JP2004028954A - Method for stabilizing reaction and enhancing measurement accuracy in immunity measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stabilize reaction and to enhance measurement accuracy by relaxing the effects of external variation factors on a reagent for an immunity measuring method comprising two liquids applicable to an autoanalyzer for biochemical inspection. <P>SOLUTION: This immunity measuring method is based on a condensation method using a first reagent including a buffer solution and a second reagent including an antibody or antigen. According to this immunity measuring method, measurement is performed by using an autoanalyzer and the first and second reagents are put in a reaction vessel. With respect to the amount of reagents added to a reaction system, the first reagent is equal to or more than 1.5 in volume relative to the second reagent, thereby stabilizing reaction and enhancing measurement accuracy. <P>COPYRIGHT: (C)2004,JPO

Description

【００２８】
また、測定値の変動をグラフで確認すると、実施例では測定回数に拘らずほぼ横這いであり、測定値の変動は分析精度に由来するものと考えられるが、比較例では測定回数１〜５回の間で測定値の上昇が見られ、その後安定する傾向が確認できる。
【００２９】
これは、測定開始前、装置が停止していた３０分間に装置内のチューブに滞留していた第２試薬が保冷状態にない部分のみ温められ、測定に影響を及ぼしたものと考えられる。このとき、試薬が設置された試薬庫の温度は約１０℃、試薬が分注されるチューブ先端部は、３０℃を超えていた。したがって、測定開始直後の測定においては約３０℃温まった試薬が分注され、その後保冷状態にあった試薬が送り出されることで試薬の温度が徐々に低下し、試薬温度の安定に伴って測定値の安定が見られるようになったものである。
【００３０】
実施例２　ＣＲＰ測定試薬における測定精度確認試験　その２
本発明が試薬の製造ロットが異なっても適用できることを確認するため、第２試薬としてロット違いで３種類（Ｌｏｔ．１０１０２、Ｌｏｔ．１４１０７およびＬｏｔ．１８１１２）を用い、実施例１と同様な方法で測定を行った。添加容積比が１：１の例は比較例である。
【００３１】
表２〜表４に第２試薬のロット毎に各条件について、第１試薬、第２試薬および検体の量ならびに連続２０回の測定を行った場合のそれぞれの平均値、標準偏差および変動係数を示した。また、図３に検量線を、図４に測定値の変動を示した。
【００３２】
【表２】

【００３３】
【表３】

【００３４】
【表４】

表２、表３、表４、図３および図４に示されるように、第２試薬のロットが異なっても同様の結果が得られた。
【００３５】
実施例３　ＣＲＰ測定試薬における測定精度確認試験　その３
第１試薬および第２試薬容積を同じにし、第２試薬を通常の試薬庫設置により８℃（比較例）、室温設置により２７℃（条件１）、恒温槽設置により３７℃（条件２）に維持し、実施例１と同様に検討を行った。
表５に各条件について、第１試薬、第２試薬および検体の量ならびに連続２０回の測定を行った場合のそれぞれの平均値、標準偏差および変動係数を示した。また、図５に検量線を、図６に測定値の変動を示した。
【００３６】
【表５】

