JP7156807B2 - Method for improving accuracy of test measurements and additive therefor - Google Patents

Description

The present invention relates to a method for improving the accuracy of test measurements when testing a specimen with an automated analyzer having a stir bar, and an additive therefor.

Conventionally, as an automatic analyzer, for example, an apparatus is known that analyzes a reaction liquid obtained by mixing a desired reagent with a sample such as serum and reacting it, and measuring the absorbance of the reaction liquid to be analyzed. This type of analyzer has a mechanism for injecting a sample and reagents into a reaction vessel, a mechanism for stirring the sample and reagents in the reaction vessel, a mechanism for analyzing the physical properties of the sample during or after the reaction, and the like. It is configured.

In the field of analyzers, miniaturization of samples and reagents is a major technical issue. In other words, as the number of analysis items increases, the amount of samples that can be allocated to a single item is becoming smaller, and the analysis of small-volume samples such as blood tests for infants, for which a large amount of samples themselves cannot be prepared, is being performed. there is In addition, as the content of analysis becomes more sophisticated, expensive reagents are generally used, and miniaturization of reagents is demanded from the standpoint of cost as well. Miniaturization of such samples and reagents promotes miniaturization of reaction vessels, and the miniaturization of reaction vessels creates new technical problems. For example, in the sample/reagent pipetting mechanism and the reaction liquid mixing mechanism, the liquid volume ratio of the reaction liquid and washing liquid brought into the next sample has increased, and it has become easy to affect the analysis results, which is one of the issues. .

A general stirring mechanism is performed by immersing a spatula-shaped stirring rod in the liquid to be stirred in the reaction vessel and rotating it. After the stirring rod is washed with the cleaning liquid, it is immersed in the liquid to be stirred in the next reaction vessel. Excess liquid is brought into the container by adhesion of washing liquid or previous liquid to be stirred or by mixing with the liquid to be stirred. In addition, when the stirring rod rotates, the liquid to be stirred scatters and adheres to the inner wall of the reaction vessel, reducing the amount of the reaction liquid in the reaction vessel. Accurate measurement becomes impossible by dropping and being brought into the reaction solution.

With such a stirring bar, it is difficult to completely eliminate the carry-over of the reaction liquid and washing liquid to the next specimen. Therefore, Patent Literature 1 discloses a technique for agitating a liquid to be stirred by irradiating it with ultrasonic waves, and Patent Literature 2 discloses a technique for agitating the liquid by injecting air into it. Such a method of stirring without physical contact with the liquid to be stirred can, in principle, eliminate the carry-over of the reaction liquid and washing liquid to the next sample via the stirring rod.

JP-A-2001-242177 Japanese Patent Application Laid-Open No. 2004-340791

A stirring method that does not use a stirring rod has the above merits, but there is a concern that the cost will be higher than stirring using a stirring rod. In addition, if a general-purpose automatic analyzer cannot be used, the inspection site must introduce a new automatic analyzer, which increases the burden.

An object of the present invention is to provide a method for improving the accuracy of test measurement values by suppressing the carry-over of the reaction liquid and cleaning liquid into the next sample via a stirring rod, and is applicable to general-purpose automatic analyzers. and to provide an additive therefor.

In order to solve the above problems, the present inventors conducted intensive research, and found that by adding a polyoxyethylene-polyoxypropylene copolymer to the reagent, the error in the measurement value caused by bringing the reaction solution and washing solution into the next sample was reduced. We have found that it can be prevented, and have completed the present invention.

That is, the present invention provides the following.
(1) When testing a specimen with an automated analyzer equipped with a stirring rod, the polyoxyethylene-polyoxypropylene block copolymer is allowed to coexist in the mixture of the reagent and the specimen, and the reaction solution and washing solution are passed through the stirring rod. A method of improving the accuracy of laboratory measurements , including reducing carry-over to a specimen .
(2) The method according to (1), wherein the polyoxyethylene-polyoxypropylene block copolymer is added to the reagent, and the reagent and the specimen are mixed.
(3) The method according to (1) or (2), wherein the reagent comprises a first reagent and a second reagent.
(4) The method according to (3), wherein the polyoxyethylene-polyoxypropylene block copolymer is contained in the first reagent.
(5) The polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 8,000 to 17,000 and an ethylene oxide content in the molecule of 75 to 95 mol% (1) to (4). A method according to any one of
(6) The polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 10,800 and an ethylene oxide content in the molecule of 80 mol%. The method according to (5), wherein the ethylene oxide content in the molecule is 85 mol%, and the weight average molecular weight is 15,500 and the ethylene oxide content in the molecule is 80 mol%.
(7) The polyoxyethylene polyoxypropylene block copolymer is polyoxyethylene (200) polyoxypropylene glycol (40), polyoxyethylene (260) polyoxypropylene glycol (35), polyoxyethylene (280) poly The method according to any one of (1) to (6), which is at least one selected from oxypropylene glycol (55).
(8) An additive for improving the accuracy of test measurements when testing a specimen with an automatic analyzer having a stirring bar according to the method described in (1) , comprising a polyoxyethylene-polyoxypropylene block copolymer.
(9) The additive according to (8), wherein the polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 8,000 to 17,000 and an ethylene oxide content in the molecule of 75 to 95 mol%. .
(10) The polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 10,800 and an ethylene oxide content in the molecule of 80 mol%. The additive according to (9), which is at least one selected from those having an ethylene oxide content of 85 mol% and those having a weight average molecular weight of 15,500 and having an ethylene oxide content in the molecule of 80 mol%.
(11) The polyoxyethylene polyoxypropylene block copolymer is polyoxyethylene (200) polyoxypropylene glycol (40), polyoxyethylene (260) polyoxypropylene glycol (35), polyoxyethylene (280) poly The additive according to any one of (8) to (10), which is at least one selected from oxypropylene glycol (55).

