JP2002048796A - Method for detecting or measuring presence of immunologically reactive material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect or measure presence of immunologically reactive material more sensitively and more accurately than a conventional measuring method. SOLUTION: In this method, immunologically reactive material is detected or measured by an immunological agglutination reaction by using a reagent formed by sensitizing an antigen or an antibody on a carrier. In the method, the reaction is measured optically by using light having two wavelengths selected from a wavelength range of 540 nm-800 nm and having the wavelength difference of 140 nm-230 nm, and a reactivity curve is formed from the difference of the absorbance measured in the two wavelengths, and the quantity of an absorbance change in an observation section from the absorbance before stabilization of a base line to the absorbance after a fixed time is determined, to thereby detect or measure the immunologically reactive material.

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 reaction is optically measured using two wavelengths, and the difference between the absorbances measured at the two wavelengths is defined as a reactivity curve. The change in absorbance from the absorbance immediately after the start of the reaction to the absorbance after a certain period of time is observed. The present invention relates to a method for detecting or measuring an immunologically reactive substance with high accuracy and high sensitivity by expanding a measurement range by obtaining an amount.

[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. is there. As a method for detecting or measuring an immunologically reactive substance, an optical analysis method for measuring a specific agglutination reaction on a carrier such as latex or liposome (turbidimetry,
The ratio measurement method and the counting method have been developed. In these analysis systems, a reaction is started by dispensing a sample sample and a sensitizing reagent into a reaction cell and mixing them with a stirring blade or the like, and is performed at a temperature of 37 ° C to 45 ° C.
At this time, the time required for the measurement (reaction) is 1 to 20 minutes. For actual measurement, a general biochemical / immunochemical automatic analyzer or a dedicated analyzer is used.

However, there are various problems in application to these automatic analyzers. In the reaction, there is a problem that the measurement accuracy is deteriorated due to the effects of optical and electric noises and the stirring effect. As a method of solving this problem, for example, in Japanese Patent Publication No. Hei 8-14581, by using two-wavelength photometry as compared with one-wavelength photometry, dirt, electrical or It proposes to absorb variations in the absorbance baseline due to optical causes. This method uses a specific range of short and long wavelengths and is not always satisfactory.

[0004]

However, it is difficult to sufficiently avoid the disturbance of the absorbance baseline due to the stirring of the stirring blade. Therefore, the amount of change in the absorbance after the baseline is stabilized is determined. The measurement accuracy was improved. In this case, since about one minute has been spent since the start of the reaction, the immunological agglutination reaction proceeds early in a sample containing a high concentration of the substance to be detected or measured. The inability to do so narrowed the measurement range and also affected the measurement accuracy of low concentrations.

[0005] The concept of setting parameters when optically measuring immunological agglutination using an automatic analyzer is already known. In order to measure low-concentration antigens or antibodies with good reproducibility, or when setting parameters for the purpose of increasing the detection limit, it is necessary to analyze absorbance in a section where the baseline of absorbance is not disturbed by the influence of stirring or the like. ing. Generally, after the start point of the absorbance observation section is delayed and the baseline is stabilized (about 1 minute after the start of the reaction)
It was a common theory to perform absorbance analysis. However, according to the present invention, by identifying the difference between the two wavelengths in photometry, an attempt was made to reverse the concept of the conventional concept. It was also found that the reproducibility was improved without affecting the reproducibility and the detection limit could be increased, or that highly sensitive and accurate measurement could be performed. The prozone can be expanded by setting the start point of the observation section immediately after the reaction.

In order to set strong parameters for the prozone, it is desirable to make the observation start point earlier (immediately) or the observation end point earlier. However, in these cases, correct measurement of a low value cannot be performed. The concept is to determine the start point of the observation section depending on whether priority is given to the low concentration area or the high concentration area. In the normal case, since the low concentration range is prioritized, the observation starting point was adopted about one minute after the start of the reaction. The present invention has been made based on an idea contrary to the conventional common sense described above. By specifying the wavelength difference between two wavelengths, the influence of baseline fluctuation which affects the measurement immediately after the start of the reaction is considered. It is an object of the present invention to provide a method of determining an amount of change in absorbance under conditions that are difficult to receive.