表５、図５および図６に示されるように、第２試薬の温度を８℃から２７℃または３７℃に上昇させることにより、反応が安定し測定精度が向上した。
【００３７】
【発明の効果】
実施例１および２が示すように、本発明の方法に従って、反応系に添加する試薬の量を第２試薬１容に対して第１試薬を１．５容以上とすることにより、生化学検査用自動分析装置における免疫測定法による試薬において、外的な変動要因の影響を軽減し、より正確な測定を実現することができる。また、実施例３が示すように、本発明の方法に従って、第１試薬を保冷しておき、第２試薬を室温から反応温度に置いておくことにより、外的な変動要因の影響を軽減し、より正確な測定を実現することができる。
【図面の簡単な説明】
【図１】実施例１および比較例におけるＣＲＰ濃度と規定時間内の吸光度変化量の関係を示した図である。
【図２】実施例１及び比較例におけるＣＲＰ濃度１０ｍｇ／ｄＬの試料を２０回連続測定した場合の変動を測定回数毎に示す図である。
【図３】実施例２および比較例におけるＣＲＰ濃度と規定時間内の吸光度変化量の関係を示した図である。
【図４】実施例２及び比較例におけるＣＲＰ濃度１０ｍｇ／ｄＬの試料を２０回連続測定した場合の変動を測定回数毎に示す図である。
【図５】実施例３および比較例におけるＣＲＰ濃度と規定時間内の吸光度変化量の関係を示した図である。
【図６】実施例３及び比較例におけるＣＲＰ濃度１０ｍｇ／ｄＬの試料を２０回連続測定した場合の変動を測定回数毎に示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention aims to stabilize the reaction and improve the measurement accuracy by mitigating the influence of external fluctuation factors in a two-solution immunoassay reagent applicable to an automatic analyzer for biochemical testing. About the method.
[0002]
[Prior art]
The immunoassay is applied to clinical tests as a method for quantifying antigenic substances or antibodies contained in body fluids such as serum, plasma, urine, and cerebrospinal fluid.Currently, methods adapted to automatic analyzers for biochemical tests are widely used. are doing. In recent years, immunoassays for clinical tests have been realized with high sensitivity by using an insoluble carrier or the like, and the measurement accuracy has been significantly improved. For example, many reagents capable of performing highly sensitive measurement using a technique using two or more types of insoluble carrier particles having different particle sizes (Japanese Patent No. 2588174) have been developed.
However, in a reagent whose measurement accuracy has been improved by increasing the sensitivity, slight fluctuation of an external factor may affect the measurement due to high sensitivity. In particular, there has been a problem that the measured values fluctuate when the temperature conditions are unstable.
[0003]
There are various types of automatic analyzers for biochemistry used for measurement, and they can be divided into two types according to the method of dispensing reagents. One is a method in which a reagent is dispensed by pipetting, in which a part called a reagent dispensing probe sucks a reagent from a reagent bottle placed in a reagent storage and discharges it to a reaction tank. That is, the reagent dispensing probe moves between the reagent storage and the reaction tank, aspirates and dispenses the reagent, and dispenses several different reagents with one probe. The other is a method in which a reagent bottle placed in a reagent storage is dispensed into a reaction tank via a tube, and a reagent is continuously sucked from the reagent bottle and continuously discharged into the reaction tank. . That is, the reagent is aspirated at one end of the tube and dispensed at the other end, and only one reagent is operated per tube. In any method, the suction and discharge of the reagent are performed by a syringe method, and the switching between suction and discharge is performed by an electromagnetic valve.
Among these two types of devices, the tube type device has a problem in that the measured values are not stable for a while after the start of the measurement and accurate quantification cannot be performed.
[0004]
[Problems to be solved by the invention]
SUMMARY OF THE INVENTION It is an object of the present invention to solve the problem that a measured value is not stable after the start of measurement in measurement by an automatic analyzer for biochemical testing. Specifically, by providing a method for stabilizing the reaction in order to suppress a decrease in measurement accuracy caused by a change in temperature of the entire reaction solution due to the addition of the second reagent in the measurement by the automatic analyzer for biochemical testing, is there.
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problem, and found the cause of the instability of the measured value. That is, in any of the reagent dispensing probe system and the tube system of the above-mentioned automatic analyzer for biochemistry, the reagent storage for installing the reagent in order to stably maintain the reagent is often cooled, and the reagent is not used. Keep at low temperature. However, in the reagent dispensing section, cooling is practically impossible. In particular, the reaction tank, which is the reaction site, is in a thermostat kept at 37 ° C., and the tip of the reagent dispensing section close to the reaction tank is relatively Has been placed in a hot condition.
[0006]
In the pipetting type apparatus, the timing from the suction to the discharge of the reagent is constant, and the reagent is always dispensed under the same conditions. However, in the tube type apparatus, the measurement is started from a state where the apparatus is stopped. The temperature of the dispensed reagent differs between the case and the case where the reagent is continuously discharged. That is, when the apparatus is stopped, the reagent in the tube connecting the reaction tank from the cool box stays in the tube and is kept at room temperature, so when the apparatus is started, it is initially heated to room temperature. The reagent is added to the reaction system, but the reagent which has been kept at low temperature in the cool box is added to the reaction system without being heated.
[0007]
When the immunoassay is performed in a tube-type apparatus, a test sample and a first reagent, which is a buffer, are first mixed in a reaction tank, and the mixture is generally incubated at 37 ° C. for about 5 minutes. Is warmed to 37 ° C. Thereafter, a second reagent containing a component directly involved in the antigen-antibody reaction is added. When the temperature of the second reagent is low, the temperature of the reaction solution as a whole is fluctuated, thereby affecting the reaction. Therefore, the reaction system is established in consideration of such a temperature change. However, as described above, if there is a difference in the temperature of the second reagent added to the reaction system immediately after the operation of the apparatus and after a certain period of time, the reaction system will not be stable, and the measured value will be different. This causes a decrease in measurement accuracy.
