JP2018066636A - Immunological measuring method, and measuring reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunological measuring method that can sufficiently restrain over a long period, even if the measured specimen contains heterophile antibodies, rheumatoid factors and the like in high concentration interferences by such contents, and a measuring reagent using this method.SOLUTION: An immunological measuring reagent containing sulfite salt has a pH not higher than 7.0. By using this immunological measuring reagent, measurement can be accomplished stably over a long period while restraining interfering reactions by heterophile antibodies, rheumatoid factors and the like.SELECTED DRAWING: None

Description

  In the measurement of a substance to be measured by an immunological method, the present invention suppresses the interference action by an interfering substance (hereinafter referred to as an interfering substance) such as a heterophilic antibody and a rheumatoid factor, and accurately measures an antigen or antibody concentration. The present invention relates to an immunological measurement method and an immunological measurement reagent.

  In recent years, immunoassays using antigen-antibody reactions between antigens and antibodies have been widely used in the diagnosis of various diseases because immunologically active substances such as proteins in living organisms can be measured easily and rapidly. ing. Such immunological assays include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), immunoturbidimetric method (TIA), latex agglutination (LA), Various methods such as immunochromatography have been put into practical use.

  However, there are cases in which an analyte that interferes with an antigen-antibody reaction is included in a sample to be measured by an immunological measurement method. If interference substances are included in these antigen-antibody reactions, the measurement results will be affected and accurate measurement will not be possible. Especially in the field of clinical examinations, accurate diagnosis will not be possible. Alternatively, a suppression method has been proposed.

  For example, in order to solve the problem of interference caused by the mixing of rheumatoid factor, an immunological measurement method that reduces the interference caused by rheumatoid factor by pretreating the specimen with an animal-derived antibody that binds to the binding site of human rheumatoid factor in advance. (Patent Document 1) and an immunological measurement method (Patent Document 2) that suppresses the interference action of a rheumatoid factor by maintaining the pH of the reaction solution at 4.0 to 6.0 have been proposed.

Also, a method of using F (ab ′) ₂ obtained by removing the Fc portion of an antibody used for immunological measurement by an enzymatic reaction has been proposed in order to reduce or suppress the interference action caused by the interfering substance (Patent Document 3). ). However, this immunological measurement method can be used only when measuring an antigen, and requires complicated steps such as an enzyme reaction and purification for preparing F (ab ′) ₂ , resulting in an increase in cost and production. There was a problem of difference between lots.

  Furthermore, a method for suppressing the interference action caused by heterophilic antibodies using polymerized or aggregated antibody molecules has also been proposed (Patent Document 4). In order to completely suppress the interference action caused by heterophilic antibodies, Requires a large amount of polymerized or aggregated antibody molecules, which increases the cost.

  Although the methods described in these documents for suppressing the interference effect of the interference substance certainly improve the influence of the interference substance, if the sample to be measured contains the interference substance at a high concentration, The effect of suppressing the interference action was insufficient.

  A method using sulfite has been proposed (Patent Document 5) that can suppress the interference action of an interference substance even if the sample to be measured contains an interference substance at a high concentration. Sulfite is very inexpensive and has the advantage that it does not increase costs even when used at a concentration that can sufficiently suppress the effects of high concentrations of interfering substances, and it does not easily affect antigen-antibody reactions. .

Japanese Patent Laid-Open No. 7-012818 JP-A-8-146000 JP 54-119292 A JP-A-4-221762 Japanese Patent Laid-Open No. 2001-074739

  However, since sulfite is gradually oxidized to sulfate by dissolved oxygen in the measurement reagent or oxygen in the air, the effect of suppressing the interference action by the interference substance decreases with time. That is, since it is used in an automatic analyzer or the like and stored in a reagent store or the like in an opened state, the sulfite concentration decreases with time, and the influence of interference substances cannot be sufficiently suppressed. In addition, when the interference substance has a high concentration, there is a problem that the interference action may not be sufficiently suppressed.

