Method of measuring c-peptide and reagent therefor

Abstract

To provide a method of measuring a C-peptide and measurement reagent, which enable accurate c-peptide measurement of a specimen with low C-peptide concentration.SOLUTION: A method of measuring a C-peptide in a sample collected from a living body is provided, the method comprising a pretreatment step of mixing the sample collected from the living body with a pretreatment liquid containing either an alkaline substance or acidifying agent. A C-peptide measurement reagent comprises the pretreating liquid containing either an acidifying agent or alkaline substance.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring C-peptide and a reagent therefor. [Background technology]

[0002] Human C-peptide is a peptide consisting of 31 amino acids and is a component of proinsulin, an insulin precursor. C-peptide is a polypeptide that is released as a degradation product when proinsulin is cleaved and insulin is released into the blood. Human proinsulin is a polypeptide consisting of 86 amino acids, mainly consisting of insulin B chain (1-30), C-peptide (33-63), and insulin A chain (66-86). C-peptide and insulin are bound via Arg (31st and 32nd), Lys (64th), and Arg (65th). C-peptide is released into the blood at the same time as insulin is released, so it serves as an indicator of insulin secretion dynamics, and the dynamics of C-peptide in the blood can be an important indicator for investigating the endogenous insulin secretion ability of diabetic patients, etc.

[0003] A method has been reported that can measure human C-peptide with low cross-reactivity with proinsulin, high reproducibility, and high sensitivity (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Annals of Laboratory Medicine 2018;38: pp.530-537 Summary of the Invention [Problem to be solved by the invention]

[0005] Conventional methods are also capable of measuring human C-peptide with high sensitivity. In order to construct a more sensitive C-peptide measurement system, the inventors have investigated the construction of a measurement system using samples containing C-peptide at a concentration lower than the lower limit of quantification (LOQ) of conventional methods. As a result, it has been found that there are samples from type I diabetes patients, particularly samples containing low concentrations of C-peptide (low-level samples), in which the concentration of C-peptide cannot be accurately measured by conventional methods.

[0006] An object of the present invention is to provide a method and reagent for measuring C-peptide, which are capable of measuring C-peptide with high accuracy even in samples with low levels. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have discovered that by mixing a sample with a pretreatment solution containing either an alkaline substance or an acidifying agent before measuring C-peptide in the sample, it is possible to accurately measure C-peptide even in low-level samples, and have thus completed the present invention.

[0008] That is, the present invention provides the following. (1) A method for measuring C-peptide in a sample isolated from a living organism, comprising a pretreatment step of mixing the sample isolated from a living organism with a pretreatment liquid containing either an alkaline substance or an acidifying agent. (2) The method according to (1), wherein C-peptide in the sample is measured by immunoassay. (3) The method according to (1) or (2), wherein the pretreatment liquid contains an alkaline substance, and the pretreatment step is carried out under conditions of an alkaline concentration of 0.01 N or more and 1 N or less. (4) The method according to (1) or (2), wherein the pretreatment solution contains an acidifying agent, and the pretreatment step is carried out under conditions of an acid concentration of 0.01 N or more and 1 N or less. (5) The method according to any one of (1) to (4), wherein the pretreatment liquid contains a surfactant. (6) The method according to (5), wherein the pretreatment liquid contains an alkaline substance and a surfactant, and the surfactant is an anionic surfactant. (7) The method according to (6), wherein the anionic surfactant is SDS or NLS. (8) The method according to (5), wherein the pretreatment liquid contains an acidifying agent and a surfactant, and the surfactant is a cationic surfactant, a nonionic surfactant, or an amphoteric surfactant. (9) The method according to (8), wherein the pretreatment liquid contains urea. (10) A reagent for measuring C-peptide, comprising a pretreatment liquid containing either an acidifying agent or an alkaline substance.

Advantages of the Invention

[0009] According to the present invention, it becomes possible to accurately measure C-peptide even in a low-value specimen.

Modes for Carrying Out the Invention

[0010] The concentration of “%” described in this specification is, unless otherwise specified, a weight / volume (w / v) concentration indication.

[0011] <Method for Measuring C-Peptide> The C-peptide measured in the present invention is a C-peptide derived from any animal, preferably a C-peptide derived from a mammal (e.g., primates such as humans, monkeys, chimpanzees; rodents such as mice, rats; lagomorphs such as rabbits; ungulates such as cows, pigs, goats, horses, sheep; carnivores such as dogs, cats); birds (e.g., chickens); more preferably a C-peptide derived from a primate, and particularly preferably a C-peptide derived from a human.

[0012] 1. Pretreatment Step The method of the present invention is a method for measuring C-peptide present in a biological sample separated from a living body. As a method for measuring C-peptide, immunoassay is preferred, but it is not limited thereto, and it can also be measured by other methods such as mass spectrometry. The following describes the case of measuring C-peptide by immunoassay, which is a preferred measurement method.

[0013] The method of the present invention is characterized by including a pretreatment step of mixing a biological sample with a pretreatment liquid prior to the immune reaction (reaction step) in the immunoassay. As shown in the Examples, it was found that there exist samples in which the concentration of C-peptide cannot be accurately measured by conventional methods, particularly in samples containing low concentrations of C-peptide (low-level samples). This was thought to be caused by reaction inhibitors that cause falsely high values. The pretreatment step makes it possible to accurately measure C-peptide even in low-level samples. The pretreatment liquid contains either an alkaline substance or an acidifying agent.

[0014] The volume ratio of the biological sample to the pretreatment solution mixed in the pretreatment step is preferably 1:10 to 10:1, particularly 1:5 to 5:1, and further preferably 1:3 to 3:1. The biological sample used in the present invention is not particularly limited as long as it is a sample that can contain C-peptide, and examples thereof include serum, plasma, whole blood, urine, stool, oral mucosa, pharyngeal mucosa, intestinal mucosa, and biopsy samples (e.g., thyroid fine needle aspiration (FNA) samples, intestinal samples, and liver samples). Preferably, the biological sample is serum or plasma.