[0008]
Based on this elucidation of the cause, the inventors of the present application conducted studies to reduce the effect of temperature change due to the addition of the second reagent. As a result, in order to stabilize the temperature conditions during the reaction, the amount of the first reagent was It has been found that the ratio of the amount of the second reagent may be modified.
That is, it has been found that by increasing the ratio of the first reagent heated to 37 ° C. prior to the reaction, it is possible to suppress a change in the temperature of the entire reaction solution caused by a temperature difference between the second reagents. The present inventors have conducted intensive studies on the volume ratio of the first reagent and the second reagent, and as a result, completed the present invention.
[0009]
Furthermore, the inventors of the present application have tried to solve the above-mentioned problem of the decrease in measurement accuracy due to the instability of the reaction system from another approach, and have not increased the temperature of the second reagent in the cool box and raised the temperature in advance. As a result, it was found that the problem of a decrease in measurement accuracy could be solved, and the present invention was completed.
[0010]
That is, the present invention is as follows.
(1) An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer, and the first reagent and the second reagent are used. In the immunoassay to be added to the reaction tank, the reaction is stabilized and the measurement accuracy is improved by setting the amount of the reagent added to the reaction system to 1.5 volumes or more of the first reagent with respect to 1 volume of the second reagent. Method,
(2) The method according to (1), wherein the second reagent comprises a particulate insoluble carrier sensitized with an antigen or an antibody.
(3) The method according to (1) or (2), wherein the measurement is performed under a temperature condition of 25 ° C. or more and 37 ° C. or less.
(4) Any of (1) to (3), wherein the automatic analyzer used for measurement has a means for continuously dispensing via a tube when dispensing the reagent to the reaction tank. Method,
(5) An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the immunoassay is performed using an automatic analyzer and added to the reaction system. By making the amount of the first reagent 1.5 volumes or more with respect to 1 volume of the second reagent, the immunization including at least the first reagent and the second reagent for stabilizing the reaction and improving the measurement accuracy is performed. A measurement kit, wherein the volume of the first reagent is larger than the volume of the second reagent,
(6) An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer, and the first reagent and the second reagent are combined. In the immunoassay method to be added to the reaction tank, the first reagent is kept cool and the second reagent is kept at a temperature ranging from room temperature to the reaction temperature during the measurement, thereby stabilizing the reaction and improving the measurement accuracy. How to make
(7) The method according to (6), wherein the second reagent comprises a particulate insoluble carrier sensitized with an antigen or an antibody.
(8) The method according to (6) or (7), wherein the measurement is performed under a temperature condition of 25 ° C. or more and 37 ° C. or less.
(9) Any of (6) to (8), wherein the automatic analyzer used for the measurement has means for continuously dispensing via a tube when dispensing the reagent to the reaction tank. Method and (10) Automatic analysis capable of performing an immunoassay in which the first reagent and the second reagent are added to a reaction tank based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen. An automatic analyzer for use in an immunoassay method, which comprises a storage means for cooling a first reagent and a storage means for holding a second reagent at a reaction temperature from room temperature to a reaction temperature, thereby stabilizing a reaction and improving measurement accuracy.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention relates to an immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer, and the first reagent and the second reagent are used. In the immunoassay method in which is added to the reaction vessel, the amount of the reagent added to the reaction system is 1.5 volumes or more of the first reagent to 1 volume of the second reagent, thereby stabilizing the reaction and improving the measurement accuracy. It is a way to make it. The present invention also relates to an immunoassay based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer and the first reagent and the second reagent are used. In the immunoassay method in which a reagent is added to a reaction tank, the first reagent is kept cool, and the second reagent is kept at a temperature in a range from room temperature to the reaction temperature, thereby stabilizing the reaction and improving the measurement accuracy. is there.
[0012]