  Therefore, the present invention has been made paying attention to the above-mentioned present situation, and even when a sample to be measured contains an interfering substance at a high concentration, the interference action due to these can be suppressed over a long period. An object of the present invention is to provide a highly stable epidemiological measurement method and a measurement reagent using the same.

  As a result of diligent research to solve the above problems, the present inventor has found that by suppressing the pH of a measurement reagent containing sulfite to 7.0 or less, a high inhibitory effect on interfering substances can be obtained over a long period of time. The present invention has been completed.

That is, the present invention has the following configuration.
(1) In an immunoassay method in which an antigen-antibody reaction is performed in the presence of sulfite using an immunoassay reagent, the pH of the immunoassay reagent containing sulfite is 7.0 or less The immunological measurement method described above.
(2) The immunological measurement method according to (1), wherein the pH of the immunological measurement reagent containing sulfite is in the range of 5.6 to 7.0.
(3) The immunological low measurement method according to (1) or (2), wherein the sulfite is 50 mM or more.
(4) The immunological measurement method according to any one of (1) to (3), wherein the sulfite is 50 mM to 600 mM.
(5) A step of mixing the first reagent containing the immunological measurement reagent with the second reagent containing the antibody or antigen that reacts with the measurement object or the antibody or antigen that reacts with the measurement object. The immunological measurement method according to any one of (1) to (4), wherein the measurement object is quantitatively or qualitatively measured.
(6) The immunological measurement method according to any one of (1) to (5), wherein the immunological measurement method is a latex agglutination method.

(7) An immunoassay reagent for performing an antigen-antibody reaction in the presence of sulfite, wherein the immunoassay reagent containing sulfite has a pH of 7.0 or less. Measurement reagent.
(8) The immunoassay reagent according to (7), wherein the pH of the reagent containing sulfite is in the range of 5.6 to 7.0.
(9) The immunological low measurement reagent according to (7) or (8), wherein the sulfite is 50 mM or more.
(10) The immunoassay reagent according to any one of (7) to (9), wherein the sulfite is 50 mM to 600 mM.
(11) The immunological measurement reagent according to any one of (7) to (10), wherein the immunological measurement method is a latex agglutination method.
(12) The immunological measurement reagent according to any one of (7) to (11), an antibody or antigen that reacts with the measurement object, or a carrier that carries an antibody or antigen that reacts with the measurement object; An immunological measurement reagent kit.

  In the present invention, the pH of an immunological measurement reagent containing sulfite is adjusted to 7.0 or less, so that even when a sample to be measured contains an interfering substance for antigen-antibody reaction at a high concentration, the interference can be prevented. Provided is an immunoassay reagent having excellent stability, which can perform immunoassay without being affected by a substance.

  The results obtained by this immunological measurement are also expected to be highly reliable.

  Surprisingly, by reducing the pH of the reagent containing sulfite slightly from the pH 7.4 described in Example of Patent Document 5, not only the inhibitory effect of interfering substances by sulfite but also sulfite. The stability of the immunological measurement reagent or pretreatment reagent containing a salt can also be improved.

Hereinafter, preferred embodiments of the present invention will be described.
In one embodiment of the present invention, in an immunoassay in which an antigen-antibody reaction is performed using an immunoassay reagent in the presence of sulfite, the pH of the immunoassay reagent containing sulfite is set to 7. This is an immunological measurement method with 0 or less.

  In the present invention, the “immunological measurement method” refers to the determination (detection) of the presence or absence of a measurement target using a substance (for example, an antibody or an antigen) that specifically binds to the measurement target. ) And the measurement (quantification) of the abundance when the measurement object exists, for example, the RIA method or ELISA method based on the principle of the sandwich method, the competitive method, or the immunity based on the immunoagglutination method. Examples thereof include a turbidimetric method (TIA method) and a latex agglutination method (LIA method).

