JP2006194746A - Measuring method of material to be measured, and reagent for immunoassay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring method of a material to be measured capable of suppressing a nonspecific reaction caused by a material other than the material to be measured in a specimen, and preventing clogging in an automatic analyzer even when using an alkali cleaning liquid in the automatic analyzer, and a reagent for immunoassay. <P>SOLUTION: In this measuring method of the material to be measured for measuring the material to be measured included in the specimen by the automatic analyzer using the cleaning liquid, an antigen-antibody reaction between the material to be measured and an antibody or an antigen corresponding to the material to be measured is generated in the presence of zinc ions, and the material to be measured is measured based on a turbidity change or a scattered-light change caused by the antigen-antibody reaction. This reagent for immunoassay for the automatic analyzer capable of cleaning by the cleaning liquid includes the antibody or the antigen corresponding to the material to be measured and zinc ions. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a measurement substance measurement method and an immunoassay reagent for measuring a measurement substance contained in a sample in an automatic analyzer using a cleaning solution.

  In fields such as clinical tests, turbidity changes or scattered light changes caused by an antigen-antibody reaction between an antibody or antigen corresponding to the substance to be measured and the substance to be measured are detected as a method for measuring the substance to be measured contained in the specimen. Thus, an immunoassay method for measuring a substance to be measured contained in a specimen is known. Examples of such an immunoassay include an immunoturbidimetric method and an immunotrophic method.

  In the immunoassay as described above, a nonspecific reaction may occur due to a substance other than the substance to be measured contained in the measurement sample. This non-specific reaction causes turbidity of the measurement sample, which prevents accurate measurement of the substance to be measured.

  As a method for measuring a substance to be measured while suppressing non-specific reactions, a method using a reagent containing a divalent metal ion is known.

  Patent Document 1 discloses a reagent containing one or more divalent metal ions such as beryllium ion, magnesium ion, strontium ion, barium ion, iron ion, zinc ion, and cadmium ion at a concentration of 0.0001 to 1.0M. Describes a method for measuring a substance to be measured while suppressing non-specific reactions.

  Patent Document 2 describes a method for measuring a substance to be measured while suppressing a non-specific reaction by using a reagent containing magnesium ions.

  By the way, conventionally, an automatic analyzer for performing the immunoassay as described above is known. Such an automatic analyzer is provided with a flow path for supplying a sample and a reagent and a reaction container in which the sample and the reagent are mixed, and an alkaline cleaning liquid is used for cleaning the flow path and the reaction container. There is a case. In an automatic analyzer using an alkaline cleaning liquid, for example, when a reagent containing magnesium ions is used, precipitates due to magnesium ions are present in a portion where waste liquid such as the cleaning liquid after cleaning and various samples and reagents after the measurement flows. There is a case that a problem occurs that the clogging is generated in the apparatus.

JP 56-2555

JP 08-211057

  It is an object of the present invention to suppress a non-specific reaction caused by a substance other than a substance to be measured in a sample, and to prevent clogging in the apparatus even when an alkaline cleaning liquid is used in an automatic analyzer. A substance measuring method and an immunoassay reagent are provided.

  In view of the above problems, the present invention is a method for measuring a substance to be measured for measuring a substance to be measured contained in a specimen in an automatic analyzer using a cleaning liquid, and corresponds to the substance to be measured and the substance to be measured in the presence of zinc ions. Provided is a method for measuring a substance to be measured, which causes an antigen-antibody reaction with an antibody or antigen and measures the substance to be measured based on a change in turbidity or a change in scattered light caused by the antigen-antibody reaction.

  The present invention also provides an immunoassay reagent for an automatic analyzer that can be washed with a washing solution, and contains an antibody or antigen corresponding to a substance to be measured and an antigen and zinc ion. .

  By using the analyte measurement method or immunoassay reagent of the present invention in immunoassay, non-specific reactions caused by substances other than the analyte in the sample are suppressed, and an alkaline cleaning solution is used in the automatic analyzer. Even in such a case, clogging in the apparatus can be prevented.