The automatic analyzer used for measurement is an automatic analyzer for biochemical tests applicable to immunoassays, and has a mechanism that dispenses reagents to the reaction tank via tubes by a continuous dispensing method. It is. In other words, the apparatus is provided with means for installing a reagent inside or outside the apparatus, and the reagent is continuously dispensed through a tube into a reaction tank provided in the apparatus. The dispensing of the reagent through the tube is performed by a combination of a pump such as a peristaltic pump and a switching valve. As such an apparatus, there are various commercially available apparatuses, for example, Hitachi automatic analyzer 7250, automatic analyzer 7450, automatic analyzer 7350 and the like manufactured by Hitachi, Ltd.
[0013]
The method of the present invention can be applied to an immunoassay by an agglutination method using an insoluble carrier such as latex particles, bentonite, collodion, kaolin, fixed sheep erythrocytes and the like. In the immunoassay by the agglutination method, the insoluble carrier may be sensitized with an antibody when the measurement target is an antigen, that is, the antibody may be bound to the surface of the carrier, and when the measurement target is an antibody, the insoluble carrier may be sensitized with the antigen. . The present invention is particularly effective in a highly sensitive measurement system which is sensitively affected even if the reaction system is slightly unstable. As a high-sensitivity measurement system, for example, a measurement system using two or more particles having different sizes described in JP-A-2001-289853 as an insoluble carrier is exemplified. An immunoassay method based on an agglutination method, in which a measurement is performed using an automatic analyzer, wherein an agglutinate formed by binding an antigen or an antibody-sensitized carrier to a measurement target antibody or a measurement target antigen in a reaction tank is optically analyzed. The antigen or antibody is measured by capturing the target. That is, the aggregate is irradiated with light in the visible to near-infrared range, and a change in absorbance or a change in intensity of scattered light is detected to measure the degree of aggregation of the carrier particles. At this time, the measuring method includes the One Point method, the Two Point method, the Three Point method, the Rate method, and the like, but the method of the present invention can be applied to any measuring method. The test sample is previously dispensed into the reaction tank, the first reagent and the second reagent are dispensed there, the reaction is performed, and the degree of agglutination is measured. Alternatively, the antibody to be measured can be quantified. The order of addition of the first reagent and the second reagent is not limited, but preferably the first reagent is dispensed prior to the second reagent, or the first reagent and the second reagent are dispensed simultaneously. At this time, by preparing a standard curve in which the concentration of the measurement target and the degree of agglutination are associated using a plurality of samples whose concentrations of the measurement target are known in advance, the target substance in the test sample is determined based on the standard curve. Can be calculated.
[0014]
The first reagent may include a buffer, other stabilizing reagents, protective reagents, and the like. Examples of the second reagent include the above-mentioned particles sensitized with an antibody or an antigen that specifically binds to the antigen or the antibody to be measured, and may include other stabilizing reagents, protective reagents, and the like.
[0015]
As a first aspect of the present invention, the volume ratio of the first reagent and the second reagent to be added to the reaction system is set so that the ratio of the second reagent is reduced. In this case, one volume of the second reagent is used. On the other hand, the volume of the first reagent may be 1.5 volumes or more, preferably 2 volumes or more. The agglutination reaction can be optimized by appropriately adjusting the amount of the carrier sensitized with the antigen or antibody, the amount of the antigen or antibody sensitized to the carrier, the ratio of the test sample to the carrier, and the like. Depending on the volume ratio of the reagent and the second reagent, these amounts can be easily optimized.
[0016]
As a second aspect of the present invention, when the first reagent and the second reagent are set in the automatic analyzer, the first reagent is put into a cool box as usual and cooled, and the second reagent is kept cool. This may be at room temperature or preheated to around the reaction temperature at which the antigen-antibody reaction takes place. A plurality of reagent chambers capable of controlling the temperature may be installed in the automatic analyzer, one of which may be cooled to store the first reagent, and the other may be heated to store the second reagent, The second reagent may be taken out of the apparatus at the time of measurement and kept at room temperature. In this case, the amount of the first reagent and the amount of the second reagent to be added may be the same, or the amount of the first reagent and the amount of the second reagent may be reduced by combining the first mode and the second mode. You may change it.
[0017]
Further, the temperature condition of the reaction at the time of measurement is not particularly limited, but is 25 ° C. to 37 ° C., preferably 37 ° C. The reaction may be performed in a thermostat controlled at the temperature. Regarding the temperature control, either a water bath method or an air bath method is possible, and the temperature of the reaction tank to which the reaction reagent is added may be controlled using the water bath or the air bath.
[0018]
The operation timing at the time of measurement, specifically, incubation of 180 seconds or more after mixing of the test sample and the first reagent, the measurement of the absorbance is started 30 to 60 seconds after the time of adding the second reagent, The measurement is performed for 60 to 300 seconds. Preferably, the incubation after the mixing of the test sample and the first reagent is started for 240 to 300 seconds, the measurement is started 30 to 60 seconds after the addition of the second reagent, and the measurement is performed for 120 to 240 seconds.
