JP2021099243A - Immunological measurement method - Google Patents

Abstract

To provide a measurement method and a kit capable of obtaining stable and highly accurate measured values without being affected by interfering substances in a test sample regardless of the concentration of a substance to be measured by improving the shape of a high concentration area of a calibration curve when measuring the substance to be measured in the test sample derived from a living body by a heterogeneous assay.SOLUTION: In the measurement by heterogeneous immunoassay of a substance to be measured existing in a sample derived from a living body, the method adds a compound having a guanidino group to a solution used as a medium for the immune reaction. The kit includes a buffer solution containing an antibody that specifically binds to the substance to be measured and the compound having the guanidino group.SELECTED DRAWING: None

Description

The present invention relates to an immunological measurement method and a kit for accurately measuring a high concentration range.

Currently, in the field of clinical diagnostic tests, it is required to measure a wide variety of substances that are indicators of disease diagnosis for a large number of samples in a short time and with high accuracy, and to quickly and accurately feed back the results to the treatment site. There is. For example, a measurement system in which a physiologically active substance (protein such as antibody, enzyme, receptor, antigen, nucleic acid substance such as DNA or RNA, sugar chain, etc.) is bound to the particle surface, or immunology using an antigen-antibody reaction. Accurate quantification of minute amounts of substances to be measured is often carried out by targeted measurement.

In general, the performance of in vitro diagnostic agents used in clinical diagnostic tests largely depends on the performance of the main ingredient. There are two main types of main ingredients. One is a physiologically active substance typified by an antibody that recognizes a target substance, and the other is particles carrying the physiologically active substance. The physical characteristics of particles used as diagnostic agents are mainly evaluated from the viewpoints of particle size, dispersibility, magnetic substance content, and non-specific reaction. It can be said that each of these items has a trade-off relationship, and it is not easy to stably produce and supply particles having an excellent balance of performance.

Further, in an in vitro diagnostic agent using an antigen-antibody reaction using the antibody, in addition to a physiologically active substance such as an antibody and particles carrying the physiologically active substance, a surfactant, a salt, a human antibody, and a preservative , Various additives such as sensitizers are used, and these additives are also factors that influence the performance of in vitro diagnostic agents.

Among clinical diagnostic tests, unreacted samples and excess labeled substances are washed by cleaning with B / F separation, compared to the homogenius assay that measures directly without washing unreacted samples and labeled substances, so-called B / F separation. The heterogeneous assay, in which the measurement is performed after removing the sample, is expected as a method for improving the detection sensitivity and accuracy.

As a method for improving the accuracy in the heterogeneous assay, for example, in Patent Document 1 (Japanese Unexamined Patent Publication No. 11-108926), it is caused by the variation in the recovery amount of the solid phase carrier after the washing operation for B / F separation. A measurement method is disclosed in which variations in measurement results are corrected, the measurement reproducibility is improved, and measurement accuracy is further improved. This is because some of the magnetic particles collected by the magnetic force flow out during the cleaning operation, and the variation in the residual amount (recovered amount) of the solid phase carrier after the B / F separation step causes poor reproducibility of the measured values. As one of the above, the residual amount of the solid phase carrier after the washing step is used as an index to correct the variation in the measurement result in the measurement step, improve the measurement reproducibility, and further improve the measurement accuracy. A method is disclosed in which the number or concentration of particles of a solid phase alone is calculated by measuring transmitted light and scattered light, and the count value of the measurement result is divided by the residual amount of the solid phase unit using this residual amount as an index. Although this technique is effective when the same sample is measured multiple times, it does not improve the reactivity of the antigen-antibody reaction itself, so that it can be used in a limited number of situations.

Further, as another method for improving the measurement accuracy of the heterogeneous assay, in Patent Document 2 (Japanese Unexamined Patent Publication No. 2018-136179), the reproduction of the heterogeneous assay method in which the occurrence of false high values in the low concentration measurement is avoided. A measurement reagent having good properties is disclosed. This is because, in the case of a heterogeneous assay method that requires high sensitivity of measurement, a slight nonspecific adsorption of the labeled substance on the carrier often affects the measurement performance, and the conventional method causes a nonspecific adsorption reaction. Focusing on the fact that the measured values of low-concentration samples suddenly become falsely high because they cannot be completely suppressed, the amount of non-specific adsorption of the labeled substance on the carrier is reduced to avoid the occurrence of false highs in low-concentration measurement. This is the measurement method.

