimetric method or counting method) has been developed as a method for measuring an immunological agglutination reaction using a carrier such as latex or ribosome. 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 measurement sensitivity, specificity, etc., is gradually improving, but inferior to the above method,
The range of application 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. Also, 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 any case, the application of these fluctuating voltages causes the carrier particles to form a pearl chain, thereby promoting the formation of aggregates.

[0005]

However, a latex reagent (particle size: 1) generally used for immunological agglutination reaction is used.
(less than μm), it is difficult to form a pearl chain, 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 compared the conventional method using a reagent consisting of carrier particles of 0.1 to 0.5 μm used in the latex turbidimetric method, which is generally evaluated as being highly sensitive, rapid and simple. The present inventors have found a method for detecting or measuring the presence of an immunologically reactive substance more quickly and with high sensitivity, 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 prepared by sensitizing a carrier to an antigen or an antibody. And a method in which a reagent to which a minute voltage is applied in advance is used as the reagent. The present invention also provides a reagent for use in the method, comprising a carrier to which a microvoltage has been applied in advance and which has been sensitized with an antigen or an antibody. In the present invention, the immunological agglutination reaction is a method utilizing an antigen-antibody reaction, a reaction with a specific receptor, and the like, and includes both a qualitative method and a quantitative method.

[0007] The immunologically reactive substance in the present invention is:
The substance can be selected from substances that can be measured by the agglutination method using latex, ribosome, or the like as a carrier by the above-described immunological agglutination reaction. For example, infectious disease markers such as CRP, HBs antigen, HBs antibody, HCV antibody, HIV antibody, TP antibody, D-dimer, protein C, protein S, A
Coagulated fibrinolytic markers such as TIII, AFP, CEA, CA
Tumor markers such as 19-9; hormones such as TSH, prolactin and insulin; tissue components such as β2m, myoglobin and myosin; and nucleic acids such as DNA. Also,
Detectable concentrations are generally between 0.05 ng / ml and 50 μg.
/ Ml, preferably 0.1 ng / ml to 10 μg / ml
It is. As the antigen or antibody to be sensitized, an antigen or antibody capable of immunologically recognizing the immunologically reactive substance is used.

[0008] The carrier particles used in the present invention include, for example, latex, liposome, colloidal gold and the like, and preferably latex particles. As the latex particles, those which are generally used in immunological agglutination and are known can be used. The average diameter of the carrier particles is preferably from 0.01 to 1 μm in the case of latex particles. More preferably, it is 0.05 to 0.5 μm. 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 or known method.

The minute voltage of the present invention is derived from a direct current, a direct current pulse or an alternating current, and is a voltage within a range in which the aqueous medium containing the reagent does not substantially cause electrolysis, for example, the electric conductivity of the aqueous medium containing the reagent. Is 17.4 mS / cm (equivalent to physiological saline), a DC pulse voltage (pulse width 10 μS, 10 KHz) with an electric field strength of 2.4 V / mm, and the like. However, the aforementioned voltage varies depending on the material, area, application time, and the like of the electrode. The waveform of the AC voltage in the present invention may be a continuous wave or a pulse wave, and may have an arbitrary shape, but is preferably a sine wave, a square wave, or a rectangular wave. The frequency of the AC voltage is within the range considered.
It does not significantly affect the speed of the immunological agglutination reaction, but preferably has a frequency of 1 KHz to 1 MHz.

In the present invention, when a pretreatment reagent in which a microvoltage is applied in advance to a reagent comprising a carrier sensitized with an antigen or an antibody is used in the measurement system, the pretreatment reagent should be used as soon as possible after the pretreatment, preferably after the pretreatment. Preferably, it is used within 10 minutes. More preferably, it is used immediately after the pretreatment.

In the present invention, as shown in FIG. 1 or FIG. 4, for example, a device for applying an arbitrary voltage such as a DC pulse voltage, an AC voltage, a DC voltage, etc. It consists of a container with electrodes with electrodes attached to the inside surface of the container for applying voltage, an oscilloscope for monitoring voltage, frequency and pulse width. As the power supply device, an arbitrary pulse generator, for example, a commercially available function generator or cell stimulator can be used. As a container with electrodes, an insulating container such as glass or plastic 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, usually, a sample is added to a solution containing a buffer as a main component, and treated at room temperature to 37 ° C. for 0.5 to 3 minutes. The above-mentioned pretreatment reagent is added, and the turbidity is measured using a spectrophotometer that measures the turbidity.