[0019]
The substance measured by the method of the present invention is not subject to any other restrictions as long as it can be measured by an immunoassay. Examples include, but are not limited to, plasma protein components such as C-reactive protein (CRP). In addition, the test sample is not limited, and examples thereof include body fluids such as serum, plasma, urine, and cerebrospinal fluid, foods and drinks, and extracts thereof, but are not limited thereto.
[0020]
Furthermore, the present invention relates to an immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the immunoassay is performed using an automatic analyzer. The amount of the first reagent is 1.5 volumes or more, preferably 2 volumes or more, per 1 volume of the second reagent, so that the amount of the reagent to be added to the first reagent is at least 1 volume for stabilizing the reaction and improving measurement accuracy. Also included is an immunoassay kit including a reagent and a second reagent, wherein the volume of the first reagent is larger than the volume of the second reagent. When the volume of the first reagent is larger than the volume of the second reagent, the volume of the first reagent and the amount of the second reagent included in the kit correspond to the volume ratio of the reagent added during measurement, It means the amount that can be used at the same time without wasting only one of the reagents. These reagents in the kit are supplied in suitable bottles.
[0021]
Furthermore, the present invention relates to an automatic analyzer used for an immunoassay based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the first reagent and the second reagent are contained in a reaction tank. An automatic analyzer used for the added immunoassay includes an accommodating means for cooling the first reagent and an accommodating means for holding the second reagent at room temperature to the reaction temperature, thereby stabilizing the reaction and improving the measurement accuracy. It also includes an automatic analyzer used for the method.
[0022]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
Example 1 Measurement accuracy confirmation test for CRP measurement reagent 1
As the first reagent, a buffer solution (pH 7.0) based on glycine is used. As the second reagent, an anti-human CRP rabbit polyclonal antibody sensitized to polystyrene latex particles is used as a dispersion suspension liquid. At this time, polystyrene latex particles having an average particle diameter of about 0.20 μm and 0.07 μm were used together. According to the method of Cambiaso et al. (Methods in Enzymology Vol. 74; Section I, B [6]: 106-139), the second reagent is prepared by mixing the antibody and polystyrene latex in a glycine buffer at room temperature. After sensitization by leaving it for 60 minutes, and removing excess antibody by centrifugation, blocking treatment of the antibody-unsensitized portion of the polystyrene latex particle surface with a glycine buffer containing bovine serum albumin was performed, followed by centrifugation again. Was prepared by dispersing and suspending antibody-sensitized latex particles in a glycine buffer. The above reagents are commonly used in Examples and Comparative Examples.
[0023]
As a standard substance for preparing a calibration curve, a CRP standard prepared by diluting a purified human CRP antigen with normal human serum was used. The CRP standard was priced based on the plasma protein international standard CRM470, and 0.5, 2, 4, 16, and 32 mg / dL were prepared. Physiological saline was used as 0 mg / dL. Similarly, a sample of about 10 mg / dL was prepared as a sample for a measurement accuracy confirmation test.
[0024]
In the embodiment of the present invention, the first reagent amount is 180 μL, the second reagent amount is 90 μL, the sample amount is 2 μL (condition 1), and the ratio of the first reagent amount to the second reagent amount is 2: 1. The first reagent amount is 240 μL, the second reagent amount is 80 μL, and the sample amount is 2 μL (condition 2) so that the ratio of the first reagent amount to the second reagent amount is 3: 1. The measurement was performed under the conditions of the first reagent amount of 150 μL, the second reagent amount of 150 μL, and the sample amount of 3 μL, where the ratio of the second reagent amount is 1: 1.
[0025]
For the measurement, using a tube type automatic analyzer for biochemical test Hitachi 7250 type, after mixing the sample and the first reagent at 37 ° C. for 5 minutes, the reaction was started by adding the second reagent, It was set to measure the reaction from 30 seconds to 180 seconds after the start of the reaction as a change in absorbance at a wavelength of 570 nm. The conditions except for the amount of the sample and the amount of the reagent were measured in the same manner as in Examples and Comparative Examples. The measurement was carried out by holding both the first reagent and the second reagent in a cold storage at 8 ° C.
The amount of change in absorbance at each concentration of the CRP standard product was determined under the above conditions for each of the conditions of the examples and comparative examples, and calibration curve graphs showing the relationship between the CRP concentration and the amount of change in absorbance were created. This is shown in FIG.
[0026]
After measuring the CRP standard, the device is stopped for 30 minutes. Then, using the sample for the measurement accuracy confirmation test as a sample, continuous 20 measurements were performed for each condition, and the average value, standard deviation, and coefficient of variation were determined. This is shown in Table 1. FIG. 2 is a graph showing the fluctuation of the measured value.
[0027]
[Table 1]