According to the present invention, it is possible to reduce the amount of liquid brought into the biological sample or from outside the measurement system through the stirring rod, thereby reducing the amount of non-target components to suppress their effects, thereby improving the accuracy of target components in the biological sample. measurement becomes possible.

The polyoxyethylene polyoxypropylene block copolymers of the present invention are block copolymers comprising a polyoxyethylene portion and a polyoxypropylene portion, preferably a hydrophobic polyoxypropylene block polymer composed of two hydrophilic It consists of a structure sandwiched by polyoxyethylene block polymers of , and behaves as a nonionic surfactant.

Specific examples of commercial products containing the polyoxyethylene-polyoxypropylene block copolymer used in the present invention include "F" among nonionic surfactants generally sold under the name of "Pluronic". Products can be listed. Nonionic surfactants sold under the name "Epan" and nonionic surfactants sold under the name "Pronon" can also be used.

In addition, there are several methods of notation as a component of polyoxyethylene polyoxypropylene block copolymer, and identity and similarity can be confirmed between each notation. The numerical value between them does not completely match, or the content mol% of polyoxyethylene (may be described as "EO") or polyoxypropylene (may be described as "PO") to the molecular weight of the entire molecule When trying to calculate , it may not exactly match the displayed value. However, these are peculiar to polymerizable macromolecules and can be understood by those skilled in the art.

In addition, polyoxyethylene polyoxypropylene block copolymers have a classification method called "Pluronic grid" in which the EO content (mol%) is on the horizontal axis and the PPG molecular weight is on the vertical axis. are also described, and the polyoxyethylene-polyoxypropylene block copolymer of the present invention can be selected with reference to these.

In the present invention, it is preferable that the polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 8,000 to 17,000 and an EO content of 75 to 95 mol% in the molecule, Further, the polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 10,800 and an EO content in the molecule of 80 mol% (commercial product name: Pluronic F88), or a weight average molecular weight of 13,800. 333 and the EO content in the molecule is 85 mol% (commercial product name: Epan 785), or the weight average molecular weight is 15,500 and the EO content in the molecule is 80 mol% (commercial product name: Pluronic F108 ) or having a weight average molecular weight of 8,000 and an EO content of 85 mol% in the molecule (commercial product name: Epan 485), and a polyoxyethylene-polyoxypropylene block copolymer The coalescence is polyoxyethylene (200) polyoxypropylene glycol (40) (commercial product name: Pluronic F88), polyoxyethylene (260) polyoxypropylene glycol (35) (commercial product name: Epan 785), polyoxyethylene (280 ) polyoxypropylene glycol (55) (commercial name: Pluronic F108).

A method for preparing a reagent for an automatic analyzer using the polyoxyethylene-polyoxypropylene copolymer used in the present invention will be described below, taking as an example a reagent using latex particles frequently used in the field of clinical examination. Reagents using latex particles that are frequently used in the field of clinical testing are often composed of a two-reagent system of a reagent that does not contain latex particles (first reagent) and a reagent that contains latex particles (second reagent). many. When this configuration is adopted, the first reagent may play the role of preparing a measurement environment suitable for the antigen-antibody reaction performed by the second reagent (for example, dilution of the target component, exposure of the antigen epitope, etc.). many.

The polyoxyethylene-polyoxypropylene copolymer used in the present invention may be added to either the first reagent or the second reagent, but in the two-reagent configuration described above, the stir bar is first added to the reagent. Since it is desirable to prevent carry-over during contact and stirring, it is preferably added to at least the first reagent.

The concentration of the polyoxyethylene-polyoxypropylene copolymer used in the present invention can be defined, for example, by the final concentration during the latex immunoagglutination reaction. The concentration is suitably 0.0001 wt % to 1 wt %, preferably 0.001 wt % to 0.5 wt %, more preferably 0.01 wt % to 0.3 wt %.

