JPH1073596A - Method for detecting or measuring immunological active substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accelerate immunological aggregation reaction and to make it possible to perform highly sensitive detection and measurement by applying a minute voltage on a reaction system for detecting immunological active substance. SOLUTION: As immunological active substance, serum protein such as material, e.g. albumin, transferrin or the like, which is measured by immunological aggregation reaction, is used. As the minute voltage, DC, DC pulses or AC is used, and the voltage in the range, wherein the reaction system does not cause electrolysis, is used. As the measuring method, a sample is added into solution, wherein buffer is the main component, e.g. the reagent (antiserum liquid) containing specified specimen is added after the processing at the specified temperature and time. Thereafter the processing is performed at the specified temperature and time. Then, after the specimen is added, the changing amount of turbidity is obtained with the minute voltage being applied.

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 a reagent containing no carrier. More specifically,
The present invention relates to a method for detecting or measuring an immunologically reactive substance quickly and simply and with high sensitivity by applying a small voltage derived from direct current, direct current pulse, or alternating 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, an immunoturbidimetric method, an immunoturbidimetric method, and an integrating sphere turbidity method in which both are combined are known. There are many epitopes on the molecular surface of the macromolecule protein that serves as an antigen.When an antibody with a complementary structure approaches the epitope, they form an insoluble aggregate due to hydrophobic bonding, hydrogen bonding, van der Waals force, etc. Since turbidity is generated, the turbidity can be optically measured and quantitatively analyzed. These methods are extremely useful as clinical testing methods in terms of simplicity, speed, economy, etc., but the sensitivity of detectable immunologically reactive substances is limited. In order to detect an immunologically reactive substance, a high sensitivity reagent in which an antibody or an antigen is supported on carrier particles such as latex, liposome, and colloidal gold is used. 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. At this time, the time required for measurement (reaction) is about 10
Minutes.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have used a reagent comprising a buffer in which an antibody or an antigen is suspended without containing a carrier used in an immunoturbidimetry or an immunoturbidimetry. Thus, the present inventors have found a method for promoting an immunological agglutination reaction more than the conventional method and detecting or measuring an immunologically reactive substance with high sensitivity, and have reached the present invention.

[0004]

SUMMARY OF THE INVENTION The present invention relates to a method for detecting or measuring an immunologically reactive substance by immunological agglutination using an antigen or an antibody as a reagent. Is provided. 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. The immunologically reactive substance in the present invention can be selected from substances that can be measured by the above-mentioned immunological agglutination reaction. Examples include serum proteins such as CRP, albumin, transferrin, myoglobin, LP (a), C4, C3, IgG, IgM, α1 antitrypsin and the like. The detection range is generally 10 μg / dl to 100 mg / dl, and the detection sensitivity is about 10 μg / dl to 1 mg / dl. As the antigen or antibody used as the reagent, for example, an antibody capable of immunologically recognizing the above serum protein is used.

In the present invention, the minute voltage is derived from direct current, direct current pulse, or alternating current. The small voltage is preferably a voltage in a range where the reaction system does not substantially cause electrolysis, for example, when the electric conductivity of the reaction system is 17.4 mS.
/ Cm (equivalent to saline), electric field strength 2.4V /
mm DC pulse voltage (pulse width 10 μS, 10 KH
z). 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 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.

A typical measuring device for an immunologically reactive substance of the present invention is, for example, as shown in FIG.
A power supply device that can apply an arbitrary voltage such as AC voltage or DC voltage, a container with electrodes attached to the inner surface of the container for applying a small voltage to a reaction system containing a reagent, and monitoring of voltage, frequency, and pulse width And an oscilloscope for measuring the turbidity. As a power supply,
An arbitrary pulse generator, for example, a commercially available function generator or cell stimulator can be used.
As the container with electrodes, an insulating container such as glass or plastic can be used, and as the 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 solution containing a buffer as a main component, and the mixture is usually treated at room temperature to 37 ° C. for 0 to 5 minutes.
Then, for example, after adding a reagent solution (antiserum solution) containing a predetermined antibody or to a solution containing a buffer as a main component, the antiserum solution is usually treated at room temperature to 37 ° C for 0 to 5 minutes. Then, after adding a specimen, while applying a minute voltage or after applying a minute voltage, for example, a method of obtaining the turbidity change amount using the above-described measuring device can be used. In the latter method of adding a specimen to a solution containing a reagent, the specimen can be added while applying a minute voltage or immediately after the application, and then a change in turbidity can be measured.