  The “immunological measurement reagent” of the present invention is a reagent used in an immunological measurement method for performing an antigen-antibody reaction, and includes sulfite. Furthermore, the immunological measurement method of the present invention can be used as a pretreatment reagent for a measurement sample containing a measurement target substance.

  The “pretreatment reagent” of the present invention is a reagent used for treating a measurement sample before measuring the measurement sample containing the substance to be measured using an immunological measurement method, and includes sulfite. The measurement sample treated with the pretreatment reagent can be used as it is as a measurement sample of a known immunological measurement method.

  The sulfite used in the present invention can be appropriately selected from known ones. Examples of the sulfite include sodium sulfite, potassium sulfite, calcium sulfite, ammonium sulfite, sodium hydrogen sulfite, and ammonium hydrogen sulfite.

  In the present invention, when sulfite is contained in the immunological measurement reagent or pretreatment reagent, the sulfite concentration in the immunological measurement reagent or pretreatment reagent is 50 mM or more, preferably 50 mM to 600 mM, more preferably 100 mM. To 600 mM, more preferably 250 mM to 500 mM. When the sulfate concentration is less than 50 mM, it is difficult to sufficiently suppress the influence of interfering substances that affect the immune reaction, and even when the concentration exceeds 600 mM, the influence of interfering substances cannot be effectively suppressed, and the increase in the viscosity of the solution. May adversely affect measurement.

  The pH of the immunoassay or pretreatment reagent containing the sulfite of the present invention is preferably 7.0 or less, and more preferably from pH 5.6 to pH 7.0. If the pH is below 5.6, denaturation of proteins and the like is likely to occur, and accurate measurement is affected. If the pH is above 7.0, the effect of suppressing the influence of interfering substances tends to decrease over time.

  In the present invention, if necessary, a known protein protective agent can be added to increase the stability of the substance to be measured. Examples of such protein protecting agents include proteins such as albumin and gelatin, synthetic polymer materials, surfactants, and the like.

  The sample in which the influence of the interfering substance is suppressed by the immunological measurement reagent or the pretreatment reagent containing the sulfite of the present invention can be directly measured by a known immunological measurement method. Examples of immunological measurement methods include the sandwich method, the RIA method and ELISA method based on the principle of the competitive method, the immunoturbidimetric method (TIA method) and the latex agglutination method (LIA method) based on the immunoagglutination method. It is done.

  Among these immunological measurement methods, in particular, the antigen (or antibody) that is the substance to be measured in the measurement sample is reacted with the antibody (or antigen) in the measurement reagent, and the resulting immune aggregate is detected. The immunoturbidimetric method and latex agglutination method, which employs the measurement principle, are preferred.

  The immunological measurement reagent of the present invention can be used in an immunoturbidimetric method or a latex agglutination method, and includes a first reagent containing a buffer, and an antibody or antigen, or a carrier carrying an antibody or an antigen in the buffer ( Preferably, it can be composed of two reagents of the second reagent containing latex particles.

  The buffer solution that can be used for the immunoassay reagent by the immunoturbidimetric method or the latex agglutination method is not particularly limited as long as the pH can be kept constant, and can be appropriately selected from known ones. For example, phosphate buffer, Tris buffer, Good buffer, etc. are mentioned.

  The immunoassay reagent containing the sulfite of the present invention can also be used as a first reagent of an immunoassay reagent such as an immunoturbidimetric method or a latex agglutination method composed of the two reagents. That is, after mixing the measurement sample with the first reagent using the method for suppressing the influence of the interfering substance of the present invention, an antibody (or antigen) or latex particles carrying the antibody (or antigen) in the mixed solution, etc. Measurement may be carried out by mixing a second reagent containing

  In addition, after mixing the pretreatment reagent and the measurement sample using the method for suppressing the influence of the interference substance of the present invention, this mixed solution is measured as a measurement sample for an immunological measurement method such as an ELISA method or a latex agglutination method. May be.