The polyoxyethylene-polyoxypropylene copolymer to be used in the present invention is a reagent for an automatic analyzer, taking into account the desired measurement sensitivity, measurement range, reproducibility, or reagent stability as a measurement system. In addition, the practically optimum type, concentration, and reagent preparation method can be selected and used as appropriate.

Since the method of the present invention is a method for improving the accuracy of test measurements by reducing the amount of liquid adhering to a stirring bar in an automatic analyzer, it can be applied to all inspections using an automatic analyzer using a stirring bar. is. Among these, immunoassays, particularly pathogens in body fluids and antibodies against pathogens can be suitably used.

EXAMPLES The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. In addition, the density|concentration is a basis of weight unless there is particular notice.

Reference example 1
(1) Preparation of Reagent Using the NATIVE antigen of HELICOBACTER PYLORI, a reagent for measurement by immunoagglutination was prepared as follows.
i) Sensitized particles obtained by supporting 5.2 mg of HELICOBACTER PYLORI NATIVE antigen per 1 mL of a polystyrene latex suspension having an average particle size of 300 nm were suspended in a buffer solution (MOPS, pH 7) at a concentration of 0.15%. , a latex suspension was prepared.
ii) BSA was added to MES buffer (pH 6.0) so as to be 2%, and this was used as the first reagent.

(2) Measurement by automatic analyzer The automatic analyzer was a 7180 type Hitachi automatic analyzer of Hitachi High-Technologies Co., Ltd., and the automatic measurement was performed by the endpoint method.

Each control sample was measured in duplicate using the reagents described above. 200 μL of the first reagent was added to 3.5 μL of the sample solution, and the mixture was stirred and mixed at 37°C. After allowing to stand for 5 minutes, 40 µL of the latex suspension prepared above was added and mixed with stirring at 37°C. Aggregation reaction for about 5 minutes was measured as absorbance change.

At the time of the first measurement, after the first reagent was added to the sample and stirred, the stirring rod used for stirring and mixing was wiped off with a dry paper waste, and then the second measurement was performed continuously. The ratio between the first measured value and the second measured value was calculated. In addition, the stirring rod used for stirring and mixing the specimen, first reagent and latex suspension was not wiped off. The results are shown in Table 1 below.

Reference example 2
(1) Preparation of Reagent A latex suspension and a first reagent were prepared in the same manner as in Reference Example 1.

(2) Measurement by automatic analyzer Measurement was carried out in the same manner as in Reference Example 1. At the time of the first measurement, after the latex suspension was added to the sample and the first reagent and stirred, the stirring rod used for stirring and mixing was wiped off with a dry paper waste, and then the second measurement was performed continuously. The ratio between the first measured value and the second measured value was calculated. In addition, the stirring rod used for stirring and mixing the sample and the first reagent was not wiped off. The results are shown in Table 1 below.

Reference example 3
(1) Preparation of Reagent A latex suspension and a first reagent were prepared in the same manner as in Reference Example 1.

(2) Measurement by automatic analyzer Measurement was carried out in the same manner as in Reference Example 1. At the time of the first measurement, after the first reagent was added to the sample and stirred, the stirring rod used for stirring and mixing was wiped off with a dry paper waste. Furthermore, after that, the latex suspension was added to the sample and the first reagent, and after stirring, the stirring bar used for stirring and mixing was wiped off with a dry paper waste, and then the second measurement was performed continuously. The ratio between the first measured value and the second measured value was calculated. The results are shown in Table 1 below.

Reference example 4
(1) Preparation of Reagent A latex suspension and a first reagent were prepared in the same manner as in Reference Example 1.

(2) Measurement by automatic analyzer Measurement was carried out in the same manner as in Reference Example 1.

Two consecutive measurements were taken without wiping off the stir bar. The ratio between the first measured value and the second measured value was calculated. The results are shown in Table 1 below.

Table 1 shows that wiping off the stir bar improves the decrease in the second measurement value. It was also shown that the improvement effect is great when the stirring rod used for mixing is wiped off after the addition of the latex reagent.

Example 1, Comparative Examples 1-4
(1) Preparation of Reagent Using the NATIVE antigen of HELICOBACTER PYLORI, a reagent for measurement by immunoagglutination was prepared as follows.

i) Sensitized particles obtained by supporting 5.2 mg of HELICOBACTER PYLORI NATIVE antigen per 1 mL of a polystyrene latex suspension having an average particle size of 300 nm were suspended in a buffer solution (MOPS, pH 7) at a concentration of 0.15%. , a latex suspension was prepared.
ii) Various surfactants were added to a buffer solution (MES, BSA, pH 6.0) to prepare the following reagents A to D. As a comparative example, Reagent E was prepared without adding a surfactant component.