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

[0014]

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 (1) Preparation of β2m latex reagent 10 mg of anti-β2m antibody was dissolved in 9.5 ml of glycine buffer (hereinafter abbreviated as GBS), and 0.5 ml of latex (particle diameter 0.12 μm, solid content 10%) Suspension) was added thereto, and the mixture was incubated at 37 ° C. overnight. After that, the sensitized latex suspension was centrifuged to remove the supernatant. The precipitate was suspended in 20 ml of GBS to prepare a β2m latex reagent. (2) DC pulse voltage applying device A DC pulse voltage was applied using the experimental device of FIG. (4mm distance between electrodes)

(3) Measuring method A β2m latex reagent is placed in a container with electrodes attached, and a DC pulse voltage (peak value 4.5 V, pulse width 1)
0 μsec, frequency 10 KHz) was applied for 2 minutes. Apply 1
Five seconds later, 300 µl of the pretreatment reagent was added to 600 µl of GBS.
Was mixed with a solution obtained by adding 45 μl of the sample to the sample. Immediately, the time course of turbidity change was measured using a spectrophotometer (Hitachi U-3200). The β2m standard was diluted with 0.2% bovine serum albumin-containing glycine buffer (hereinafter abbreviated as 0.2% BSA-GBS) to give a β2m concentration of 0 ng.
/ Ml and 12.5 ng / ml samples were prepared and used for measurement. (4) Results The results are shown in Table 1 and FIG. In FIG. 2, the first embodiment
Β2m concentration 12.5ng / m with DC pulse voltage applied
1 is a change in turbidity of a sample, and Reference Example 1 is a change in turbidity of a sample having a β2m concentration of 0 ng / ml to which a DC pulse voltage is applied.

Comparative Example 1 The same specimens and the β2m latex reagent as used in Example 1 were used. The time course of turbidity change was measured in the same manner as in Example 1 except that the electrical pretreatment of the reagent was not performed. The results are shown in Table 1 and FIG. Comparative Example 1 had a β2m concentration of 12.5 ng where no DC pulse voltage was applied.
/ Ml of the turbidity of the sample (conventional method), and Comparative Reference Example 1 shows β2m without applying a DC pulse voltage.
It is a turbidity change about the test substance of the concentration of 0 ng / ml.

[0017]

[Table 1]

Comparative Example 2 The same specimens and the β2m latex reagent as used in Example 1 were used. 45 μl of the sample is added to 600 μl of GBS,
The electrode was placed in a container with the electrodes attached, and a DC pulse voltage (peak value 4.5 V, pulse width 10 μsec, frequency 10 KHz) was applied for 2 minutes. 300 μl of a β2m latex reagent was added to the sample test solution subjected to the electrical pretreatment, and the time course of turbidity change was measured immediately using a spectrophotometer (Hitachi U-3200). The results are shown in Table 2 and FIG. FIG. 3 also shows the results of Example 1.

[0019]

[Table 2]

From the results shown in FIG. 2, it can be seen that the present invention can be measured in a shorter time and with higher sensitivity than the conventional method (Comparative Example 1 in which the reagent is not subjected to the electrical pretreatment and is used as in the conventional method). You can see that it can be done. Further, the results shown in FIG. 3 indicate that the immunological agglutination reaction can be promoted and the sensitivity can be increased by subjecting the test solution containing latex to electrical pretreatment.

Example 2 (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 (particle diameter 0.22 μm, solid 10% suspension), 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 device An AC voltage was applied using the experimental device of FIG. (3mm distance between electrodes)

(3) Measuring method The AFP latex reagent was placed in a container having electrodes attached thereto, and an AC voltage (0 V to ± 15 V, frequency 10 KHz) was applied for 2 minutes. 15 seconds after application, 300 µl of pretreatment reagent
In a phosphate buffer (15 mM phosphate, 0.4% polyethylene glycol 6000, 2% sodium chloride, 0.1%
600 μl containing sodium azide (hereinafter abbreviated as PBS)
Was mixed with a solution obtained by adding 60 μl of a sample to the sample. Immediately, the time course of turbidity change was measured using a spectrophotometer (Hitachi U-3200), and the turbidity difference for 4 minutes (ΔOD 572 nm)
I asked. In addition, using 0.2% BSA-GBS, A
A specimen having an AFP concentration of 2.5 ng / ml was prepared by diluting the FP standard and used for measurement. (4) Results From the results shown in Table 3 and FIG. 5, it can be seen that, as the applied voltage, the higher the electric field strength, the more the effect of promoting the immunological agglutination can be obtained.