Japanese Unexamined Patent Publication No. 11-108926 Japanese Unexamined Patent Publication No. 2018-136179

In addition to the above-mentioned patent documents, various attempts have been made to suppress non-specific adsorption, but a large amount of various impurities are contained in a test sample derived from a living body, and non-specific adsorption is performed. It is difficult to completely suppress it, and it must be said that the effect of improving the measurement accuracy is insufficient.
In the first place, the heterogeneous assay is often used when measuring a small amount of a substance to be measured, and many of the methods for improving the measurement accuracy of the heterogeneous assay focus on the reactivity in the low concentration range to improve the measurement sensitivity. There is no document that discloses a method for improving the accuracy of the high concentration region in the heterogeneous assay. Moreover, it was not sufficient as a technique capable of stably measuring clinical specimens.

As will be described in detail in Examples described later, the present inventors attempted to measure a substance to be measured in a test sample derived from a living body by an immunological heterogeneous assay, and found that the measurement accuracy in a high concentration range was obtained. It was found that it may not be stable and the cause is that the shape of the calibration curve is not accurate. That is, the calibration curve that is approximated and complemented is different from the behavior of the test substance, and the reproducibility of the measured value represented by the coefficient of variation (CV (%)) deteriorates.
Therefore, the present invention improves the shape of the high concentration region of the calibration curve when the substance to be measured in the test sample derived from the living body is measured by the heterogeneous assay, regardless of the concentration of the substance to be measured. It is an object of the present invention to provide a measurement method and a kit capable of obtaining stable and highly accurate measurement values without being affected by an interfering substance in a test sample.

As a result of diligent studies in view of the above-mentioned current situation, the present inventors have conducted a solution to be used as a reaction medium when measuring a substance to be measured in serum and heparinized plasma by a heterogeneous immunoassay method. The present invention has been completed by finding that the shape of the high concentration region of the calibration curve can be improved by adding a compound having a guanidino group therein. In particular, it is surprising that the above-mentioned effect cannot be obtained when a compound having no guanidino group is coexisted, and the above-mentioned high effect cannot be obtained unless a compound having a guanidino group is coexisted. there were.

Therefore, the present invention
[1] In the measurement of a substance to be measured present in a sample derived from a living body by a heterogeneous immunoassay method, a compound having a guanidino group is added to a solution used as a medium of an immune reaction to obtain a calibration curve. How to improve the shape of the high concentration range,
[2] A method for measuring a substance to be measured existing in a sample derived from a living body by a heterogeneous immunoassay.
(1) (a) a test sample that may contain a substance to be measured, (b) a solution containing a labeled immunological partner, (c) a solution used as a medium for an immune reaction, and , A solution containing a compound having a guanidino group (provided that the solution (b) and the solution (c) are the same reagent (that is, a solution used as a medium for an immunoreaction, and a compound having a guanidino group and a label). The step of preparing a solution) containing a modified immunological partner) or different reagents);
(2) A step of mixing the above (a), the above (b), and the above (c) to form an immunological partner containing a substance to be measured and a labeled immunological partner;
(3) Step of removing unreacted test sample and labeled immunological partner;
(4) The method, which comprises a step of analyzing a signal derived from the immunological partner and determining the amount of substance to be measured.
[3] The method of [1] or [2], wherein the compound having a guanidine group is contained in a sample diluent and / or an antibody solution.
[4] The method according to any one of [1] to [3], wherein the compound having a guanidine group is a basic amino acid, histamine, spermidine, or creatine.
[5] The method of [4], wherein the basic amino acid is arginine, lysine, or histidine.
[6] The method of [4] or [5], wherein the basic amino acid concentration is 0.5 mol / L or less.
[7] The method according to any one of [1] to [6], wherein the compound having a guanidino group is present in the immune reaction solution in a state of being electrically close to neutral.
[8] The method of [7], wherein the pH of the immune reaction solution is 9 or more.
[9] Measurement of the substance to be measured, which comprises an antibody that specifically binds to the substance to be measured and a buffer solution containing the compound having a guanidino group for any of the methods [1] to [8]. Regarding the kit.