  In the present invention, the antibody or antigen carried on the carrier is not particularly limited and can be appropriately selected from known ones. Examples of antibodies or antigens carried on a carrier include antibodies to plasma proteins such as C-reactive protein (CRP) and transferrin, antibodies to hormones such as thyroid stimulating hormone (TSH), thyroxine, insulin, and human placental lactogen, cancer Antibodies against tumor-related substances such as fetal antigen (CEA), β2-microglobulin, α-fetoprotein (AFP), antibodies against viral hepatitis antigens such as HBs antigen and HBe antigen, and viral properties such as HBs antibody and HBe antibody Examples include antigens against hepatitis antibodies, viruses such as herpes, measles and rubella, antibodies or antigens against various biological components, antibodies against various drugs such as phenobarbital, acetaminophen, and cyclosporine.

  In the present invention, the carrier for supporting the antibody or antigen is not particularly limited and can be appropriately selected from known ones. Examples of the carrier for supporting the antibody or antigen include polystyrene plates, polystyrene beads, latex particles, metal colloid particles, silica colloid particles, and the like.

  The above-mentioned antibodies that can be used in the present invention are not particularly limited, and examples thereof include antibodies derived from animals such as rabbits, goats, mice, rats, horses, sheep, etc. Either a polyclonal antibody obtained from animal serum or a monoclonal antibody obtained by cell fusion of the spleen of an animal immunized with a measurement object with myeloma cells may be used.

  In the present invention, the measurement sample is not particularly limited, and examples thereof include blood, serum, plasma, urine, lymph fluid, stab fluid, spinal fluid, sweat, saliva, gastric fluid, lung lavage fluid, feces and the like. Of these, serum and plasma are particularly preferred.

  In the present invention, the substance to be measured is not particularly limited. For example, plasma proteins such as C-reactive protein (CRP) and transferrin, thyroid stimulating hormone (TSH), thyroxine, insulin, human placental lactogen and other hormones Tumor-related substances such as carcinoembryonic antigen (CEA), β2-microglobulin, α-fetoprotein (AFP), viral hepatitis antigens such as HBs antigen and HBe antigen, and viral hepatitis such as HBs antibody and HBe antibody Examples include antibodies, viruses such as herpes, measles and rubella, antibodies or antigens against various biological components, various drugs such as phenobarbital, acetaminophen, and cyclosporine.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples.

Example 1 Effect of Sulphite Concentration on Interference Reaction Suppression Effect The effect of sulfite concentration on the suppression of interference reaction by adding sodium sulfate was evaluated. Further, the stability was evaluated by storage under conditions that promote the oxidation of sulfite (half-filled reagent container and shaken at 4 ° C. for 3 weeks).

(1) Reagent
First reagent: 50 mM HEPES buffer containing 150 mM NaCl, 0.1% NaN ₃ , 0.1% polyoxyethylene polyoxypropylene cetyl ether, 0 mM, 50 mM, 100 mM, 250 mM, 350 mM, 500 mM, or 600 mM sodium sulfite ( pH 7.4) was prepared. Further, half of the prepared first reagent was filled in a reagent container and shaken at 4 ° C. for 3 weeks to promote oxidation of sulfite by oxygen in the air.
Second reagent: A 50 mM HEPES buffer (pH 7.4) containing an anti-KL-6 antibody-supported polystyrene latex solution, 400 mM arginine hydrochloride, and 0.1% NaN ₃ was prepared.

  Said anti-KL-6 carrying | support latex liquid was prepared by the well-known method. That is, it was prepared by mixing an anti-KL-6 antibody and polystyrene latex and supporting the anti-KL-6 antibody on the polystyrene latex surface.

(2) RF sample is prepared by adding interference check RF plus (Sysmex Corporation) to the measurement sample pool serum so that the rheumatoid factor concentration is 0, 100, 200, 300, 400, 500, or 1000 IU / mL. did.