  The immunoassay reagent of the present embodiment measures a substance to be measured contained in a specimen based on a change in turbidity or a change in scattered light caused by an antigen-antibody reaction between an antibody or antigen corresponding to the substance to be measured and the substance to be measured. It is a reagent for immunoassay used for the automatic analyzer which implements an immunoassay method. Examples of such immunoassay methods include immunoturbidimetry and immunotrophic method. Furthermore, an immunoturbidimetric method and an immunotrophic method using carrier particles sensitized with an antibody or antigen corresponding to the substance to be measured can be mentioned.

  FIG. 1 is a diagram showing a configuration of an automatic analyzer. The automatic analyzer 1 includes a specimen setting section 2 for setting a specimen, a reagent setting section 3 for setting a reagent, a cleaning liquid setting section 4 for setting a cleaning liquid, a cleaning mechanism 5 for cleaning using the cleaning liquid, and dispensing of the specimen and reagents. A dispensing mechanism 6, a reaction container 7 in which a specimen and a reagent are mixed, a reaction section 8 in which the reaction container 7 is set, a measurement mechanism 9, a computer 10 for controlling the whole, and the like, and further, a specimen and a reagent And a flow path through which various liquids such as waste liquid flow. Here, the reaction container 7 is a place where a specimen or a reagent is dispensed, mixed, or a measurement sample is prepared. An alkaline cleaning liquid is often used for cleaning the reaction vessel 7 and the flow path. As an alkaline cleaning solution, a cleaning solution having a pH of 8 or higher is generally used, and examples thereof include Hitachi Ltd. high alkali-D and HITERGENT, and Toshiba Corporation Bianch 14.

  The reagent for immunoassay contains zinc ions in order to suppress non-specific reactions and prevent clogging in the apparatus even when an alkaline cleaning solution is used in the automatic analyzer. Here, the non-specific reaction is a reaction other than the antigen-antibody reaction and is known to be caused by a substance other than the substance to be measured contained in the measurement sample. When such a non-specific reaction occurs, the measurement sample becomes turbid, and accurate measurement of the substance to be measured is hindered. The causative substance of the nonspecific reaction whose influence is suppressed by the immunoassay reagent of the present embodiment is not particularly limited, and examples thereof include coagulation factors such as fibrinogen. Coagulation factors such as fibrinogen are not contained in serum but are contained in plasma, and thus cause a non-specific reaction specific to plasma. Due to the causative substance of the non-specific reaction peculiar to plasma, there is a problem that a divergence occurs between the measured value when using serum and the measured value when using plasma. In such a case, the above problem can be solved by using the immunoassay reagent of the present embodiment. In addition, when preparing serum, the clotting factor cannot be completely eliminated, and a part of the clotting factor may remain in the serum. Even in such a case, by using the immunoassay reagent of the present embodiment, it is possible to eliminate the influence of the coagulation factor remaining in the serum and perform an accurate measurement.

  The zinc ion contained in the immunoassay reagent is not particularly limited, and examples thereof include zinc ions derived from compounds such as zinc chloride and zinc sulfate. The reagent for immunoassay may be one obtained by dissolving the compound in an appropriate liquid such as water or a buffer solution. The buffer solution is not particularly limited, and examples thereof include Hepes buffer solution, Tris buffer solution, and phosphate buffer solution. The pH of the immunoassay reagent is not particularly limited as long as it does not suppress the antigen-antibody reaction, but is preferably 4 to 10 and more preferably 6 to 8.

  The reagent for immunoassay further contains an antibody or an antigen corresponding to the substance to be measured. Here, the antibody or antigen corresponding to the substance to be measured represents an antibody against these when the substance to be measured is a protein, enzyme, or hormone, and represents the antigen if the substance to be measured is an antibody. The antibody or antigen against the substance to be measured may be a natural one, or those obtained from the substance to be measured by a known method. The antibody or antigen may be composed of a plurality of antibodies or antigens, and may be those obtained by limited degradation or protein modification of antibodies or antigens. Further, the antibody or antigen may be sensitized to carrier particles. In this case, for example, if the measurement item is C-reactive protein (CRP), the anti-CRP antibody is sensitized to the carrier particles.