By using the method of the present invention and the reagent kit, when the substance to be measured in a test sample derived from a living body is measured by a heterogeneous immunoassay, a guanidino group is added to a solution used as a reaction medium. By adding the compound, the shape of the high concentration region of the calibration curve can be improved, so that stable and accurate measurement can be performed.

It is a graph which shows the relationship between the pH of a sample diluent and the shape of a calibration curve. It is a graph which shows the relationship between the concentration of the surfactant in the B / F cleaning liquid, and the coefficient of variation (CV (%)).

The test sample derived from a living body in the present invention is a sample derived from a living body and may contain a substance to be measured collected from a human or the like. Examples of the test sample derived from a living body include blood samples such as whole blood, serum, and plasma.

The test sample (sample) that can be used in the present invention may be a measurement sample that can be prepared from the test sample derived from the living body. For example, in the case of a blood sample, blood coagulation Whole blood obtained using an inhibitor, after blood collection, standing to allow sufficient coagulation, and then centrifuging to remove blood clots to obtain serum, after blood collection using a blood coagulation inhibitor , Specimens such as plasma from which blood cell components have been removed by centrifugation. As the blood coagulation inhibitor, for example, heparin, EDTA, citric acid and the like can be used. These can be preferably added to a blood collection tube or the like in advance when blood is collected from a measurement target such as a human.

The substance to be measured in the present invention is not particularly limited as long as it is a substance that can be measured by an immunoassay method. For example, soluble interleukin 2 receptor, prostate-specific antigen, myocardial troponin, preceptin, brain natriuretic peptide, human brain natriuretic peptide precursor N-terminal fragment, myoglobin, CK-MB, D-dimer, thrombin-antithrombin complex. , Estrogen, progesterone, human chorionic gonadotropin, luteinizing hormone, follicular stimulating hormone, prolactin, testosterone, procalcitonin, CRP, thrombomodulin, thyroid stimulating hormone, FT ₄ , FT _3, etc. Any substance that can be measured by the assay is sufficient, and is not particularly limited.

The heterogeneous assay immunoassays that can be used in the present invention include, for example, radioimmunosassay (RIA), enzyme immunoassay (EIA), chemiluminescence / enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA). ), Electrochemiluminescent immunoassay (ECLIA), etc., but is not limited thereto. Specifically, in the reaction solution, an immunocomplex of the substance to be measured in the test sample and an immunological partner (for example, an antibody or an antigen) specific to the substance to be measured is formed and formed. It is a method of detecting the presence of the substance to be measured by appropriately detecting the signal due to the above, and is a method including a step of performing measurement after removing an unreacted sample or an excess labeling substance by washing. When the immunological partner is an antibody, the antibody to be used can be determined by the measurement system, but for highly sensitive quantitative measurement, sandwich immunology using two or more kinds of antibodies. The measurement method (sandwich immunoassay) can be selected. The method of sandwich immunoassay may be carried out in one or more steps (two steps, three steps, etc.).

For example, when procalcitonin contained in a test sample derived from a living body is used as a measurement target substance and this is measured, a sample prepared from the test sample derived from the living body as described above is further sampled. An insoluble carrier prepared as a sample for measurement in a diluted solution and carrying an antibody (first antibody) that specifically binds to procalcitonin, and a procalcitonin that is different from the first antibody labeled with a labeling substance. The amount of the labeling substance bound to the insoluble carrier after the unreacted antibody and procalcitonin were removed (B / F separation) by washing to form an immune complex by mixing with the antibody (second antibody) that binds to. Can be done by measuring.