(3) Evaluation method After 120 μL of the first reagent is mixed with 2.0 μL of the RF sample and incubated at 37 ° C. for 5 minutes, 40 μL of the second reagent is mixed with this mixed solution and reacted at 37 ° C. After mixing the second reagent The change in absorbance at 660 nm for about 5 minutes was measured. A series of measurements was performed using a Hitachi 7180 automatic analyzer (Hitachi High-Technologies Corporation).
In order to quantify the concentration of KL-6, the concentration of KL-6 in the serum sample was calculated from a calibration curve obtained by measuring the standard substance of KL-6. The results immediately after the preparation are shown in Table 1, and the results when the oxidation of sulfite is promoted are shown in Table 2.

  From Table 1, the effect of suppressing interference reaction by RF is seen at a sulfite concentration of 50 mM or more, and even at a sulfite concentration of 50 mM, the interference reaction by RF can be suppressed to about ½ of that when no sulfite is added (0 mM). all right. In addition, it was found that the suppression effect of the interference reaction was improved depending on the sulfite concentration, the suppression effect was maximized at a sulfite concentration of 250 mM to 500 mM, and decreased at a sulfite concentration of 600 mM. The sulfite concentration is preferably 50 mM or more, more preferably 50 mM to 600 mM, still more preferably 100 mM to 600 mM, and most preferably 250 mM to 500 mM.

  Also, from Tables 1 and 2, when the sulfite concentration is 50 mM, the interference reaction suppression effect is reduced due to oxidation of sulfite. The effect of sulfite oxidation on the inhibition effect of interference reaction is reduced depending on the sulfite concentration, and the effect is minimized and the stability is improved at a sulfite concentration of 250 mM to 500 mM. However, when the sulfite concentration is 600 mM, It was found that the effect of suppressing interference reaction is reduced by oxidation of sulfite.

Example 2 Influence of pH on inhibition of interference reaction of sulfite Sodium sulfite was set to 250 mM, and the influence of pH on inhibition of interference reaction of an interference substance was evaluated.

(1) Reagent
First reagent: 50 mM PIPES buffer (pH 6.4 or 6.8) or 50 mM HEPES buffer containing 150 mM NaCl, 0.1% NaN ₃ , 0.1% polyoxyethylene polyoxypropylene cetyl ether, 250 mM sodium sulfite (PH 7.4 or 7.8) was prepared.
Second reagent: The same as in Example 1.
(2) Measurement sample RF prepared by adding interference check RF plus (Sysmex Corporation) to the pooled serum so that the rheumatoid factor concentration is 0, 100, 200, 300, 400, or 500 IU / mL. In addition to the samples, normal samples (heterophilic antibodies and RF negative human serum samples) and abnormal samples (heterophilic antibodies and RF positive human serum samples) were used as samples.
(3) Evaluation method A sample was measured in the same manner as in Example 1. Table 3 shows the results of the RF sample, and Table 4 shows the results of the human serum sample.

  As is apparent from Table 3, the effect of suppressing the RF interference reaction by sulfite is significantly reduced at higher pH than 7.8, and the positive error of the measured value is increased. However, there is no significant difference in the suppression effect of RF interference reaction by sulfite at pH 7.4 or lower.

  Further, from Table 4, almost no influence of pH on suppression of interference reaction by sulfite is observed in normal specimens. In contrast, in the case of an abnormal specimen in which an interfering substance is present, the suppression effect of the sulfite interference reaction was remarkably reduced and the measured value increased at pH 7.8 or higher, but the measured values at pH 6.4 and 6.8 were measured. It can be seen that the fluctuations in are small and the interference reaction is effectively suppressed.

Example 3 Effect of pH on inhibition of interference reaction of sulfite (detailed evaluation)
Sodium sulfite was set to 250 mM, and the influence of pH on the suppression of the interference reaction of the interference substance was evaluated in more detail. Further, the stability was evaluated by storage under conditions that promote the oxidation of sulfite (half-filled reagent container and shaken at 4 ° C. for 3 weeks).