  The immunoassay reagent may contain a sensitizer in order to obtain higher sensitivity. Examples of the sensitizer include water-soluble polymers such as polyethylene glycol, polyvinyl alcohol, and dextran. Further, the molecular weight of polyethylene glycol used as a sensitizer is preferably 1000 to 70000, more preferably 5000 to 30000.

  The reagent for immunoassay may contain, together with zinc ions, a substance that suppresses nonspecific reactions other than zinc ions. For example, a nonionic surfactant is mentioned as a substance which suppresses the nonspecific reaction by a lipid protein. Among such nonionic surfactants, polyoxyethylene nonionic surfactants are preferred, and examples of such nonionic surfactants include polyoxyethylene lauryl ether, polyoxyethylene polypropylene lauryl ether, octylphenyl ether, and nonyl. Examples include phenyl ether.

  Moreover, a reducing agent is mentioned as a substance which suppresses the nonspecific reaction by a rheumatoid factor. Examples of the reducing agent include thiol compounds, thiol urea derivatives, and phosphine derivatives. Specific examples include α-thioglycerol, 2-mercaptoethylamine hydrochloride, 2-aminoethylisothiouronium hydrobromide, tris (2-carboxyethyl) phosphine, and the like.

  Furthermore, reagents for immunoassays are particularly limited as long as they do not inhibit the stability of the reagents or the antigen-antibody reaction. You may contain without.

  The reagent for immunoassay may be composed of a first reagent containing zinc ions and a second reagent containing an antibody or antigen corresponding to the substance to be measured. From the viewpoint of effectively reducing the influence of non-specific reactions, first, a sample and a first reagent containing zinc ions are mixed to suppress non-specific reactions, and antibodies or antigens are added to the mixture. It is desirable to prepare a measurement sample by mixing the second reagent containing.

  In addition, for substances that suppress non-specific reactions other than zinc ions, such as the nonionic surfactants and reducing agents, from the viewpoint of effectively reducing the influence of non-specific reactions, It is desirable that they are contained together in one reagent.

  Furthermore, the sensitizer may be contained in either the first reagent or the second reagent. The stabilizer, preservative and the like may be contained in the first reagent or the second reagent according to the action effect of each substance.

  In addition, when the immunoassay reagent is composed of the first reagent and the second reagent, and the sample for measurement is prepared by mixing 15 μL of the sample, 250 μL of the first reagent, and 50 μL of the second reagent, The concentration of zinc ions contained is preferably 0.2 to 50 mM, more preferably 1 to 10 mM.

  The zinc ion concentration is determined when the immunoassay reagent consists of the first reagent and the second reagent, and the sample for measurement is prepared by mixing 15 μL of the sample, 250 μL of the first reagent, and 50 μL of the second reagent. Although the density | concentration of was described, it is not limited to this. When the mixing ratio of the sample and each reagent is different, the concentration of zinc ions contained in the immunoassay reagent is such that the final concentration of zinc ions after mixing the reagent and sample is in the same range as above. do it.

  Further, the immunoassay reagent may contain both zinc ions and an antibody or antigen corresponding to the substance to be measured in one reagent. In this case, the concentration of zinc ions contained in the immunoassay reagent may be such that the final concentration of zinc ions after mixing the reagent and the sample is in the same range as in the above case.

  Examples of the specimen include body fluids such as plasma, serum, and urine. In addition, the substance to be measured in the specimen that can be measured by the immunoassay reagent is not particularly limited as long as it is a substance to be measured by the immunoassay as described above. For example, immunoglobulin (IgG, IgA , IgM, IgD, IgE), complement (C3, C4, C5, C1q), C-reactive protein (CRP), α1-antitrypsin, α1-microglobulin, β2-microglobulin, haptoglobulin, transferrin, ceruloplasmin , Ferritin, albumin, hemoglobin A1, hemoglobin A1C, myoglobin, myosin, dupan-2, α-fetoprotein (AFP), tissue polypeptide antigen (TPA), apolipoprotein A1, apolipoprotein E, rheumatoid factor, anti-streptolysin O ( ASO), antithrombin-III (AT-III) and the like.