As the insoluble carrier, those usually used in the art can be used. Examples of the insoluble carrier material include latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, and polyglycidyl methacrylate. , Polymer materials such as achlorine-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone; agarose; gelatin; erythrocytes; inorganic materials such as silica gel, glass, inert alumina, magnetic material and the like. .. One or more of these may be combined.

Examples of the shape of the insoluble carrier include microtiter plates, test tubes, beads, particles, nanoparticles and the like. Examples of the particles include magnetic particles, hydrophobic particles such as polystyrene latex, copolymerized latex particles having a hydrophilic group such as an amino group and a carboxyl group on the particle surface, erythrocytes, gelatin particles and the like. Among them, magnetic particles are particularly preferable from the viewpoint of realizing quick and simple B / F separation, and specifically, for example, triiron tetroxide (Fe ₃ O ₄ ), diiron trioxide (Fe ₂ O ₃ ), and the like. Magnetic particles such as fine particles made of various metals such as ferrite, iron, manganese, nickel, cobalt and chromium, and alloys such as cobalt, nickel and manganese are preferably used. Further, these magnetic particles can be preferably prepared in a form contained inside a high molecular weight latex such as polystyrene, gelatin, liposomes or the like, or those immobilized on the surface can be preferably used.
Methods of immobilizing the first antibody on these insoluble carriers are known in the art. The immobilization can be performed by, for example, a physical adsorption method, a covalent bond method, an ionic bond method, a combination thereof, or the like.

The labeling substance is not particularly limited as long as it is a labeling substance that can be used in ordinary immunological measurement methods, and examples thereof include enzymes, fluorescent substances, radioisotopes, and insoluble granular substances. Examples of the labeling enzyme include alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, and acid phosphatase. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), and luciferin. Radioisotopes include ¹²⁵ I, ¹⁴ C, ³² P and the like.

When the labeling substance is an enzyme, the labeling substance can be measured by performing a luminescence, fluorescence, or color development reaction using a substrate for the enzyme. For example, when the enzyme is alkaline phosphatase, the substrate used is CDP-star® (4-chloro-3- (methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro) tricyclo). [3.3.1.1 ^3,7 ] Decane} -4-yl) disodium phenylphosphate), CSPD® (registered trademark) (3- (4-methoxyspiro {1,2-dioxetane-3,2-) (5'-Chloro) tricyclo [3.3.1.1 ^3,7 ] decane} -4-yl) disodium phenylphosphate), AMPPD® (adamantylmethoxyphenylphosphoryldioxycetan), APS- Chemiluminescent substrates such as 5; fluorescent substrates such as 4-methylumbelliferylphospate; p-nitrophenyl phosphate, BCIP (5-bromo-4-chloro-3-indrill-phosphate), NBT A chemiluminescent substrate such as (4-nitroblue tetrazolium chloride) or INT (iodonitrotetrazolium) can be used.

As the antibody that specifically binds to the substance to be measured used in the present invention, those known to those skilled in the art can be appropriately selected and used. For example, the antibody that specifically binds to procalcitonin may be a monoclonal antibody that recognizes the amino acid sequence or three-dimensional structure of procalcitonin as an epitope, or a polyclonal antibody. For example, antibodies such as M18612PC1, M18612PC2, M18612PC3, and M18612PC4 (manufactured by Fitzgerald) can be purchased and used as appropriate, but are not limited thereto.

These antibodies are prepared by immunizing an animal according to a known method, for example, in the case of procalcitonin, using procalcitonin purified from a human sample or recombinant procalcitonin prepared in vitro as an antigen. In addition, the epitope can be determined. Epitope means not only the smallest region recognized by an antibody, but also one identified as a region recognized by an antibody. Further, it may be an antibody fragment such as Fab, Fab', F (ab') ₂ , or Fv that can be prepared by a known method.

When two types of antibodies are used in the measurement of the present invention, they are limited if it is possible to form an immune complex with a substance to be measured (for example, procalcitonin) in a test sample derived from a living body. However, it is preferable that the epitopes recognized by the first antibody and the second antibody are different. Further, the monoclonal antibody and the polyclonal antibody can be used in an appropriate combination. Further, the first antibody and the second antibody are not limited to one type, and two or more types can be used in combination.