Ａ～Ｄ：非イオン性界面活性剤(Ａ：「プロノン２０８」（日油）、Ｂ：「エマルゲン７０７」（花王）、Ｃ：「Ｔｗｅｅｎ８０」（和光純薬）Ｄ：「エマルゲンＢ６６」（花王）をそれぞれ使用
Ｅ：緩衝剤のみ

A to D: Nonionic surfactants (A: "Pronon 208" (NOF), B: "Emulgen 707" (Kao), C: "Tween 80" (Wako Pure Chemical Industries) D: "Emulgen B66" (Kao ) are used respectively E: Buffer only

(2) Measurement by automatic analyzer The automatic analyzer was a 7180 type Hitachi automatic analyzer of Hitachi High-Technologies Co., Ltd., and the automatic measurement was performed by the endpoint method.

A control sample (40 U/mL) was measured five times in succession using the buffer solutions of reagents A to E described above. To 3.5 μL of the specimen solution, 200 μL of the reagent A to H buffer solutions were added, and the mixture was stirred and mixed at 37°C. After allowing to stand for 5 minutes, 40 µL of the latex suspension prepared above was added and mixed with stirring at 37°C. The agglutination reaction for about 5 minutes was measured as the amount of change in absorbance, and the ratio between the first measured value and the fifth measured value was calculated. In addition, the measured values of Comparative Example 1 and Example were compared. The results are shown in Table 2 below.

From Table 3, it was shown that the decrease in measured value was improved by adding a surfactant. In addition, since there is little difference between the measured value of the reagent without added surfactant (the first measured value is the correct value) and the measured value of the reagent with the addition of polyoxyethylene-polyoxypropylene condensate, polyoxyethylene -Polyoxypropylene condensate was shown to prevent the measured values from deteriorating and not to affect the sensitivity. On the other hand, in Comparative Examples 1 to 3, the ratio of the 5th measured value / 1st measured value (%) is as excellent as in Example 1, but Comparative Example 4 (currently used conventional method) The deviation from the first measured value (correct measured value) of is large, and it can be seen that the accuracy of the measured value is impaired by the surfactant.

Claims (11)

When testing a sample with an automatic analyzer equipped with a stirring bar, polyoxyethylene-polyoxypropylene block copolymer is allowed to coexist in the mixture of reagent and sample, and the reaction solution and washing solution are passed through the stirring bar to the next sample. A method of improving the accuracy of test measurements , including reducing carry -over. 2. The method of claim 1, wherein said polyoxyethylene polyoxypropylene block copolymer is added to said reagent and said reagent and said specimen are mixed. 3. The method of claim 1 or 2, wherein said reagent consists of a first reagent and a second reagent. 4. The method of claim 3, wherein said polyoxyethylene polyoxypropylene block copolymer is contained in said first reagent. 5. Any one of claims 1 to 4, wherein the polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 8,000 to 17,000 and an ethylene oxide content in the molecule of 75 to 95 mol%. The method described in . The polyoxyethylene polyoxypropylene block copolymer has a weight average molecular weight of 10,800 and an ethylene oxide content in the molecule of 80 mol%, a weight average molecular weight of 13,333 and ethylene oxide in the molecule 6. The method according to claim 5, wherein at least one selected from those having a content of 85 mol % and those having a weight average molecular weight of 15,500 and an ethylene oxide content in the molecule of 80 mol %. Polyoxyethylene polyoxypropylene block copolymers are polyoxyethylene (200) polyoxypropylene glycol (40), polyoxyethylene (260) polyoxypropylene glycol (35), polyoxyethylene (280) polyoxypropylene glycol The method according to any one of claims 1 to 6, wherein at least one selected from (55). An additive for improving the accuracy of test measurements when testing a specimen in an automated analyzer having a stir bar according to the method of claim 1 , comprising a polyoxyethylene-polyoxypropylene block copolymer. The additive according to claim 8, wherein the polyoxyethylene-polyoxypropylene block copolymer has a weight average molecular weight of 8,000 to 17,000 and an ethylene oxide content in the molecule of 75 to 95 mol%. The polyoxyethylene polyoxypropylene block copolymer has a weight average molecular weight of 10,800 and an ethylene oxide content in the molecule of 80 mol%, a weight average molecular weight of 13,333 and ethylene oxide in the molecule 10. The additive according to claim 9, which is at least one kind selected from those having a content of 85 mol %, having a weight average molecular weight of 15,500 and having an ethylene oxide content in the molecule of 80 mol %. Polyoxyethylene polyoxypropylene block copolymers are polyoxyethylene (200) polyoxypropylene glycol (40), polyoxyethylene (260) polyoxypropylene glycol (35), polyoxyethylene (280) polyoxypropylene glycol The additive according to any one of claims 8 to 10, which is at least one selected from (55).