Example 1
Using plasma as a specimen, six types of immunoassay reagents with different zinc ion concentrations were used to verify the inhibitory effect of nonspecific reactions caused by the addition of zinc ions.

  The reagent for immunoassay is composed of R1 reagent and R2 reagent. As R1 reagent, the concentration of zinc chloride is 0 mM (R1 reagent A), 2 mM (R1 reagent B), 4 mM (R1 reagent C), 6 mM (R1 reagent D), 8 mM (R1 reagent E) or 10 mM (R1 reagent F). 6 types of buffer solutions different from the above were used. The composition of R1 reagent A is shown below. The R1 reagents B to F are obtained by adding zinc chloride to the R1 reagent A so as to have the aforementioned concentrations.

Reagent composition (R1 reagent A)
Hepes buffer 50 mM
Sodium chloride 100mM
Polyethylene glycol (molecular weight 20000) 2.3% (v / v)
Polyoxyethylene polyoxypropylene lauryl ether 1.0% (v / v)
1-thioglycerol 40 mM
EDTA ・ 2Na 1mM
Sodium hydroxide The amount that makes the pH 7.95

  As the R2 reagent, a buffer solution having the following composition was used.

Reagent composition (R2 reagent)
Hepes buffer 25mM
Sodium chloride 500mM
Sodium hydroxide The amount that makes the pH 7.95

  First, 250 μL of R1 reagent was added to 15 μL of specimen, and pre-warmed for 5 minutes. Thereafter, 50 μL of R2 reagent was further added to the mixture. The R1 reagent and the R2 reagent do not contain an antibody or an antigen corresponding to the substance to be measured, and no antigen-antibody reaction occurs even when these reagents and the sample are mixed. Therefore, by using an immunoassay reagent that does not contain an antibody or antigen corresponding to the substance to be measured, a nonspecific reaction can be detected. The measurement was performed for 10 minutes in total from immediately after the addition of the R1 reagent to 5 minutes after the addition of the R2 reagent. A Hitachi 7170 automatic analyzer manufactured by Hitachi, Ltd. was used as the measuring device, and the measurement wavelength was a main wavelength of 340 nm and a sub wavelength of 700 nm, and measurement was performed at 37 ° C.

  Figure 2 shows R1 reagent A with 0 mM zinc chloride, Figure 3 shows R1 reagent B with 2 mM zinc chloride, Figure 4 shows R1 reagent C with 4 mM zinc chloride, and Figure 5 shows zinc chloride. Fig. 6 shows the change in absorbance when measured using R1 reagent D with a concentration of 6 mM, Fig. 6 shows R1 reagent E with a zinc chloride concentration of 8 mM, and Fig. 7 shows R1 reagent F with a zinc chloride concentration of 10 mM. Is shown. The Hitachi 7170 automatic analyzer divides 10 minutes into 0 to 34 points and measures the absorbance at each point. In each figure, the horizontal axis represents measurement points, and the vertical axis represents absorbance.

  In FIG. 2, it was confirmed that the absorbance increased with the passage of time from the addition of the R1 reagent to the addition of the R2 reagent and after the addition of the R2 reagent. This increase in absorbance is due to a reaction other than the antigen-antibody reaction, that is, a non-specific reaction. On the other hand, in FIGS. 3 to 7, no increase in absorbance was observed from the addition of the R1 reagent to the addition of the R2 reagent, and after the addition of the R2 reagent, and a constant value was observed over time. It was confirmed to show. From the above, it was found that non-specific reactions were suppressed by adding zinc chloride to the immunoassay reagent. The plasma used as a specimen contains a coagulation factor such as fibrinogen. Therefore, it is considered that by adding zinc chloride to the immunoassay reagent, non-specific reactions caused by coagulation factors such as fibrinogen are suppressed.