The high concentration range may differ for each substance to be measured, but refers to a region having a relatively high concentration within the range of the calibration curve created for each substance to be measured, but can be quantified when the calibration curve is created. The concentration range may be more than half of the above concentration range. For example, when 0 to 100 ng / mL is set as the quantifiable range in the procalcitonin measurement using the CLEIA method, the concentration range may be set to 50 ng / mL or more. it can.

The addition of the compound having a guanidino group in the present invention is performed at least during the first immune complex formation (reaction solution) of the substance to be measured and the antibody that specifically binds to the substance to be measured in the above immunological measurement method. It should be done so that it exists in (middle). Specifically, it may be added at the same time as the immune complex is formed (in the reaction solution), but it is added to the sample before the complex is formed (before the antibody and the sample containing the substance to be measured come into contact with each other). Is also good.

In the present invention, a compound having a guanidino group can be added to a solution used as a medium for an immune reaction (for example, a sample diluent, a solution containing an immunological partner (for example, an antibody or an antigen)). In addition, the compound having a guanidino group to be added can be dissolved in a known buffer solution and prepared as a solution. In addition to the buffer, it may be prepared alone, or it may be prepared together with a known substance necessary for pretreatment of a sample or reaction. As described above, the compound having a guanidine group may be added at least at the time of the first immune complex formation (in the reaction solution), and thus is contained in a buffer solution or the like that can be present at that time. Can be made to. The buffer solution and the like that can be present at the time of immune complex formation can be appropriately set depending on the measurement method and device used, and for example, a sample diluent and an antibody solution (antibody solid phase particle solution and labeled antibody). Liquid, etc.). Further, it may be added to a plurality of buffer solutions.

Examples of the compound having a guanidine group in the present invention include basic amino acids, histamine, spermidine, creatine and the like. Preferably, the compound having a guanidine group is a basic amino acid, and specific examples thereof include arginine, lysine, and histidine. The basic amino acids also include variants, adducts, and multimers to which amino acids are bound, in which some of these functional groups are modified, and the multimers include peptides composed of a plurality of different amino acids, which are the same. Contains both peptides composed of the amino acids of. In addition, salts of the basic amino acids and the like are also included. The salt is not particularly limited, and examples thereof include hydrochlorides in addition to sodium salts and potassium salts, and specific examples thereof include basic amino acid salts such as arginine hydrochloride and lysine hydrochloride.

The ionized state of the compound having a guanidino group is that when the substance to be measured in the sample is measured by a heterogeneous immunoassay, the compound having the guanidino group is present in the solution used as a reaction medium. , The effect of improving the shape of the high concentration range of the calibration curve may be obtained, and the pH range is not particularly limited, but it is preferably added under basic pH conditions, preferably pH 9 or higher, and pH 9.5 or higher. Is more preferably added at a pH of 10 or more, preferably pH 14 or less, further preferably pH 13 or less, and most preferably added at a pH of 12 or less. It may also be configured to use various buffers to achieve the desired pH and maintain the pH during the assay. For example, boric acid buffer solution, phosphate buffer solution, carbonic acid buffer solution, Tris buffer solution, HEPES buffer solution and the like may be used. The particular buffer used is not particularly important to the present invention, but a particular buffer can be preferably selected and used in individual assays.

As the concentration of the compound having a guanidino group in the present invention, those skilled in the art should appropriately set the concentration at which the effect of improving the shape of the high concentration region of the calibration curve is observed when measured by the heterogeneous immunoassay method. Can be done. The upper and lower limits of the addition concentration may be any concentration that has the effect of improving the shape of the high concentration region of the target calibration curve, and the addition concentration in the solution used as a medium for the immune reaction is preferably, for example, 0. It may be in the range of 0.01 mol / L or more and 0.5 mol / L or less, preferably 0.1 mol / L or more and 0.2 mol / L or less, and more preferably 0.12 mol / L or more and 0.18 mol. It is in the range of / L or less. The concentration in the reaction solution during the antigen-antibody reaction is preferably in the range of 0.0017 mol / L or more and 0.083 mol / L or less, and preferably 0.017 mol / L or more and 0. It is 033 mol / L or less, more preferably 0.02 mol / L or more and 0.03 mol / L or less. The upper and lower limits may be appropriately combined as desired.