[0007]

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. Is measured optically using two wavelengths, and the difference between the absorbances measured at the two wavelengths is defined as a reactivity curve, and the observed section is the absorbance change from the absorbance before the stabilization of the baseline to the absorbance after a certain time. The present invention relates to a method for detecting or measuring an immunologically reactive substance by obtaining the same.

[0008] The two wavelength light used in the present invention has a wavelength of
It is selected from the range of 540 nm to 800 nm and the difference between the two wavelengths is 140 to 260 nm, preferably 200 to 230 nm. 540nm
One wavelength (dominant wavelength) in the range of ~ 600nm and 700 ~ 800n
One wavelength (sub-wavelength) having a range of m, and the difference between the two wavelengths is preferably 200 to 230 nm. In the observation section, the observation start point is before the stabilization of the absorbance baseline, and specifically, it is desirable that the observation section calculates the absorbance change amount from the absorbance within 40 seconds from the start of the reaction to the absorbance after a certain period of time. It is less than 30 seconds immediately after the start of the reaction. In the present invention, the term "before stabilization of the absorbance baseline" refers to the time until the disturbance of the absorbance baseline due to the influence of stirring or the like observed in the measurement of absorbance at one wavelength stabilizes, usually 60 seconds to 40 seconds. Less than time.

In the present invention, the immunological agglutination is
This method utilizes 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-described immunological agglutination reaction by an agglutination method using latex, liposome, or the like as a carrier. For example, CRP, HBs antigen, HBs antibody, HC
Infectious disease markers such as V antibody, HIV antibody, TP antibody, D
-Coagulation / fibrinolysis markers such as dimer, protein C, protein S, ATIII, AFP, CEA, CA19-9
Tumor markers such as TSH, prolactin and insulin, tissue components such as β2m, myoglobin and myosin, and nucleic acids such as DNA.

The carrier particles of the present invention include latex, liposome, colloidal gold and the like, and are preferably latex particles. As latex particles,
Those generally used for immunological agglutination can be used. The average diameter of the carrier particles is 0.
01 to 1 μm is preferred. More preferably, 0.05 to
0.5 μm. As the reagent composition, a composition well known to those skilled in the art can be used.Examples include a protein such as albumin based on a glycine buffer / Tris-HCl buffer or a phosphate buffer, and a polymer such as polyethylene glycol. , Tween, Triton and the like, chlorides such as choline chloride, amino acids such as arginine and asparagine, and preservatives such as sodium azide.

The sensitization of latex particles with an antibody or an antigen can be carried out, for example, by adsorbing or binding by a conventionally known method. As the reagent that can be used in the present invention, a commercially available reagent that is applied to an automatic analyzer based on immunological agglutination can be used, and generally, a two-reagent system (a diluent and a latex reagent sensitized with an antigen or antibody) is used. )
Often it is.

Next, the actual measuring method will be described in detail along the flow of a general automatic analyzer. First, a specimen sample and a first reagent (diluent) are dispensed into a reaction cell and mixed by a stirring blade. After a few minutes, the second reagent (latex reagent sensitized with the antigen or antibody) is dispensed and mixed by a stirring blade. One wavelength (main wavelength) having a wavelength of 540 nm to 660 nm and one wavelength (sub wavelength) having a wavelength of 700 to 800 nm
The absorbance is measured from immediately after (or immediately before) the sample sample and the first reagent are mixed to the end of the reaction using the two wavelengths. The measurement conditions differ slightly depending on the device,
The time until the end of the reaction is generally about 10 to 20 minutes. Also, since the wavelength range that can be measured also differs depending on the device, the specified range of the two wavelengths is not limited to the above, but in the measurement according to the present invention, the difference between the two wavelengths is preferably 140 to 260 nm, more preferably Is 200-230 nm. Wavelength difference is 100n
If it is smaller than m, the value obtained by subtracting the absorbance measured at two wavelengths will be small, which will hinder the measurement of low concentration.
If the difference between the two wavelengths is 300 nm or more, it is not possible to absorb the baseline fluctuation within 1 minute of the observation start point, so the start point had to be delayed. However, in this case, it is not preferable because the measurement range is narrowed because it hinders the measurement in the high concentration range, and the prozone is easily affected by the prozone.