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.
The concentration is 100 mS / cm, more preferably 2 mS / cm.
６０60 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 the composition of the aqueous medium containing the reagent, a composition well known to those skilled in the art can be used. Reagents include:
For example, proteins such as albumin, polymers such as polyethylene glycol, surfactants such as Tween and Triton,
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.

[0009]

The present invention will be described in detail with reference to the following Examples and Comparative Examples, which do not limit the present invention.

Example 1 (1) CRP immunoturbidimetric reagent N-assay TIA CRP-S (Nitto Bo Medical) was used. Reagents consist of Tris buffer and antiserum (20% anti-CRP goat serum antibody). (2) DC pulse voltage applying device Using the experimental device of FIG. 1, a DC pulse voltage was applied by setting the condition of the potential applied between the electrodes using an oscilloscope. (3 mm between electrodes) (3) Measurement method A Tris buffer 840 μm was placed in a glass cell with electrodes attached.
36 μl of the sample was dispensed to each sample, incubated at 37 ° C. for 5 minutes, and the absorbance (OD1) was measured. 6 V, pulse width 10 μs,
(Frequency 10 KHz) was applied for 30 seconds. Incubation was further performed for 5 minutes, and the absorbance (OD2) was measured again. ΔOD value (OD2-OD1) for various samples
And a calibration curve was prepared. The specimen was a phosphate buffer containing 0.2% bovine serum albumin (hereinafter referred to as 0.2% bovine serum albumin).
% BSA-PBS), a commercially available CRP standard solution was diluted to prepare a sample having a CRP concentration of 0 to 4.1 mg / dl, and used for measurement. (4) Results The results are shown in Table 1 and FIG.

[0011]

[Table 1]

Example 2 Using the same samples and reagents as used in Example 1, the pulse voltage applied to the reaction system was set to a peak value of 4.6 V and a pulse width of 2
The operation was performed in the same manner as in Example 1 except that the frequency was set to 0 μs, the frequency was set to 10 KHz, and the voltage was applied for 18 seconds, and the turbidity change was measured to prepare a calibration curve. The results are shown in Table 2 and FIG.

[0013]

[Table 2]

Comparative Example 1 The same samples and reagents as used in Example 1 were used. Except that no pulse voltage was applied to the reaction system, the operation was performed in the same manner as in Example 1, and the turbidity change was measured to prepare a calibration curve. The results are shown in FIG. 2 and FIG. From the results shown in FIGS. 2 and 3, it can be seen that the method of the present invention promotes an immunological agglutination reaction and can detect or measure an immunologically reactive substance with high sensitivity compared to the conventional method. Is shown.

Example 3 Measurement of Voltage Inducing Electrolysis Sodium chloride was dissolved in purified water and adjusted to a sodium chloride concentration of 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 3, FIG. 4, and Table 4.
And FIG.

[0016]

[Table 3]

[0017]

[Table 4]

From FIG. 4, the electric conductivity and the salt concentration are in a proportional relationship, and from FIG. 5, the voltage that induces electrolysis is:
There is a logarithmic function relationship with the electric conductivity, and it can be seen that the lower the electric conductivity of the reagent, the more difficult it is to cause electrolysis.

[0019]

According to the present invention, immunological nephelometry or immunoturbidimetric reagents are used to promote immunological agglutination more rapidly than conventional methods,
Further, the immunologically reactive substance can be detected or measured with high sensitivity.

[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 calibration curves in a low concentration range between a conventional method and a method according to the present invention.

FIG. 3 is a graph comparing a calibration curve in a low concentration range between the conventional method and the method according to the present invention.

FIG. 4 is a graph showing the relationship between salt concentration and electric conductivity.

FIG. 5 is a graph showing a relationship between electric conductivity and induced voltage of electrolysis.

 ──────────────────────────────────────────────────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) using an antigen or an antibody as a reagent,
A method for detecting or measuring an immunologically reactive substance by an immunological agglutination reaction, wherein a minute voltage is applied to the reaction system. 2. The method of claim 1, wherein the minute voltage is derived from direct current, direct current pulses, or alternating current. 3. The method according to claim 1, wherein the minute voltage is a voltage at which the reaction system does not substantially cause electrolysis.