[0028]
Also, when the fluctuation of the measured value is confirmed by a graph, it is almost flat regardless of the number of measurements in the example, and it is considered that the fluctuation of the measured value is derived from the analysis accuracy. The measured value increases during the period, and then a tendency to stabilize can be confirmed.
[0029]
This is considered to be due to the fact that only the part where the second reagent stayed in the tube in the apparatus was not kept in a cold state for 30 minutes while the apparatus was stopped before the measurement was started, which affected the measurement. At this time, the temperature of the reagent storage in which the reagent was set was about 10 ° C., and the temperature at the tip of the tube into which the reagent was dispensed exceeded 30 ° C. Therefore, in the measurement immediately after the start of the measurement, the reagent warmed at about 30 ° C. is dispensed, and then the reagent that has been kept cool is sent out, so that the temperature of the reagent gradually decreases, and the measured value is reduced as the reagent temperature stabilizes. It is now that stability has been seen.
[0030]
Example 2 Measurement accuracy confirmation test for CRP measurement reagent Part 2
In order to confirm that the present invention can be applied even when the production lot of the reagent is different, a method similar to that of Example 1 was used by using three different lots (Lot. 10102, Lot. 14107 and Lot. 18112) as the second reagent. Was measured. An example in which the addition volume ratio is 1: 1 is a comparative example.
[0031]
Tables 2 to 4 show, for each condition of each lot of the second reagent, the amounts of the first reagent, the second reagent, and the sample, and the respective average values, standard deviations, and coefficients of variation when performing 20 consecutive measurements. Indicated. FIG. 3 shows the calibration curve, and FIG. 4 shows the fluctuation of the measured value.
[0032]
[Table 2]