[0015] As the alkaline substance contained in the pretreatment liquid, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth hydroxides such as magnesium hydroxide, etc. can be suitably used. When an alkaline substance is used, the normality of the alkaline substance in the pretreatment liquid is preferably 0.01N or more and 1N or less, particularly 0.1N or more and 0.4N or less, in terms of the concentration during pretreatment (after mixing). By setting the normality of the alkaline substance to 0.01N or more and 1N or less, it is possible to obtain a sufficient effect of the pretreatment and minimize the influence on the subsequent reaction step.

[0016] In the present invention, the pH when the pretreatment liquid containing an alkaline substance is mixed with the sample is, for example, pH 10.0 or more, preferably pH 11.0 or more, more preferably pH 12.0 or more, depending on the alkaline substance added. In addition, in the present invention, the pH when the pretreatment liquid containing an alkaline substance is mixed with the sample is, for example, pH 13.7 or less, preferably pH 13.5 or less, more preferably pH 13.3 or less, depending on the alkaline substance added. Specifically, the pH when the pretreatment liquid containing an alkaline substance is mixed with the sample is, for example, pH 10.0 to 13.7, preferably pH 11.0 to 13.5, more preferably pH 12.0 to 13.3. By setting the pH in the pretreatment step within these ranges, the effect of the pretreatment can be sufficiently obtained and the influence on the subsequent reaction step can be minimized.

[0017] As the acidifying agent contained in the pretreatment liquid, hydrochloric acid, sulfuric acid, acetic acid, etc. can be suitably used. When an acidifying agent is used, the normality of the acid in the pretreatment liquid is preferably 0.01 N or more and 1 N or less, particularly 0.1 N or more and 0.4 N or less, in terms of the concentration during pretreatment. By setting the normality of the acid to 0.01 N or more and 1 N or less, it is possible to obtain a sufficient effect of the pretreatment and minimize the influence on the subsequent reaction step.

[0018] In the present invention, the pH when the pretreatment liquid containing an acidic substance is mixed with the sample is, for example, pH 4.8 or less, preferably pH 4.5 or less, more preferably pH 4.2 or less, depending on the acidic substance added. In addition, in the present invention, the pH when the pretreatment liquid containing an acidic substance is mixed with the sample is, for example, pH 0.3 or more, preferably pH 0.4 or more, more preferably pH 0.5 or more, depending on the acidic substance added. Specifically, the pH when the pretreatment liquid containing an acidic substance is mixed with the sample is, for example, pH 0.3 to 4.8, preferably pH 0.4 to 4.5, more preferably pH 0.5 to 4.2. By setting the pH in the pretreatment step within these ranges, the effect of the pretreatment can be sufficiently obtained and the influence on the subsequent reaction step can be minimized.

[0019] In the present invention, the pH when the pretreatment solution containing an acidic substance is mixed with the sample can be set to a more optimal pH within the above-mentioned exemplary range depending on the type and concentration of the surfactant or denaturant added.

[0020] The pretreatment liquid may contain a surfactant. When the pretreatment liquid contains an alkaline substance, the surfactant is preferably an anionic surfactant. As the anionic surfactant, sulfate ester surfactants such as sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate, carboxylic acid surfactants such as sodium N-lauroyl sarcosine (NLS), sulfonic acid surfactants such as sodium dodecylbenzenesulfonate, bile acids such as deoxycholic acid and cholic acid or derivatives thereof, or salts thereof, etc. can be preferably used, and SDS and NLS can be particularly preferably used. The concentration of the surfactant is not particularly limited and can be appropriately set, but for example, when SDS or NLS is used, the concentration of the mixture mixed with the biological sample during pretreatment is preferably 0.01 to 12.5%, particularly 0.05 to 10%, and further preferably 0.1 to 7.5%. When SDS is used, the effect of the present invention can be further enhanced by setting the concentration of SDS to 0.05 to 10%.

[0021] When the pretreatment liquid contains an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant may be added alone or in combination after the pretreatment in order to reduce the effect of the anionic surfactant carried over into the reaction system. The cationic surfactant, amphoteric surfactant, and nonionic surfactant added after the pretreatment may be added to the neutralization liquid described below.

[0022] When the pretreatment liquid contains an acidifier and a surfactant, the surfactant is preferably a cationic surfactant, a nonionic surfactant, an amphoteric surfactant, or a combination thereof. The cationic surfactant is preferably a cationic surfactant having a single-chain alkyl group having 10 or more carbon atoms and a tertiary amine or a quaternary ammonium salt in the same molecule. Examples of such cationic surfactants include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, laurylpyridinium chloride, tetradecylpyridinium chloride, cetylpyridinium chloride, etc. Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., Tween (trade name, registered trademark) series) such as polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene sorbitan monooleate (Tween 80), and polyoxyethylene alkylphenyl ethers (e.g., Triton (trade name, registered trademark) series) such as polyoxyethylene octylphenyl ether. Examples of amphoteric surfactants include sulfobetaine surfactants such as CHAPS, CHAPSO, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS). The amount of these surfactants added is preferably 0.1% to 15%, and more preferably 0.5% to 10%, in terms of the concentration when mixed with the sample.

[0023] The pretreatment solution may contain other protein denaturants such as urea, thiourea, etc., as necessary. The concentration of the denaturant is preferably 0.1 M or more, more preferably 0.5 M or more and less than 4 M, at the time of treatment. In order to enhance the treatment effect, the pretreatment solution may contain any one or a combination of monosaccharides, disaccharides, citric acid, and citrate salts. Furthermore, the pretreatment solution may contain a chelating agent such as EDTA.

[0024] The pretreatment step can be carried out by mixing the biological sample with a pretreatment solution and leaving the mixture at room temperature. The pretreatment time is not particularly limited as long as it is a time that allows the sample to be treated so that C-peptide can be measured accurately, and can be, for example, 1 second or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 3 minutes or more, or 5 minutes or more. There is no particular upper limit to the pretreatment time, but it can be 60 minutes or less, 30 minutes or less, and particularly 15 minutes or less.

[0025] In the pretreatment step, the biological sample may be further heated after mixing with the pretreatment solution. The heating temperature is preferably 30 to 50°C. The heating time is included in the pretreatment time and may be any time that allows the sample to be treated so that C-peptide can be measured with high accuracy. The heating time is not particularly limited, and may be, for example, 1 second or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 3 minutes or more, or 5 minutes or more. There is no particular upper limit to the heating time, but it may usually be 60 minutes or less, 30 minutes or less, and particularly 15 minutes or less.