(Example 2)
Serum aj and plasma aj prepared from each blood aj collected from 10 subjects were used as specimens. In addition, CRP contained in each specimen was measured using CRP as a substance to be measured.

  The reagent for immunoassay is composed of R1 reagent and R2 reagent. As the R1 reagent, R1 reagent A (zinc chloride 0 Mm) used in Example 1 or R1 reagent G having a zinc chloride concentration of 5 mM was used. The R1 reagent G is obtained by adding 5 mM zinc chloride to the R1 reagent A.

  As the R2 reagent, a reagent having the following composition containing a CRP antibody was used.

Reagent composition (R2 reagent)
Hepes buffer 25mM
Sodium chloride 500mM
Anti-human CRP goat serum 1.95mg / mL
Sodium hydroxide The amount that makes the pH 7.95

  First, 250 μL of R1 reagent was added to 15 μL of specimen, and pre-warmed for 5 minutes. Thereafter, 50 μL of R2 reagent was further added to the mixture to initiate an antigen-antibody reaction. The measurement was performed for 10 minutes in total from immediately after the addition of the R1 reagent to 5 minutes after the addition of the R2 reagent. In addition, the Hitachi 7170 type | mold automatic analyzer made from Hitachi, Ltd. was used for the measuring apparatus, the measurement wavelength was 340 nm of main wavelengths, and 700 nm of sub wavelengths, and it measured at 37 degreeC.

FIG. 8 is a scatter showing serum a to j and plasma a to j as specimens, measurement using R1 reagent A with a zinc chloride concentration of 0 mM, and the correlation between serum and plasma for the resulting CRP concentration. FIG. FIG. 9 is a scatter showing serum a to j and plasma a to j as specimens, measurement using R1 reagent G having a zinc chloride concentration of 5 mM, and the correlation between serum and plasma with respect to the resulting CRP concentration. FIG. 8 and 9, the horizontal axis indicates the CRP concentration (mg / dL) obtained by measuring the serum sample, and the vertical axis indicates the CRP concentration (mg / dL) obtained by measuring the plasma sample. Indicated. Each figure shows a correlation coefficient (R ² ) indicating the degree of correlation between the CRP concentration obtained by measuring the serum sample and the CRP concentration obtained by measuring the plasma sample.

Comparing FIG. 8 and FIG. 9, it can be seen that the data variation in FIG. 9 is lower than that in FIG. Further, comparing the correlation coefficients (R ² ), it can be seen that the value of R ² is larger in FIG. 9 than in FIG. From this, it was found that the correlation between serum and plasma was improved by adding zinc chloride to the reagent for immunoassay.

  FIG. 10 shows changes in absorbance when measurement is performed using R1 reagent A using serum c and plasma c as specimens. FIG. 11 shows changes in absorbance when measurement is performed using R1 reagent G using serum c and plasma c as specimens. 10 and 11, the horizontal axis indicates the measurement point, and the vertical axis indicates the absorbance.

  Serum c and plasma c are serum and plasma obtained from the same subject. In FIG. 10, when zinc chloride is not contained in the R1 reagent, the absorbance change of serum c and the absorbance change of plasma c are greatly different. On the other hand, in FIG. 11, when zinc chloride is contained in the R1 reagent, the absorbance change of serum c and the absorbance change of plasma c are almost the same. From the above, it has been found that by adding zinc chloride to the immunoassay reagent, it is possible to eliminate the discrepancy in the measured value between serum and plasma.

(Example 3)
Each of the immunoassay reagent not containing metal ions and the immunoassay reagent containing metal ions was mixed with an alkaline cleaning solution, and the presence or absence of precipitates was verified.

  The R1 reagent A used in Example 1 was used as an immunoassay reagent not containing metal ions. As an immunoassay reagent containing metal ions, R1 reagent F (zinc chloride 10 mM) used in Example 1, R1 reagent G (zinc chloride 5 mM) used in Example 2, and R1 reagent containing 5 mM magnesium chloride H was used. And it was confirmed whether the precipitate was produced | generated by comparing the light absorbency obtained when using the reagent containing a metal ion with the light absorbency obtained when using the reagent which does not contain a metal ion. The R1 reagent H is obtained by adding 5 mM magnesium chloride to the R1 reagent A. As the alkaline cleaning liquid, High Alkali-D, which is used as a cleaning liquid in an automatic analyzer manufactured by Hitachi, Ltd., was used.