From the absorbance measured as described above, a reactivity curve (time course) consisting of a difference in absorbance at two wavelengths is obtained. From the reactivity curve, the presence or absence of an immunological reactant is detected or measured by calculating the amount of change in absorbance in an observation section in which parameters are set in advance. It is preferably within 40 seconds immediately after the start, more preferably within 30 seconds. In addition, the end point of the observation section is determined in consideration of the linear area of the time course and the like. These calculation processes may be slightly different depending on the device, but the concept is the same. The amount of change in absorbance can be calculated by the end method, the rate method, or the like. For example, in the end method, it can be obtained from the absorbance difference between the observation start point and the end point. At this time, the observation start point and / or end point is an average value or a median value of one to several points. In the rate method, it can be obtained by the least square method or the like from the observation start point to the end point. The present invention is particularly useful when the reactivity curve in the observation section includes a point out of the linear range.

[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. In the embodiment, for example, a main wavelength of 405 nm and a sub wavelength of 800 nm
Are referred to as wavelengths 405-800 nm.

Example 1 (1) Preparation of CRP latex reagent 10 mg of anti-CRP antibody was added to 9.5 ml of 50 mM glycine buffer, pH 8.2 (containing 150 mM NaCl, 0.1% NaN ₃ , hereinafter referred to as G
BS), and 0.5 ml of latex (particle diameter: 0.12 μm, solid content: 10% suspension) was added thereto.
After overnight incubation at, the sensitized latex suspension was centrifuged to remove the supernatant. 1% B precipitation
The suspension was suspended in 20 ml of SA-GBS to prepare a CRP latex reagent. 1% BSA-GBS was prepared as a diluent.

(2) Measurement method Measurement was performed using a Hitachi 7150 automatic analyzer with the following parameters. Sample volume: 3 μL, first reagent (diluent), 180 μL, second
Reagent (CRP latex reagent) 60 μL, wavelength: 405-800,
505-800, 546-800, 570-800, 600-800, 660-800nm 2
Wavelength, observation interval: 27-35P, 28-35P, 29-35P, 30-35P.
Formula: Endpoint. CRP control serum (concentration 0.20mg
/ dL) was used as the sample for the simultaneous reproducibility test (N = 1
0). Next, CRP high concentration control serum (concentration 30-60mg / dL)
Was measured as a specimen sample.

(3) Results From the results shown in Table 1, the present invention has improved simultaneous reproducibility as compared with the conventional method (the observation starting point is about one minute later), and the difference between the two wavelengths is reduced from 140 to 260 nm. It can be seen that the measurement can be performed with high accuracy by limiting. Also, from the results shown in FIG. 1, it can be seen that by limiting the difference between the two wavelengths to 200 to 230 nm, the prozone can be improved and a wide measurement range can be measured.

Example 2 (1) Preparation of CRP Latex Reagent A CRP latex reagent was prepared in the same manner as in Example 1. (2) Measurement method Measurement was performed with the following parameters using a Hitachi 7170S automatic analyzer. Sample volume: 3 μL, first reagent (diluent), 150 μL, second
Reagent (CRP latex reagent) 50μL, wavelength: 570-800nm
2 wavelengths, observation interval: 17-34P, 18-34P, 19-34P, 20-34
P, 21-34P. Formula: Endpoint. A simultaneous reproducibility test was performed using CRP control serum (concentration: 0 to 0.10 mg / dL) as a sample (N = 10). Next, medium to high concentration control serum of CRP (concentration 0 to 100 mg / dL) was measured as a sample.

(3) Results From the results shown in Tables 2 and 3, in the present invention, the start point of the observation section is made as short as 40 seconds immediately after the reaction as compared with the conventional method (the observation start point is about 1 minute later). This shows that simultaneous reproducibility is improved, and measurement can be performed with high sensitivity and high accuracy. Further, from the results shown in FIG. 2, it can be seen that the prozone can be improved and the measurement in a wide measurement range can be performed by setting the observation section start point as early as 40 seconds immediately after the reaction.