[0026] When the pretreatment liquid contains an alkaline substance, the method may further include a step of adding a neutralizing liquid containing an acidifying agent to neutralize the pH after the pretreatment. Examples of the acidifying agent include those mentioned above. When the method does not include a step of adding a neutralizing liquid, for example, in the reaction step described below, the buffer capacity and pH of the buffer solution to be mixed may be adjusted to neutralize the alkaline substance in the pretreatment liquid and reduce the effect in the reaction step. The pH in the reaction step can be appropriately set depending on the components contained in the reaction step, and the amount of the acidifying agent or buffer solution contained in the reaction step can be appropriately set to, for example, pH 5.5 to 9.5. When the pH after the addition of the neutralizing liquid is higher or lower than the pH in the above reaction step, the buffer capacity and pH of the buffer solution to be mixed in the reaction step may be adjusted to neutralize the alkaline substance in the pretreatment liquid and reduce the effect in the reaction step.

[0027] When the pretreatment liquid contains an acidifying agent, the method may further include a step of adding a neutralizing liquid containing an alkaline substance to neutralize the pH after the pretreatment. Examples of the alkaline substance include those mentioned above. When the method does not include a step of adding a neutralizing liquid, the acidifying agent in the pretreatment liquid may be neutralized and its influence in the reaction step may be alleviated, for example, by adjusting the buffer capacity and pH of the buffer solution to be mixed in the reaction step described below. The pH in the reaction step may be appropriately set depending on the components contained in the reaction step, and the amount of the acidifying agent or buffer solution contained in the reaction step may be appropriately set so that the pH is, for example, 5.5 to 9.5. When the pH after the addition of the neutralizing liquid is higher or lower than the pH in the above reaction step, the buffer capacity and pH of the buffer solution to be mixed in the reaction step may be adjusted to neutralize the acidifying agent in the pretreatment liquid and its influence in the reaction step.

[0028] 2.Reaction process The biological sample mixture obtained in the above pretreatment step of the method of the present invention (a sample obtained by pretreatment by mixing a biological sample with a pretreatment solution, or a sample obtained by neutralizing the mixture) is then subjected to a reaction step of an immunoassay. In the reaction step, the biological sample mixture is mixed with a buffer solution, and the antigen in the mixture is reacted with an antibody against C-peptide. Various methods for the immunoassay of C-peptide are well known, and any immunoassay capable of quantifying C-peptide can be used.

[0029] Examples of the buffer solution include those based on MES buffer, phosphate buffer, Tris buffer, and carbonate buffer, and particularly those based on Tris buffer can be preferably used. When a pretreatment solution containing a surfactant is used, it is preferable to use a buffer solution containing a water-soluble polymer such as BSA, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), dextran sodium sulfate, etc., at a final concentration of 0.01 to 10.0%, particularly about 0.05 to 5.0%, when mixed with the mixture after pretreatment, in order to absorb unreacted surfactant. When a pretreatment solution containing an alkaline substance is used in the pretreatment step, the buffer solution may have a buffer capacity capable of alleviating the effects of the alkaline substance. As described above, when the neutralization step is not included or when the pH after neutralization is high, it is preferable to use a buffer solution having a buffer capacity capable of alleviating the effects of the alkaline substance. When a pretreatment solution containing an acidifier is used, a buffer solution having a buffer capacity capable of alleviating the effects of the acid of the pretreatment solution may be used. As described above, when the neutralization step is not included or when the pH after neutralization is low, it is preferable to use a buffer solution having a buffering capacity capable of mitigating the effects of the acidifying agent. The mixture of the mixture in the pretreatment step and the buffer solution may be, for example, 1:10 to 10:1, particularly 1:5 to 5:1, and further 1:3 to 3:1 in volume ratio.

[0030] The antibody against C-peptide used in the method of the present invention is an antibody that recognizes at least a part of the amino acid sequence of C-peptide as an epitope. The antibody against C-peptide is not particularly limited, and any antibody that recognizes a known epitope can be used, but preferably, the antibody against C-peptide is an antibody that recognizes a C-peptide-specific epitope (particularly, a human C-peptide-specific epitope).

[0031] The antibody against C-peptide may be either a polyclonal antibody or a monoclonal antibody. The antibody against C-peptide may be any isotype of immunoglobulin (e.g., IgG, IgM, IgA, IgD, IgE, IgY). The antibody against C-peptide may also be a full-length antibody. A full-length antibody refers to an antibody comprising a heavy chain and a light chain each comprising a variable region and a constant region (e.g., an antibody comprising two Fab portions and an Fc portion). The antibody against C-peptide may also be an antibody fragment derived from such a full-length antibody. An antibody fragment is a part of a full-length antibody, and examples of such antibodies include constant region-deleted antibodies (e.g., F(ab')2, Fab', Fab, Fv). The antibody against C-peptide may also be a modified antibody such as a single-chain antibody.

[0032] Antibodies against C-peptide can be produced using a method known in the art. For example, antibodies against C-peptide can be produced using the above-mentioned epitopes as antigens. In addition, since many antibodies against C-peptide that recognize the above-mentioned epitopes are commercially available, such commercially available products can also be used.

[0033] The antibody against C-peptide may be immobilized on a solid phase. The antibody against C-peptide may be an antibody that can be immobilized on a solid phase in a reaction step. In this specification, an antibody immobilized on a solid phase and an antibody that can be immobilized on a solid phase in a reaction step may be simply referred to as an immobilized antibody. Examples of the solid phase include a solid phase that can accommodate or mount a liquid phase (e.g., a support such as a plate, a membrane, or a test tube, and a container such as a well plate, a microchannel, a glass capillary, a nanopillar, or a monolith column), and a solid phase that can be suspended or dispersed in a liquid phase (e.g., a solid phase carrier such as particles). Examples of the material of the solid phase include glass, plastic, metal, and carbon. As the material of the solid phase, a non-magnetic material or a magnetic material can also be used, but a magnetic material is preferable from the viewpoint of ease of operation, etc. The solid phase is preferably a solid phase carrier, more preferably a magnetic solid phase carrier, and even more preferably a magnetic particle. As a method for immobilizing an antibody, a method previously known in the art can be used. Such methods include, for example, physical adsorption methods, covalent binding methods, methods using affinity substances (e.g., biotin, streptavidin), and ionic binding methods. In a specific embodiment, the antibody against C-peptide is an antibody immobilized on a solid phase, preferably an antibody immobilized on a magnetic solid phase, and more preferably an antibody immobilized on a magnetic particle.