(1) Reagent
First reagent: 50 mM PIPES buffer (pH 5.6, 6.0, 6.2, 6 containing 150 mM NaCl, 0.1% NaN ₃ , 0.1% polyoxyethylene polyoxypropylene cetyl ether, 250 mM sodium sulfite .4, 6.6, 6.7, 6.8 or 7.0) or 50 mM HEPES buffer (pH 7.4) was prepared. The prepared first reagent was half filled in a reagent container and shaken at 4 ° C. for 3 weeks to promote oxidation of sulfite by oxygen in the air.
Second reagent: The same as in Example 1.
(2) Measurement sample An RF sample was prepared in the same manner as in Example 1.
(3) Evaluation method A sample was measured in the same manner as in Example 1. The results immediately after the preparation are shown in Table 5, and the results when the oxidation of sulfite is promoted are shown in Table 6.

  As can be seen from Table 5, when the pH is 7.4, the interference suppression effect of high concentration RF (1000 IU / mL) is insufficient, whereas when the pH is 7.0 or less, the RF It was found that there was no difference in pH with respect to the inhibitory effect of the interference reaction, and the inhibitory effect was high even for high concentrations of RF.

  Further, from Table 5 and Table 6, when pH is 7.4, a decrease in the effect of suppressing interference reaction due to oxidation of sulfite is observed, whereas when pH is 7.0 or less, RF interference is observed. The influence of sulfite oxidation on the reaction inhibitory effect was not observed, and it was found that the stability of the reagent containing sulfite was improved. From the above results and the present results, the pH of the immunoassay reagent or pretreatment reagent containing sulfite is preferably 7.0 or less, and more preferably in the range of pH 5.6 to pH 7.0.

  As described above, according to the present invention, even when a sample to be measured contains a high concentration of interfering substances such as heterophilic antibodies and rheumatoid factors, the interference action due to these is sufficiently suppressed for a long period of time. Therefore, it is possible to provide a highly stable immunoassay method and immunoassay reagent that can accurately measure a measurement object.

Claims (12)

  In an immunoassay method in which an antigen-antibody reaction is performed in the presence of sulfite using an immunoassay reagent, the pH of the immunoassay reagent containing sulfite is 7.0 or less. The immunological measurement method.   The immunoassay method according to claim 1, wherein the immunoassay reagent containing sulfite has a pH in the range of 5.6 to 7.0.   The immunological low measurement method according to claim 1 or 2, wherein the sulfite is 50 mM or more.   The immunological measurement method according to any one of claims 1 to 3, wherein the sulfite is 50 mM to 600 mM.   Mixing a first reagent containing the immunological measurement reagent with an antibody or antigen that reacts with the measurement object, or a second reagent that contains a carrier carrying an antibody or antigen that reacts with the measurement object, The immunological measurement method according to any one of claims 1 to 4, wherein the measurement object is quantitatively or qualitatively measured.   The immunological measurement method according to any one of claims 1 to 5, wherein the immunological measurement method is a latex agglutination method.   An immunological measurement reagent for performing an antigen-antibody reaction in the presence of sulfite, wherein the pH of the immunological measurement reagent containing sulfite is 7.0 or less. reagent.   The immunoassay reagent according to claim 7, wherein the reagent containing the sulfite has a pH in the range of 5.6 to 7.0.   The immunological low measurement reagent according to claim 7 or 8, wherein the sulfite is 50 mM or more.   The immunoassay reagent according to any one of claims 7 to 9, wherein the sulfite is 50 mM to 600 mM.   The immunoassay reagent according to any one of claims 7 to 10, wherein the immunoassay is a latex agglutination method. An immunoassay comprising the immunological measurement reagent according to any one of claims 7 to 11 and an antibody or antigen that reacts with the measurement object, or an antibody or antigen that reacts with the measurement object or a carrier carrying the antigen. Reagent kit.