[0023]

[Table 3]

Example 3 (1) Preparation of AFP latex reagent In the same manner as in Example 2, an AFP latex reagent was prepared. (2) AC voltage applying device An AC voltage was applied using the experimental device of FIG. (3mm distance between electrodes)

(3) Measuring Method An AFP latex reagent was placed in a container having electrodes attached thereto, and an AC voltage (± 15 V, frequency 10 KHz) was applied for 2 minutes. 15 seconds after application, 300 µl of pretreatment reagent is
It mixed with the solution which added 60 microliters of samples to 600 microliters of S. Immediately, the time course of turbidity change was measured using a spectrophotometer (Hitachi U-3200), and the turbidity difference (ΔOD
572 nm). In addition, 0.2% BSA-GBS
The AFP standard was diluted to a concentration of 0 ng / m
1, 0.625 ng / ml, 1.25 ng / ml, 2.5
An ng / ml sample was prepared and used for measurement. (4) Results From the results shown in Table 4 and FIG. 6, the present invention measures in a very short time and with high sensitivity compared to the conventional method (using a reagent that has not been subjected to a pretreatment by an electric method). You can see that it can be done.

Comparative Example 3 Using each sample of Example 3 and an AFP latex reagent,
The time course of the turbidity change was measured in the same manner as in Example 2 except that the electrical pretreatment of the reagent was not performed, and the turbidity difference for 5 minutes was determined. The results are shown in Table 4 and FIG.

[0027]

[Table 4]

Example 4 Measurement of voltage for inducing electrolysis Sodium chloride was dissolved in purified water, and the concentration of sodium chloride was adjusted to 0 to 600 mM. The electrical conductivity and the electrolysis onset voltage of the sodium chloride aqueous solution of each concentration were measured. The applied voltage is a DC pulse voltage (pulse width 10 μsec, frequency 1
0 KHz). The results are shown in Table 5 and FIG.

[0029]

[Table 5]

From FIG. 7 (A), the electrical conductivity and the salt concentration are in a proportional relationship, and from FIG. 7 (B), the voltage inducing electrolysis is in a logarithmic relationship with the electrical conductivity. It can be seen that the smaller the electrical conductivity of the sample, the less likely it is to cause electrolysis.

[0031]

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

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a DC pulse voltage application experimental device used in the present invention.

FIG. 2 is a graph comparing the time course of turbidity change due to immunological agglutination reaction between the method of the present invention using a pretreatment reagent in which a DC pulse voltage is applied to a latex reagent in advance and a conventional method.

FIG. 3 shows the results of an immunological agglutination reaction between the method of the present invention in which a pretreatment in which a DC pulse voltage was previously applied was performed on a reagent containing latex and the case of performing a test solution containing no latex (Comparative Example 3). It is the graph which compared the time course of the turbidity change.

FIG. 4 is a schematic diagram of an AC voltage application experimental device used in the present invention.

FIG. 5 is a graph showing a relationship between a magnitude of an applied voltage and a change in turbidity when performing a pretreatment of applying an AC voltage to a latex reagent in advance.

FIG. 6 is a graph showing a comparison between the method of the present invention using a pretreatment reagent in which an AC voltage is applied to a latex reagent in advance and the conventional method with respect to the relationship between human AFP concentration and turbidity change due to immunological agglutination. is there.

FIG. 7A is a graph showing a relationship between salt concentration and electric conductivity. (B) It is a graph showing the relationship between the electric conductivity and the induced voltage of electrolysis.

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

Claims (5)

[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 a minute voltage is applied in advance as the reagent. A method comprising using a reagent. 2. The method according to claim 1, wherein the minute voltage is derived from a direct current, a direct current pulse or an alternating current. 3. The method according to claim 1, wherein the minute voltage is a voltage at which the aqueous medium containing the reagent does not substantially cause electrolysis. 4. The method according to claim 1, wherein the carrier has an average particle size of 0.01 to 1 μm. 5. A reagent comprising a carrier to which a microvoltage has been applied in advance and which has been sensitized with an antibody or an antigen.