[0034] In the reaction step, the mixture from the pretreatment step may be mixed with a buffer solution and then contacted with the immobilized antibody, or the mixture may be mixed with a buffer solution containing the immobilized antibody. For example, the antibody immobilized on the particles may be added in advance to a buffer solution to prepare a particle solution, and the mixture may be mixed with the particle solution. The reaction step may be performed with only a primary reaction step, such as in the immune agglutination method or competitive method, or a secondary reaction step may be provided, such as in the sandwich method. When a secondary reaction step is provided, a washing step for removing unreacted components may be provided between the primary and secondary reaction steps. The primary and secondary reactions may also be performed simultaneously, such as in the sandwich one-step method.

[0035] The antibody against C-peptide may be labeled with a labeling substance. In this specification, an antibody labeled with a labeling substance may be simply referred to as a labeled antibody. Examples of the labeling substance include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin), fluorescent substances or fluorescent proteins (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances or light-absorbing substances (e.g., luciferin, aequorin, acridinium, ruthenium), radioactive substances (e.g., 3 H, 14 C. 32 P, 35 S, 125 In the case where a secondary reaction is carried out in the method of the present invention, the antibody used in the secondary reaction may be labeled with such a labeling substance.

[0036] In a specific embodiment, the method of the present invention includes, as the antibody used in the secondary reaction, another antibody against C-peptide that recognizes an epitope different from that of the antibody against C-peptide used in the primary reaction. The details of the epitope recognized by such another antibody are the same as those described in detail for the antibody against C-peptide above (however, when used in combination, the type of epitope is different). The combination of the epitope recognized by the antibody against C-peptide and the epitope recognized by the other antibody against C-peptide is not particularly limited. The use of such another antibody is preferable, for example, when the sandwich method is used.

[0037] 3. Detection process When a label is used for the primary or secondary antibody, it can be detected by a method appropriate for the label used, for example, by adding an enzyme substrate when an enzyme label is used. For example, when alkaline phosphatase (ALP) is used as the labeled antibody, a chemiluminescent enzyme immunoassay (CLEIA) system can be used with 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) as the enzyme substrate.

[0038] The method of the present invention is an immunoassay using an antibody against C-peptide. Examples of such immunoassays include direct competitive assays, indirect competitive assays, and sandwich assays. Examples of such immunoassays include chemiluminescent enzyme immunoassays (CLEIA), chemiluminescent immunoassays (CLIA), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), and immunochromatography. These immunoassays themselves are well known and need not be described in detail here, but each will be briefly described.

[0039] The direct competitive method is, for example, a method in which an antibody against a target antigen to be measured (C-peptide in the present invention) is immobilized on a solid phase (the solid phase and immobilization are as described above), and after a blocking treatment (treating the solid phase with a protein solution such as serum albumin) to prevent non-specific adsorption, this antibody is reacted with a test sample containing the target antigen (in the present invention, a biological sample that has been subjected to a pretreatment step as described above) and a certain amount of labeled antigen (the label is as described above), and after washing, the label bound to the solid phase is quantified. Since the antigen and the labeled antigen in the test sample competitively bind to the antibody, the greater the amount of antigen in the test sample, the smaller the amount of label bound to the solid phase. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase (absorbance, luminescence intensity, fluorescence intensity, etc., depending on the nature of the label, the same applies below) is measured for each, and a calibration curve is created with the antigen concentration on the horizontal axis and the label amount on the vertical axis. The amount of the label of an unknown test sample is measured, and the amount of the antigen in the unknown test sample can be measured by applying the measured amount of the label to the calibration curve. The direct competition method itself is well known in the art and is described, for example, in US 20150166678A.

[0040] In the indirect competitive method, for example, a target antigen (C-peptide in the present invention) is immobilized on a solid phase (the solid phase and immobilization are as described above). Then, after blocking of the solid phase, a test sample containing a target antigen (in the present invention, a biological sample subjected to a pretreatment step as described above) is mixed with a certain amount of an anti-target antigen antibody and reacted with the immobilized antigen. After washing, the anti-target antigen antibody bound to the solid phase is quantified. This can be performed by reacting a labeled secondary antibody (labeled as described above) against the anti-target antigen antibody, washing, and measuring the amount of label. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase is measured for each, to create a calibration curve. The amount of label is measured for an unknown test sample, and the amount of antigen in the unknown test sample can be measured by applying the measured amount of label to the calibration curve. It is also possible to use a labeled primary antibody without using a labeled secondary antibody. The indirect competitive method itself is well known in the art and is described, for example, in the above-mentioned US 20150166678A.

[0041] The sandwich method is, for example, a method in which an anti-target antigen antibody is immobilized on a solid phase (the solid phase and immobilization are as described above), and after blocking, a test sample containing a target antigen (in the present invention, a biological sample subjected to a pretreatment step as described above) is reacted, and after washing, a labeled secondary antibody (labeled as described above) against the target antigen is reacted, and after washing, the label bound to the solid phase is quantified. Various antigen standard solutions of known concentrations are prepared, and the amount of label immobilized on the solid phase for each is measured to create a calibration curve. The amount of label for an unknown test sample is measured, and the measured amount of label is applied to the calibration curve, thereby measuring the amount of antigen in the unknown test sample. The sandwich method itself is well known in the art and is described, for example, in US 20150309016A.