[0033]
[Table 3]

[0034]
[Table 4]

As shown in Table 2, Table 3, Table 4, FIGS. 3 and 4, similar results were obtained even when the lot of the second reagent was different.
[0035]
Example 3 Measurement accuracy confirmation test for CRP measurement reagent Part 3
The first reagent volume and the second reagent volume are made the same, and the second reagent is set to 8 ° C. (comparative example) by installing a normal reagent storage, 27 ° C. (condition 1) by installing at room temperature, and 37 ° C. (condition 2) by installing a constant temperature bath. It was maintained and examined in the same manner as in Example 1.
Table 5 shows, for each condition, the amounts of the first reagent, the second reagent, and the sample, as well as the average value, standard deviation, and coefficient of variation when performing 20 consecutive measurements. FIG. 5 shows a calibration curve, and FIG. 6 shows fluctuations of measured values.
[0036]
[Table 5]

As shown in Table 5, FIG. 5 and FIG. 6, by raising the temperature of the second reagent from 8 ° C. to 27 ° C. or 37 ° C., the reaction was stabilized and the measurement accuracy was improved.
[0037]
【The invention's effect】
As shown in Examples 1 and 2, according to the method of the present invention, the amount of the reagent added to the reaction system is set to 1.5 volumes or more of the first reagent with respect to 1 volume of the second reagent, whereby the biochemical test is performed. In the reagent by the immunoassay method in the automatic analyzer for use, the influence of external fluctuation factors can be reduced, and more accurate measurement can be realized. In addition, as shown in Example 3, by keeping the first reagent cold and keeping the second reagent at room temperature to the reaction temperature according to the method of the present invention, the influence of external fluctuation factors is reduced. , More accurate measurement can be realized.
[Brief description of the drawings]
FIG. 1 is a diagram showing the relationship between the CRP concentration and the amount of change in absorbance within a specified time in Example 1 and Comparative Example.
FIG. 2 is a diagram showing, for each of the number of measurements, fluctuations in a case where a sample having a CRP concentration of 10 mg / dL was continuously measured 20 times in Example 1 and Comparative Example.
FIG. 3 is a diagram showing the relationship between the CRP concentration and the amount of change in absorbance within a specified time in Example 2 and Comparative Example.
FIG. 4 is a diagram showing, for each of the number of measurements, fluctuations in a case where samples having a CRP concentration of 10 mg / dL in Example 2 and Comparative Example were continuously measured 20 times.
FIG. 5 is a graph showing the relationship between the CRP concentration and the amount of change in absorbance within a specified time in Example 3 and Comparative Example.
FIG. 6 is a diagram showing, for each of the number of measurements, fluctuations in a case where a sample having a CRP concentration of 10 mg / dL was continuously measured 20 times in Example 3 and Comparative Example.

Claims (10)

An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer, and the first reagent and the second reagent are placed in a reaction tank. In the immunoassay method to be added, a method in which the amount of the reagent added to the reaction system is 1.5 volumes or more of the first reagent to 1 volume of the second reagent, thereby stabilizing the reaction and improving the measurement accuracy. The method according to claim 1, wherein the second reagent comprises a particulate insoluble carrier sensitized with an antigen or an antibody. The method according to claim 1, wherein the measurement is performed under a temperature condition of 25 ° C. or more and 37 ° C. or less. The method according to any one of claims 1 to 3, wherein the automatic analyzer used for measurement has means for continuously dispensing via a tube when dispensing the reagent to the reaction tank. . An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen. An immunoassay kit containing at least a first reagent and a second reagent for stabilizing the reaction and improving the measurement accuracy by setting the amount of the first reagent to 1.5 volumes or more per 1 volume of the second reagent A kit wherein the volume of the first reagent is larger than the volume of the second reagent. An immunoassay method based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, wherein the measurement is performed using an automatic analyzer, and the first reagent and the second reagent are placed in a reaction tank. In the immunoassay to be added, a method in which the first reagent is kept cool and the second reagent is kept at a temperature in a range from room temperature to the reaction temperature during the measurement, thereby stabilizing the reaction and improving the measurement accuracy. 7. The method of claim 6, wherein the second reagent comprises a particulate insoluble carrier sensitized with an antigen or an antibody. The method according to claim 6, wherein the measurement is performed under a temperature condition of 25 ° C. or more and 37 ° C. or less. The method according to any one of claims 6 to 8, wherein the automatic analyzer used for the measurement has means for continuously dispensing via a tube when dispensing the reagent to the reaction tank. . In an automatic analyzer capable of performing an immunoassay in which a first reagent and a second reagent are added to a reaction tank based on an agglutination method using a first reagent containing a buffer and a second reagent containing an antibody or an antigen, the first reagent An automatic analyzer for use in an immunoassay method, comprising a storage means for cooling the sample and a storage means for holding the second reagent at a reaction temperature from room temperature to stabilize the reaction and improve the measurement accuracy.

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