  Reagent for immunoassay (R1 reagent A, R1 reagent F, R1 reagent G or R1 reagent H) and alkaline washing liquid are mixed at a ratio of 10: 1, and after a predetermined time at 25 ° C. (1 hour, 72 hours) After or one week later, the absorbance (660 nm) of the mixed solution was measured. The measurement results are shown in Table 1.

Each numerical value in Table 1 uses R1 reagent A (no metal ion), R1 reagent F (Zn ²⁺ 10 mM), R1 reagent G (Zn ²⁺ 5 mM), R1 reagent H (Mg ²⁺ 10 mM) as reagents. It is the value of the absorbance (660 nm) obtained when

  From Table 1, in the case of R1 reagent H, the absorbance obtained by measurement after 1 hour is 0.076. Furthermore, when this absorbance is compared with the absorbance in the case of R1 reagent A, the absorbance of R1 reagent H is clearly higher. From this, it was found that a precipitate was formed when a reagent containing magnesium chloride and an alkaline cleaning solution were mixed. In addition, about the R1 reagent H, since the production | generation of the precipitate was confirmed in the measurement after 1 hour, the measurement after 72 hours and 1 week was not implemented.

  On the other hand, when the absorbance in the case of R1 reagent F was compared with the absorbance in the case of R1 reagent A, similar results were obtained in all measurements after 1 hour, 72 hours, and 1 week. Similarly, in the case of R1 reagent G, when this absorbance and the absorbance of R1 reagent A were compared, similar results were obtained. From the above, it was found that no precipitate was generated even when a reagent containing zinc chloride and an alkaline cleaning liquid were mixed.

1 is a diagram showing a configuration of an automatic analyzer 1. FIG. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent A. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent B. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent C. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent D. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent E. In Example 1, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent F. In Example 2, it is a graph which shows the correlation of serum and plasma at the time of measuring using R1 reagent A. In Example 2, it is a graph which shows the correlation of serum and plasma at the time of measuring using R1 reagent G. In Example 2, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent A. In Example 2, it is a graph which shows the change of the light absorbency at the time of measuring using R1 reagent G.

Claims (10)

A measurement substance measurement method for measuring a measurement substance contained in a specimen in an automatic analyzer using a cleaning liquid, wherein an antigen-antibody reaction between a measurement substance and an antibody corresponding to the measurement substance or an antigen in the presence of zinc ions A measured substance measurement method for measuring a measured substance based on a change in turbidity or a change in scattered light caused by the antigen-antibody reaction. The method for measuring a substance to be measured according to claim 1, wherein the automatic analyzer includes a reaction container for causing the antigen-antibody reaction to be performed, and the cleaning liquid cleans the reaction container. The method for measuring a substance to be measured according to claim 1, wherein the cleaning liquid is alkaline. An immunoassay reagent for an automatic analyzer that can be washed with a washing solution, comprising an antibody or an antigen corresponding to a substance to be measured, and a zinc ion. The reagent for immunoassay according to claim 4, wherein the washing solution is alkaline. The immunoassay reagent comprises a first reagent and a second reagent, the first reagent contains zinc ions, and the second reagent contains an antibody or an antigen corresponding to the substance to be measured. 5. The immunoassay reagent according to 5. The reagent for immunoassay according to any one of claims 4 to 6, wherein the first reagent further contains a sensitizer. The reagent for immunoassay according to any one of claims 4 to 7, wherein the first reagent further contains a nonionic surfactant. The reagent for immunoassay according to any one of claims 4 to 8, wherein the first reagent further contains a reducing agent. The reagent for immunoassay according to any one of claims 4 to 9, wherein the substance to be measured is C-reactive protein (CRP).

Images