[0042] Among the various immunoassays mentioned above, chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescent immunoassay (FIA) are immunoassays classified based on the type of label used in the above-mentioned direct competitive method, indirect competitive method, sandwich method, etc. Chemiluminescent enzyme immunoassay (CLEIA) is an immunoassay that uses an enzyme (e.g., the above-mentioned alkaline phosphatase) as a label and a substrate (e.g., the above-mentioned AMPPD) that generates a chemiluminescent compound as a substrate. Enzyme immunoassay (EIA) is an immunoassay that uses an enzyme (e.g., the above-mentioned peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc.) as a label. As the substrate for each enzyme, a compound that can be quantified by absorbance measurement, etc. is used. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) or 3,3'5,5'-tetramethylbenzidine (TMB) is used, in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP) is used, in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside, NG: nitrophenyl galactoside is used, and in the case of luciferase, luciferin is used. Radioimmunoassay (RIA) is a method that uses a radioactive substance as a label, and the radioactive substance can be, as described above, 3 H, 14 C. 32 P, 35 S, 125 Examples of the label include radioactive elements such as I. Fluorescence immunoassay (FIA) is a method using a fluorescent substance or a fluorescent protein as a label, and examples of the fluorescent substance or fluorescent protein include fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein, etc., as described above. Immunoassays using these labels are well known in the art and are described, for example, in US 8039223B and US 20150309016A.

[0043] The immunoturbidimetric assay (TIA) is an immunoassay that utilizes the phenomenon that turbidity increases due to an antigen-antibody complex generated by an antigen-antibody reaction between a target antigen (C-peptide in the present invention) to be measured and an antibody against the antigen. Various known concentrations of antigens are added to an anti-target antigen-antibody solution, and the turbidity is measured for each, and a calibration curve is created. The turbidity of an unknown test sample is similarly measured, and the measured turbidity is applied to the calibration curve, so that the amount of antigen in the unknown test sample can be measured. The immunoturbidimetric assay itself is well known, and is described, for example, in US 20140186238A. The latex agglutination method is similar to the immunoturbidimetric assay, but instead of the antibody solution in the immunoturbidimetric assay, a suspension of latex particles with anti-target antigen-antibody immobilized on the surface is used. The immunoturbidimetric assay and the latex agglutination method themselves are well known in the art, and are described, for example, in US7820398B.

[0044] The immunochromatography method is a method of performing the above-described sandwich method or competitive method on a substrate (also called a matrix or strip) formed of a porous material such as filter paper, cellulose membrane, glass fiber, or non-woven fabric. For example, in the case of the immunochromatography method by the sandwich method, a detection zone immobilized with an anti-target antigen antibody is provided on the above substrate, and a test sample containing a target antigen (in the present invention, a biological sample that has undergone the pretreatment step as described above) is added to the substrate, and a developing solution is flowed from the upstream side to move the target antigen to the detection zone and immobilize it in the detection zone. The immobilized target antigen is sandwiched with a labeled secondary antibody, and the label immobilized in the detection zone is detected to detect the target antigen in the test sample. By forming a labeling zone containing the labeled secondary antibody upstream of the detection zone, the conjugate of the target antigen and the labeled secondary antibody is immobilized in the detection zone. When the label is an enzyme, a substrate zone containing the substrate of the enzyme is also provided upstream of the detection zone. In the case of the competitive method, for example, the target antigen can be immobilized in the detection zone, and the target antigen in the test sample can be made to compete with the target antigen immobilized in the detection zone. A labeled antibody zone is provided upstream of the detection zone, the target antigen in the test sample is reacted with the labeled antibody, and the unreacted labeled antibody is immobilized in the detection zone to detect or quantify the label, whereby the target antigen in the test sample can be detected or quantified. The immunochromatography method itself is well known in this field and is described, for example, in US6210898B.

[0045] <C-Peptide Measurement Reagent> The C-peptide measurement reagent of the present invention is a measurement reagent capable of realizing the above-described C-peptide measurement method. The measurement reagent of the present invention is characterized in that, in addition to the configuration used in a normal immunoassay, it contains a pretreatment solution containing either an alkaline substance or an acidifying agent as a constituent component.

[0046] The reagent of the present invention contains each component in a form isolated from each other or in the form of a composition. Specifically, each component may be provided in a form contained in a different container (e.g., a tube, a plate), but some components may be provided in the form of a composition (e.g., in the same solution). Alternatively, the reagent of the present invention may be provided in the form of a device. Specifically, all of the components may be provided in a form contained in the device. Alternatively, some of the components may be provided in a form contained in the device, and the remaining components may be provided in a form not contained in the device (e.g., in a form contained in a different container). In this case, the components not contained in the device may be used by being injected into the device when measuring the target substance.

[0047] In a preferred embodiment, the reagent of the present invention may have a configuration according to the type of immunoassay to be adopted. For example, when the sandwich method is adopted, the reagent of the present invention may contain, as essential components, i) a pretreatment solution, ii) an antibody against C-peptide, iii) a buffer solution, and, as optional components, iv) another antibody against C-peptide, v) a labeling substance, vi) a diluent, and, if necessary, vii) a substrate that reacts with the labeling substance. The components ii) and iii) may be contained in the same solution. The component iv) may be labeled with v) a labeling substance. Preferably, the antibody against C-peptide may be immobilized on magnetic particles.

[0048] The present invention will be described in detail below with reference to examples, although the present invention is not limited to the following examples.

[0049] (Reference Example 1) Preparation of anti-C-peptide antibody immobilized magnetic particle solution Anti-C-peptide antibody was added to the magnetic particles in 10 mM MES buffer (pH 5.0) to obtain a suspension containing 0.04 mg / mL anti-C-peptide antibody and 5 mg / mL magnetic particles. This suspension was incubated at 25°C for 1 hour with gentle stirring to immobilize the anti-C-peptide antibody on the magnetic particles. The magnetic particles were then collected with a magnet and washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain particles with immobilized anti-C-peptide antibody. In the measurement, the particles with immobilized anti-C-peptide antibody were suspended in a particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN3, 2.0% BSA, pH 7.2). This was used as the antibody-bound particle solution.

[0050] Example 1: Dilution linearity confirmation test of alkaline treatment (1) Preparation of diluted samples Purchased specimens (1) and (2) of type I diabetes were diluted 2-fold, 4-fold, and 8-fold using Lumipulse (registered trademark) specimen dilution solution (manufactured by Fujirebio Inc.). The 1-fold diluted specimen was defined as an undiluted specimen, and the specimens diluted 1-fold to 8-fold were referred to as serially diluted specimens.