Generally, a method of adding arginine to the reaction solution of an antigen-antibody reaction for the purpose of suppressing a non-specific reaction is known, but this is because the aggregation inhibitory effect of arginine, that is, the improvement of dispersibility is the reaction of the antigen-antibody reaction. It is presumed that the effect is obtained by affecting the speed. The effective concentration suitable for arginine to have such an effect of improving dispersibility is when a high concentration of arginine such as 0.5 to 1.0 mol / L is added, and is about 0.1 mol / L. It is said that such an effect cannot be obtained when a low concentration of arginine is added. However, in the present invention, it is a surprising effect that the addition of arginine at a concentration other than such a high concentration affects the antigen-antibody reaction, and in particular, the effect of improving the shape of the calibration curve in the high concentration range is obtained. there were. It is considered that this effect was obtained by changing the reaction rate of the antigen-antibody reaction by improving the dispersibility by arginine.

In addition to the above-mentioned arginine addition concentration, the pH of the reaction solution is also considered to be important. The affinity of an antibody for an antigen represents the strength of binding between the antibody forming the complex and the antigen, and the higher the affinity, the more antigens and antibodies are bound in an equilibrium state. It is known that the effect of pH on the equilibrium constant of the antigen-antibody complex occurs when the pH is 6.5 to 8.4, and the antigen-antibody reaction is strongly inhibited at pH below 6.5 and above pH 8.4. ing. For example, the equilibrium constant of the antigen-antibody reaction at pH 5.0 or pH 9.5 is said to be 100 times smaller than the equilibrium constant of the antigen-antibody reaction at pH 6.5-7.0. As described above, it is common knowledge for those skilled in the art that under extreme pH conditions, the three-dimensional structure of the antibody is changed and the complementarity with the antigen is impaired. Further, since the isoelectric point of arginine is 10.76 and is electrically near neutral at pH 10, the effect of improving the dispersibility of arginine is usually observed when arginine is added at a low concentration and near the isoelectric point. It is unlikely that the effect of stably causing the antigen-antibody reaction and improving the shape of the calibration curve in the high concentration range was obtained at pH 10 after the addition of arginine as in the present invention. It was surprising.

In addition, IgG added to suppress a non-specific reaction may be appropriately selected so as to be present in the reaction solution. In particular, in order to maintain reproducibility in the measurement in the high value region, a surfactant may be added to the reaction reagent and used. The surfactant that can be used in the reagent of the present invention can be appropriately selected and used by those skilled in the art, and for example, Triton X-100 can be used. The concentration of Triton X-100 at the time of immune complex formation is preferably 0.001% or more and 0.4% or less, more preferably 0.003% or more and 0.3% or less, and most preferably 0.008% or more. It is selected from the range of 0.25% in an appropriate combination.

As for the concentration and type of the compound having a guanidino group in the present invention, as shown in Examples described later, the substance to be measured in each blood sample is measured, and the shape of the high concentration range of the calibration curve is improved. By grasping it, a person skilled in the art can easily determine it. According to CLSI (The Clinical & Laboratory Standards Institute) Guideline EP06-A, it is clinically determined whether the calibration curve obtained statistically has linearity or conforms to the quadratic or cubic equation. It can be judged by confirming whether it is useful or not. For example, as the measured value of each concentration, an optical count provided by the measuring device, a concentration calculated using a standard substance, or the like may be used as an index.

The kit of the present invention can be used to carry out the method of the present invention and contains a buffer solution containing an antibody that specifically binds to the substance to be measured and a compound having a guanidino group.