Example 3 (1) Preparation of CRP Latex Reagent A CRP latex reagent was prepared in the same manner as in Example 1. (2) Measurement method Measurement was performed with the following parameters using a Toshiba 200FR automatic analyzer. Sample volume: 3 μL, first reagent (diluent), 150 μL, second
Reagent (CRP latex reagent) 50μL, wavelength: 574-804nm
2 wavelengths, observation interval: 18-23P, 19-23P, 20-23P, 19-24
P, 20-25P. Arithmetic formula: Rate A simultaneous reproducibility test was performed using CRP control serum (concentration: 0 to 0.20 mg / dL) as a sample (N = 10). (3) Results From the results shown in Table 4, the present invention sets the observation section start point immediately after the reaction as compared with the conventional method (observation start point is about 1 minute later).
It can be seen that simultaneous reproducibility is improved by setting the time as short as 40 seconds or less, and measurement can be performed with high sensitivity and high accuracy.

Example 4 (1) Preparation of ferritin latex reagent 9.5 ml of 10 mg of anti-ferritin antibody (F (ab ') ₂ )
0.1 M Tris-HCl buffer pH 7.6 (150 mM NaCl, 0.1% NaN
₃ containing, hereinafter abbreviated as Tris-HCl), add 0.5 ml of latex (particle diameter 0.34 μm, solid content 10% suspension), incubate overnight at 37 ° C., and sensitize latex The suspension was centrifuged to remove the supernatant. The precipitate was suspended in 20 ml of 1% BSA-Tris-HCl to prepare a CRP latex reagent. As a diluent,
1% BSA-GBS was prepared.

(2) Measurement method Measurement was performed using the Hitachi 7170S automatic analyzer with the following parameters. Sample volume: 9 μL, first reagent (diluent), 180 μL, second
Reagent (ferritin latex reagent) 30μL, wavelength: 600-8
Two wavelengths of 00 nm, observation sections: 17-33P, 18-33P, 19-33P.
Formula: Endpoint. Ferritin control serum (concentration 7.
5, 10, 15 ng / mL) were used as specimen samples for simultaneous reproducibility tests (N = 10). (3) Results From the results shown in Table 5, the present invention sets the observation start point 30 minutes after the reaction immediately after the conventional method (the observation start point is about 1 minute later).
It can be seen that simultaneous reproducibility is improved by setting the time as short as within seconds, and measurement can be performed with high sensitivity and high accuracy.

[0023]

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

[0024]

[Table 1]

[0025]

[Table 2]

[0026]

[Table 3]

[0027]

[Table 4]

[0028]

[Table 5]

[Brief description of the drawings]

FIG. 1 is a graph showing an amount of change in absorbance obtained from a reactivity curve based on a difference between two wavelengths when a high-concentration sample is measured at different wavelengths.

FIG. 2 is a graph showing the amount of change in absorbance when the observation start point is changed when a sample specimen of each concentration is measured.

Claims (4)

[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 the reaction is performed optically.
By measuring the difference in absorbance measured at two wavelengths using the light of the wavelength, the difference between the absorbance measured at the two wavelengths is defined as the reactivity curve. A method comprising detecting or measuring an immunologically reactive substance. 2. The method according to claim 1, wherein the light of two wavelengths is selected from the range of 540 nm to 800 nm, and the observation section calculates the amount of change in absorbance from the absorbance within 40 seconds after the start of the reaction to the absorbance after a predetermined time. The method of claim 1. 3. The method of claim 1, wherein the difference between the two wavelengths is between 140 and 260 nm. 4. One wavelength (main wavelength) in the range of 540 nm to 600 nm and one wavelength (sub wavelength) in the range of 700 to 800 nm.
The method according to claim 1, wherein the difference between the two wavelengths is from 200 to 230 nm, and the observation start point is less than 30 seconds immediately after the start of the reaction.