[0051] (2) Alkaline treatment of specimens and calibrators 45 μL of each sample was mixed with 75 μL of an alkaline treatment solution (condition 3: 0.2 M NaOH, 0.8% N-lauroyl sarcosine sodium (NLS), pH 12.8 at pretreatment; condition 4: 0.2 M NaOH, 0.16% SDS, pH 12.7 at pretreatment) and incubated at 37°C for 7 minutes. After that, 75 μL of a neutralizing solution (0.2 M HCl) was added and quickly stirred to obtain an alkaline-treated sample (condition 3: pH 7.2 at neutralization; condition 4: pH 9.8 at neutralization).

[0052] As a calibrator for the measurement, Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio, concentrations: 0, 0.03, 0.3, 3, 30 ng / mL) was used. This calibrator was also treated in the same manner as above under conditions 3 and 4 to obtain an alkali-treated calibrator.

[0053] (3) Measurement of C-peptide in samples The C-peptide concentration of the obtained alkali-treated samples and calibrators was measured using an automatic analyzer Lumipulse L2400.

[0054] 50 μL of antibody-bound particle solution and 10 μL of sample (serial diluted specimen or calibrator) were dispensed into a cuvette. After stirring, the mixture was incubated at 37°C for 8 minutes (condition 3: pH 7.15 during the first reaction, condition 4: pH 7.47 during the first reaction). The particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a washing solution (0.05% Tween20) / PBS). 50 μL of enzyme-labeled antibody solution (alkaline phosphatase (ALP)-labeled anti-C-peptide monoclonal antibody) included with Lumipulse Presto C-peptide was added to the washed cuvette, and incubated at 37°C for 8 minutes. The particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a washing solution, and then 200 μL of substrate solution (Lumipulse Presto substrate solution (common reagent)) containing AMPPD as a substrate was added, and the reaction was allowed to proceed at 37°C for 4 minutes. The amount of light emitted (counts) with a maximum absorption wavelength of 463 nm was measured. Using the counts of the calibrators, calibration curves were created for each, and the C-peptide concentrations in the diluted samples were calculated.

[0055] As controls, the C-peptide concentrations of untreated samples and calibrators were similarly measured (Condition 1).

[0056] In condition 2, in order to confirm the effect on the measurement system when only a surfactant (NLS) was present during the primary reaction, NLS was further added to the particle dilution solution described in Reference Example 1 to a final concentration of 0.0615%, and the serially diluted samples and calibrators were measured in the same manner as in condition 1.

[0057] In condition 3, serially diluted samples that had been alkaline-treated with the alkaline processing solution containing the above-mentioned NLS and an alkaline-treated calibrator were measured in the same manner as in condition 1.

[0058] Condition 4 was performed in the same manner as condition 1, except that the sample volume of the stepwise diluted specimens that had been alkaline-treated with the alkaline treatment solution containing SDS described above and the alkaline-treated calibrator mixed with the particle liquid was 30 μL.

[0059] Table 1 shows the actual measurement values ​​of the samples diluted stepwise, the conversion values ​​calculated by multiplying the actual measurement values ​​by the dilution factor, and the percentages (recovery rates) of the conversion values ​​of each dilution factor divided by the conversion value of 1. The closer the recovery rate is to 100%, the better the dilution linearity is, and the target substance in the sample is properly quantified. On the other hand, a low recovery rate indicates that the value is higher than the actual concentration of the target substance in the sample (false high value), and a high recovery rate indicates that the value is lower than the actual concentration (false low value). Under conditions 1 and 2, the recovery rates of both sample (1) and sample (2) were below 50% when diluted 2-fold. On the other hand, an improvement in the recovery rate was confirmed under conditions 3 and 4, in which alkaline treatment was performed. Under condition 3, which was treated with an alkaline treatment solution containing NLS, the recovery rate was 100 ± 50% up to 4-fold dilution for sample (1) and up to 8-fold dilution for sample (2). Under condition 4, in which samples were treated with an alkaline treatment solution containing SDS, both samples (1) and (2) showed a recovery rate of 100±40% up to an 8-fold dilution. These results demonstrate that treatment with a pretreatment solution containing an alkaline substance reduces reactions that cause falsely high values, allowing accurate measurement even when the C-peptide in the sample is at a low level.

[0060] [Table 1]

[0061] Example 2: Specificity confirmation test using an absorption test by alkali treatment for samples showing false high values When the C-peptide concentration was measured for purchased type I diabetes samples (purchased from IIC-Japan) under the condition of untreated samples (condition 1) in the same manner as in Example 1, five samples (samples (3) to (7)) were extracted that showed measured values ​​of 0.002 ng / mL or more. When these five samples were measured under the methods of conditions 1 and 4 in Example 1, two samples (samples (3) and (4)) showed values ​​more than twice as high as the method of untreated alkali in the measurement under condition 4 in which alkaline pretreatment was performed, and three samples (samples (5), (6), and (7)) showed values ​​less than half (see the measured values ​​without antibody addition in Table 2 in Example 1). The following absorption test was performed on these samples to confirm their specificity.

[0062] The absorption test was performed by measuring the concentration of C-peptide in the same manner as in Example 1, except that an absorption test particle liquid in which the solid phase antibody (antibody used for antibody-bound particles) and labeling antibody (antibody used for alkaline phosphatase-labeled antibody) used in the measurement were added to the antibody-bound particle liquid so that each antibody was 100 μg / mL was used as an absorbent instead of the antibody-bound particle liquid. Condition 5 was the same as condition 1 in Example 1, and condition 6 was the same as condition 4 in Example 1, and the calibrator and the above five samples (3) to (7) were measured.

[0063] Table 2 shows the measurement values ​​of samples (3) to (7) measured using a reagent in which no absorption antibody was added to the antibody-bound particle liquid (measurement values ​​without antibody added), the measurement values ​​measured using a reagent in which the absorption antibody was added to the antibody-bound particle liquid (particle liquid for absorption test) (measurement values ​​with antibody added), and the value (absorption rate) expressed as a percentage of "1-(measurement values ​​with antibody added / measurement values ​​without antibody added)".