As the antibody that specifically binds to the substance to be measured, the above-mentioned known antibody can be used, or either a monoclonal antibody or a polyclonal antibody can be used. It can also be used in the kit as an antibody fragment having a specific binding ability to the substance to be measured, for example, Fab, Fab', F (ab') _{2, or Fv.}
Furthermore, the antibody can be used in the kit as it is, or in an insoluble carrier such as magnetic particles if a suitable form, for example, CLEIA, is used based on the immunological method to be used. In the case of a method using a substrate such as an immunochromatography method in a fixed state, labeling with a labeling substance (for example, enzyme, fluorescent substance, chemical luminescent substance, radioisotope, biotin, avidin) while fixed on the substrate If necessary, it can be used in the kit in a labeled state.

As the buffer solution containing the compound having a guanidine group, the above may be referred to, and examples thereof include a sample diluent and an antibody solution (antibody solid phase particle solution and labeled antibody solution). Preferably, it is prepared as a sample diluent.

In addition, the kit of the present invention may include an instruction manual in which an explanation regarding the procedure for carrying out an immunological measurement method using the kit of the present invention, precautions regarding storage and handling of the kit itself, and the like are described. ..

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.

<< Reference Example 1: Preparation and measurement of reagents for measuring procalcitonin (PCT) >>
Reference Example 1-1: Preparation of reagent for procalcitonin measurement and preparation of test sample The following reagents were prepared as reagents for procalcitonin measurement.
-Sample diluted solution: A buffer solution (pH 8.0) containing 0.1 mol / L HEPES, 0.45 mol / L NaCl, 0.05% Tween 20, and 0.001% Triton X-100 was used.
-First antibody solution (magnetic latex reagent): A magnetic particle solution in which a mouse monoclonal antibody (manufactured by EastCoast) that recognizes procalcitonin was bound to magnetic latex particles (JSR) was used.
-Second antibody solution (enzyme-labeled antibody reagent): A labeled antibody solution in which another mouse monoclonal antibody (manufactured by Hytest) that recognizes procalcitonin was alkaline phosphatase (ALP) -labeled by the maleimide method was used.
B / F wash: A buffer containing 0.01 mol / L MES, 0.9% NaCl, 0.001% Triton X-100, 0.05% Tween 20 was used.
-Luminous substrate solution: CDP-star (manufactured by Applied Biosystems) was used.
-Test sample (sample): Serum obtained from a healthy person was used according to a conventional method.

Reference Example 1-2: Measurement with a reagent for measuring procalcitonin The measurement of procalcitonin was carried out by the following method using an immunoluminescence measuring device (PATHFAST: manufactured by LSI Medience Corporation).
The PATH FAST dedicated cartridge is filled with the sample diluent prepared in Reference Example 1-2, the first antibody solution (magnetic latex reagent), the second antibody solution (enzyme-labeled antibody reagent), the B / F washing solution, and the luminescent substrate solution, respectively. Then, the measurement was performed according to the operation method of the device.
Specifically, 25 μL of the sample diluent, 50 μL of the magnetic latex reagent, and 50 μL of the labeled antibody reagent were added to 25 μL of the sample and reacted at 37 ° C. for 5 minutes to form a magnetic latex-PCT-ALP-labeled antibody complex. Then, the B / F washing liquids (1), (2), and (3) were washed three times with 400 mL each. 100 μL of a luminescent substrate was added to the washed complex, and the amount of luminescence was measured while allowing an enzymatic reaction at 37 ° C. for 3 minutes. CDP-Star is hydrolyzed by alkaline phosphatase in the complex and emits light. For the detection, the luminescence count of the chemiluminescent substrate detected by the photomultiplier tube (PMT) was used as the measurement result.

<< Example 1: Effect of change in pH of reaction solution on calibration curve shape >>
The pH of the sample diluent obtained by further adding 0.175 mol / L arginine to the sample diluent described in Reference Example 1-1 was changed to pH 8 to 10, and the procedure was carried out according to the method of Reference Example 1.
As a standard product, recombinant procalcitonin antigen diluted to 93.1 ng / mL, 46.1 ng / mL, 1.16 ng / mL, 0.21 ng / mL, and 0 ng / mL was used. A buffer solution containing 0.05 mol / L HEPES (pH 7.0), 2% lactose, 1% bovine serum albumin (BSA), and 0.05% Tween 20 was used for the dilution.
The result is shown in FIG. It was confirmed that the calibration curve is curved when the pH of the sample diluent is 8.0 to 9.0, whereas the calibration curve is linear when the pH is 9.5 to 10.0, so that the calibration curve is linear. Showd that the pH of the sample diluent needs to be high.