[0064] [Table 2]

[0065] Absorption tests were performed on two samples (samples (3) and (4)) that showed higher values ​​with the alkali treatment method than with the non-alkali treatment method. Under condition 6, in which alkali treatment was performed, all samples showed an absorption rate of 90% or more. This result indicates that C-peptide can be specifically detected by alkali treatment. Absorption tests were performed on three samples (samples (5), (6), and (7)) that showed higher values ​​with the non-alkali treatment method than with the alkali pretreatment method. Under condition 5, in which alkali treatment was not performed, no absorption by the measurement antibody occurred. In other words, these three samples would show falsely high values ​​if alkali treatment was not performed, and this shows that even with such samples, alkali treatment can avoid nonspecific reactions and more accurately detect C-peptide.

[0066] Example 3 Calculation of limit of detection (LOD) and limit of quantification (LOQ) when treated with alkali (1) Preparation of diluted samples Diluted samples containing C-peptide at concentrations of 0, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.008, and 0.01 ng / mL were used.

[0067] (2) Alkaline treatment of samples and calibrators 30 μL of each diluted sample was mixed with 75 μL of alkaline treatment solution (0.2 M NaOH, 0.8% N-lauroyl sarcosine sodium (NLS)) and incubated at 37°C for 7 minutes. After that, 50 μL of neutralization solution (0.2 M HCl) was added and quickly stirred to obtain an alkaline-treated diluted sample.

[0068] As a calibrator for the measurement, Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio Inc., concentrations: 0, 0.03, 0.3, 3, 30 ng / mL) was used. This calibrator was also treated in the same manner as above to obtain an alkali-treated calibrator.

[0069] (3) Measurement of C-peptide in samples The obtained alkali-treated diluted samples and calibrators were subjected to measurement of C-peptide concentration using an automatic analyzer Lumipulse L2400 in the same manner as in Example 1, Condition 3.

[0070] The alkali-treated diluted samples were measured 10 times (N=10) and the calibrators were measured twice (N=2) per concentration.

[0071] Among the alkaline-treated diluted samples measured at N=10, the average value + 3SD value of the 0 ng / mL sample (detection limit (LOD)) was calculated. The calculated detection limit (LOD) was 0.0011 ng / mL. Furthermore, the percentage (coefficient of variation (CV)) of the standard deviation of the alkaline-treated diluted samples divided by the average value was calculated. The CV of each concentration was calculated, and the minimum concentration (quantification limit (LOQ)) at which the CV was 10% or less was calculated. The calculated quantification limit was 0.008 ng / mL. From these results, it was shown that by treating with a pretreatment solution containing an alkaline substance, C-peptide can be measured or quantified with high accuracy even if the concentration of C-peptide contained in the sample is less than 0.02 ng / mL. Furthermore, it was shown that C-peptide can be measured or quantified with high accuracy even if the concentration of C-peptide contained in the sample is 0.008 ng / mL to 0.015 ng / mL.

[0072] Example 4: Dilution linearity confirmation test by acid treatment in samples showing false high values (1) Preparation of diluted samples The diluted samples used were the same as the serially diluted samples described in Example 1(1).

[0073] (2) Acid treatment of the sample and detection of C-peptide in the sample Acid treatment under conditions 7, 8, and 9 and detection of C-peptide in samples were performed as follows. Under condition 7, a dilution series of samples and a calibrator were measured in the same manner as under condition 1 in Example 1. Under condition 8, 45 μL of each sample was mixed with 75 μL of a pretreatment solution (0.5 M HCl, 2 M urea, 4% TritonX-100) (pH 1.02 at pretreatment) and incubated at 37° C. for 7 minutes, after which 75 μL of a neutralization solution (0.5 M NaOH) was added and quickly stirred (pH 10.14 at neutralization) to obtain an acid-treated sample.

[0074] The concentration of C-peptide in 30 μL of the resulting acid-treated sample was measured in the same manner as in Example 1 (pH 7.44 during the primary reaction).

[0075] Regarding Lumipulse C-peptide calibrator (manufactured by Fujirebio, concentrations: 0, 0.03, 0.3, 3, 30 ng / mL), an acid-treated calibrator was prepared in the same manner as above and measured in the same manner as in Example 1.

[0076] In condition 9, the acid treatment and neutralization of the sample were carried out on a Lumipulse L2400 instrument.

[0077] Specifically, condition 9 involved mixing 90 μL of acid treatment solution (2.4 M urea, 0.16 M HCl, 4% Tween 80, 6.4% N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS), pH 3.43) with 30 μL of sample or calibrator, incubating at 37° C. for 6.5 minutes (pretreatment pH 4.13), and then adding 80 μL of particle solution in which C-peptide antibody immobilized magnetic particles were diluted with particle dilution solution (1 M Tris buffer, 20 mM EDTA, 3% BSA, 0.1% Tween 80, pH 7.9), and reacting at 37° C. for 8 minutes (primary reaction pH 7.67). After washing with 0.05% Tween20 / PBS, 50μL of enzyme-labeled antibody solution (alkaline phosphatase (ALP)-labeled anti-C-peptide monoclonal antibody) included with Lumipulse Presto C-peptide was added and reacted at 37℃ for 8 minutes. After washing with 0.05% Tween20 / PBS, 200μL of substrate solution was added and reacted at 37℃ for 4 minutes. The amount of light emitted (counts) with a maximum absorption wavelength of 463nm was measured. Using the counts of the calibrators, a calibration curve was created for each, and the C-peptide concentration in the serially diluted samples was calculated.

[0078] Table 3 shows the actual measurement values ​​of the serially diluted samples, the converted values ​​calculated by multiplying the actual measurement values ​​by the dilution factors, and the percentage (recovery rate) calculated by dividing the converted values ​​for each dilution factor by the converted value for 1x.

[0079] [Table 3]

[0080] Under condition 7, the recovery rates of both specimens (1) and (2) were below 50% at 2-fold dilution. On the other hand, under conditions 8 and 9, in which acid treatment was performed, an improvement in recovery rate was confirmed. Under condition 8, specimen (1) showed a recovery rate of 100±40% up to 4-fold dilution, and specimen (2) showed a recovery rate of 100±30% up to 8-fold dilution. Under condition 9, in which treatment was performed with an acid treatment solution containing a cationic surfactant and neutralization was performed with a Tris buffer, both specimens (1) and (2) showed a recovery rate of 100±10% up to 8-fold dilution. These results show that treatment with a pretreatment solution containing an acidifier reduces reactions that cause false high values, and C-peptide in the sample can be accurately measured even if it is at a low level.

[0081] Example 5 Confirmation of specificity of samples showing falsely high values ​​using an absorption test with acid treatment As in Example 2, purchased specimens of type I diabetes mellitus described in Example 2 were measured under the methods of Conditions 7 and 8 of Example 4. Under Condition 8, in which acid treatment was performed, two specimens (Sample (3) and Sample (4)) showed values ​​1.7 times higher than those under Condition 7, in which the specimens were not treated, and three specimens (Sample (5), Sample (6), and Sample (7)) showed values ​​half or lower (see the measured values ​​without antibody added in Table 4 of Example 4). For these specimens, an absorption test was performed in which the measurement antibody was added to the particle liquid to confirm the specificity.

[0082] The absorption test was performed by measuring the concentration of C-peptide in the same manner as in Example 4, except that an absorption test particle liquid in which the solid phase antibody (antibody used for antibody-bound particles) and labeling antibody (antibody used for alkaline phosphatase-labeled antibody) used in the measurement were added to the antibody-bound particle liquid so that each antibody was 100 μg / mL was used as an absorbent instead of the antibody-bound particle liquid. Condition 10 was the same as condition 7 in Example 4, condition 11 was the same as condition 8 in Example 4, and condition 12 was the same as condition 9 in Example 4, and the calibrator and the above five samples (3) to (7) were measured.

[0083] Table 4 shows the measurement values ​​of samples (3) to (7) measured using a reagent in which no absorption antibody was added to the antibody-bound particle liquid (measurement values ​​without antibody added), the measurement values ​​measured using a reagent in which the absorption antibody was added to the antibody-bound particle liquid (particle liquid for absorption test) (measurement values ​​with antibody added), and the value (absorption rate) expressed as a percentage of "1-(measurement values ​​with antibody added / measurement values ​​without antibody added)".

[0084] [Table 4]

[0085] The results of the absorption test for the two samples that showed higher values ​​with the acid treatment method than with the untreated method showed an absorption rate of 95% or more for all samples under conditions 11 and 12, in which acid treatment was performed. This result indicates that C-peptide can be specifically detected by acid treatment. The results of the absorption test for the three samples (samples (5), (6), and (7)) that showed higher values ​​with the untreated method than with the acid pretreatment method showed that no absorption by the measurement antibody occurred under condition 10, in which no acid treatment was performed. In other words, these three samples were samples that showed falsely high values ​​if acid treatment was not performed, and this shows that even with such samples, nonspecific reactions can be avoided by performing acid treatment, and C-peptide can be detected more accurately.

[0086] Example 6 Calculation of limit of detection (LOD) and limit of quantification (LOQ) when treated with acid (1) Preparation of diluted samples Diluted samples containing C-peptide at concentrations of 0, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.008, and 0.01 ng / mL were used.

[0087] (2) Acid treatment of samples and calibrators and C-peptide measurement Using an automatic analyzer Lumipulse L2400, the C-peptide concentration in the sample was measured in the same manner as in Example 4, Condition 9. That is, 30 μL of each diluted sample was mixed with 90 μL of an acid treatment solution (2.4 M urea, 0.16 M HCl, 4% Tween 80, 6.4% N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16APS), pH 3.43), and incubated at 37° C. for 6.5 minutes, after which 80 μL of a particle solution in which C-peptide antibody immobilized magnetic particles were diluted with a particle dilution solution (1 M Tris buffer, 20 mM EDTA, 3% BSA, 0.1% Tween 80, pH 7.9) was added, and the reaction was allowed to proceed at 37° C. for 8 minutes.

[0088] As a calibrator for the measurement, Lumipulse Presto (registered trademark) C-peptide calibrator (manufactured by Fujirebio Inc., concentrations: 0, 0.03, 0.3, 3, 30 ng / mL) was used. C-peptide was also measured for this calibrator in the same manner as above.

[0089] The acid-treated diluted samples were measured 10 times (N=10) and the calibrators were measured twice (N=2) per concentration.

[0090] Among the acid-treated diluted samples measured at N=10, the average value + 3SD value of the 0 ng / mL sample (detection limit (LOD)) was calculated. The calculated detection limit (LOD) was 0.0002 ng / mL. Furthermore, the percentage (coefficient of variation (CV)) of the standard deviation of the alkali-treated diluted samples divided by the average value was calculated. The CV of each concentration was calculated, and the minimum concentration (quantification limit (LOQ)) at which the CV was 10% or less was calculated. The calculated quantification limit was 0.001 ng / mL. From these results, it was shown that by performing treatment with a pretreatment solution containing an alkaline substance, C-peptide can be measured or quantified with high accuracy even if the concentration of C-peptide contained in the sample is less than 0.02 ng / mL. Furthermore, it was shown that C-peptide can be measured or quantified with high accuracy even if the concentration of C-peptide contained in the sample is 0.001 ng / mL to 0.015 ng / mL.

Claims

1. A method for measuring C-peptide in a sample separated from a living body, comprising a pretreatment step of mixing the sample separated from a living body with a pretreatment liquid containing either an alkaline substance or an acidifying agent.

2. The method of claim 1, wherein C-peptide in the sample is measured by immunoassay.

3. The method according to claim 1 or 2, wherein the pretreatment liquid contains an alkaline substance, and the pretreatment step is carried out under conditions of an alkaline concentration of 0.01N or more and 1N or less.

4. The method according to claim 1 or 2, wherein the pretreatment solution contains an acidifying agent, and the pretreatment step is carried out under conditions of an acid concentration of 0.01N or more and 1N or less.

5. The method of claim 1 or 2, wherein the pretreatment solution comprises a surfactant.

6. The method of claim 5 , wherein the pretreatment liquid comprises an alkaline substance and a surfactant, the surfactant being an anionic surfactant.

7. The method of claim 6 , wherein the anionic surfactant is SDS or NLS.

8. 6. The method of claim 5, wherein the pretreatment solution comprises an acidifying agent and a surfactant, the surfactant being a cationic surfactant, a nonionic surfactant, or a zwitterionic surfactant.

9. The method of claim 8 , wherein the pretreatment solution comprises urea.

10. A reagent for measuring C-peptide, comprising a pretreatment liquid containing either an acidifying agent or an alkaline substance.