<< Example 2: Effect of change in surfactant concentration of B / F cleaning solution on simultaneous reproducibility >>
Except for the following, the method of Reference Example 1 was followed.
-Sample diluted solution: A buffer solution (pH 10.0) containing 0.1 mol / L HEPES, 0.45 mol / L NaCl, 0.05% Tween 20, 0.001% Triton X-100, 0.175 mol / L arginine. used.
B / F wash: A buffer containing 0.01 mol / L MES, 0.9% NaCl, 0.001% to 0.4% Triton X-100, 0.05% Tween 20 was used.
-The calibration curve was measured with a multiplicity of n = 18 using a sample between the stored 40 ng / mL and 100 ng / mL as a high-concentration sample, and the coefficient of variation (C) was determined from the results measured under each condition. .V (%)).
The result is shown in FIG. It was confirmed that the coefficient of variation (CV (%)) improved up to a Triton X-100 concentration of 0.15% and became almost constant thereafter.

In the present invention, when the substance to be measured is detected, the substance to be measured in a test sample (for example, serum and heparinized plasma) is measured by a heterogeneous immunoassay, in a solution used as a reaction medium. By adding a compound having a guanidino group to the sample, the shape of the high concentration region of the calibration curve can be improved. Therefore, the measurement result can be obtained stably and with high accuracy. Clinical tests need to be measured easily and quickly, and are measured not only in laboratories but also in the POCT field and the like. For example, when a blood sample is targeted, considering the possibility that it can be measured in various situations, since the containers used for collecting the sample are diverse, stable and highly accurate regardless of the type of the obtained sample. The present invention, which makes it possible to obtain measurement results, is particularly useful.

Claims (9)

In the measurement by the heterogeneous immunoassay method of the substance to be measured existing in the sample derived from the living body, the high concentration range of the calibration curve is obtained by adding the compound having a guanidino group to the solution used as the medium of the immune reaction. How to improve the shape of. It is a method of measuring a substance to be measured existing in a sample derived from a living body by a heterogeneous immunoassay.
(1) (a) a test sample that may contain a substance to be measured, (b) a solution containing a labeled immunological partner, (c) a solution used as a medium for an immune reaction, and , A solution containing a compound having a guanidino group (provided that the solution (b) and the solution (c) are the same reagent (that is, a solution used as a medium for an immunoreaction, and a compound having a guanidino group and a label). The step of preparing a solution) containing a modified immunological partner) or different reagents);
(2) A step of mixing the above (a), the above (b), and the above (c) to form an immunological partner containing a substance to be measured and a labeled immunological partner;
(3) Step of removing unreacted test sample and labeled immunological partner;
(4) The method comprising the step of analyzing a signal derived from the immunological partner and determining the amount of substance to be measured. The method according to claim 1 or 2, wherein the compound having a guanidine group is contained in a sample diluent and / or an antibody solution. The method according to any one of claims 1 to 3, wherein the compound having a guanidine group is a basic amino acid, histamine, spermidine, or creatine. The method according to claim 4, wherein the basic amino acid is arginine, lysine, or histidine. The method according to claim 4 or 5, wherein the basic amino acid concentration is 0.5 mol / L or less. The method according to any one of claims 1 to 6, wherein the compound having a guanidino group is present in the immune reaction solution in a state of being electrically close to neutral. The method according to claim 7, wherein the pH of the immune reaction solution is 9 or more. The substance measurement kit for measurement, which comprises an antibody that specifically binds to the substance to be measured and a buffer solution containing the compound having a guanidino group for the method according to any one of claims 1 to 8. ..

Images