Standard substance for ferritin measurement and ferritin measurement method

Abstract

To provide a method for stabilizing a measurement value of a ferritin concentration in a sample.SOLUTION: A method for reducing the difference in ferritin measurement results in a sample between at least two or more lots of ferritin measurement standard substances uses ferritin that satisfies the following (1) or (2) in the standard substance: (1) a ratio of ferritin monomer in ferritin in the standard substance is 90% or more; (2) the difference in a ratio of ferritin oligomer equal to or larger than trimmer in ferritin in the standard substance is 8 points or less between the standard substances.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a standard substance for measuring ferritin, a method for producing the standard substance, and a method for measuring ferritin using the standard substance. The present invention also relates to a method for reducing differences in ferritin measurement results. [Background technology]

[0002] Ferritin is a globular protein complex containing 24 polypeptide subunits and a molecular weight of approximately 480 kDa. Two types of polypeptide subunits with different molecular weights, H chain (21 kDa) and L chain (19 kDa), are known. The inside of a ferritin molecule is hollow, and a large number of iron ions can be stored in this space. One ferritin molecule contains 4,500 ferric ions (Fe 3+ ) can be stored.

[0003] In humans, ferritin is present in organs such as the liver, kidneys, spleen, and placenta, as well as in serum, and is thought to function as an iron transport protein. As a method for treating iron deficiency disorders, a method of administering a ferritin-iron complex to a patient has been proposed (for example, Patent Document 1).

[0004] In addition, it is known that the concentration of ferritin in serum is closely related to the amount of iron stored in the body, and is an important measurement item in clinical tests for iron deficiency and the like (for example, Non-Patent Document 1). Various immunological measurement methods are known for measuring ferritin, but in recent years, immunoassays using the latex agglutination method have been widely used because they are simpler and faster than other methods. In order to improve the detection sensitivity of the latex agglutination method, a method has been proposed in which a reaction solution contains one selected from the group consisting of polyvinylpyrrolidone, pullulan, and polyethylene glycol, each of which has a predetermined molecular weight (Patent Document 2), and a method has been proposed in which a quaternary ammonium salt is contained in the reaction solution to suppress the influence of the freshness of the serum sample (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2011-516419 [Patent Document 2] Japanese Patent Application Publication No. 10-115615 [Patent Document 3] JP 2000-88849 A [Non-patent literature]

[0006] [Non-Patent Document 1] Guidelines for quantitative test evaluation methods and samples for clinical laboratory quality control surveys, Medical Testing, 2008, 57:109-117 Summary of the Invention [Problem to be solved by the invention]

[0007] However, in the above-mentioned Non-Patent Document 1, ferritin as a measurement item is classified as "Classification 3) Items for which no standard measurement procedure or standard substance has been established and for which there is a large difference between facilities." Thus, in the immunological measurement of ferritin, differences in the measurement results may occur when the measurement facilities are different, and quality control has been an important issue.

[0008] The above Patent Document 2 proposes a highly sensitive, rapid and simple ferritin quantification method, and the above Patent Document 3 proposes a ferritin measurement method that is less affected by the freshness of a ferritin sample. However, even these methods are still not sufficient from the viewpoint of quality control.

[0009] Therefore, an object of the present invention is to provide a method for stabilizing the measured value of ferritin concentration in a sample. [Means for solving the problem]

[0010] As a result of research conducted by the present inventors to solve the above problems, it was found that when ferritin, which contains 24 polypeptide subunits and has a molecular weight of approximately 480 kDa, is referred to as a monomer, ferritin monomers associate to form ferritin dimers, ferritin oligomers, etc., and that the reactivities of these in immunological measurements differ, and that the differences in the reactivities of these in standard substances are one of the causes of the differences in measurement values. That is, in preparing a standard substance (calibrator) for immunoassay in measuring ferritin in a sample, ferritin derived from biological tissue is often used as the standard substance, but when using ferritin from different lots, the ratio of ferritin monomers, dimers, oligomers, etc. may differ, and therefore the standard substance may also differ in their constituent ratios. Furthermore, since ferritin monomers, dimers, oligomers, etc. have different reactivities in immunoassay, it has been found that the constituent ratios of ferritin monomers, dimers, oligomers, etc. differ between multiple lots of standard substances, which is the cause of the variation in the calibration curve and, ultimately, is one of the factors that cause the measured values ​​of the sample to change. The present inventors then discovered that the above problems can be solved by using ferritin in which the ratio of ferritin monomers, dimers, oligomers, etc. falls within a specified range in a standard substance, and thus completed the present invention. Specifically, the present invention is as follows.

[0011] [1] A method for reducing a difference in a ferritin measurement result in a sample between at least two or more lots of a standard substance for ferritin measurement, comprising: A method for using ferritin that satisfies the following (1) or (2) as the standard substance: (1) The proportion of ferritin monomers in the ferritin in the standard material is 90% or more; (2) The difference in the proportion of ferritin oligomers of trimers or higher in ferritin in the standard substances is 8 points or less between the standard substances. [2] The method according to [1], wherein in (2), the difference in the proportion of ferritin dimer in the ferritin in the standard substances is 10 points or less between the standard substances. [3] The method according to [1] or [2], wherein the ratio is adjusted for ferritin before it is prepared as a standard substance. [4] A method for producing a standard substance for ferritin measurement, comprising the steps of: A method for using ferritin that satisfies the following (1) as the standard substance: (1) The proportion of ferritin monomers in the ferritin is 90% or more. [5] A method for producing a second lot of a standard substance for ferritin measurement by referring to a first lot of a standard substance for ferritin measurement, comprising: A method in which ferritin that satisfies the following (2) is used as the second standard substance: (2) The difference in the proportion of ferritin oligomers of trimers or higher in ferritin in the said standard substances is 8 points or less between the said first standard substance and the said second standard substance. [6] The method for producing a standard substance for measuring ferritin according to [5], wherein in (2), the difference in the proportion of ferritin dimer in the ferritin in the standard substance is 10 points or less between the first standard substance and the second standard substance. [7] The method for producing a standard substance for measuring ferritin according to any one of [4] to [6], wherein in (1) or (2), the ratio is adjusted for ferritin before it is prepared as a standard substance. [8] A standard substance for measuring ferritin, comprising: A standard substance for measuring ferritin, characterized in that the proportion of ferritin monomers in the ferritin in the standard substance is 90% or more. [9] The standard substance for measuring ferritin according to [8], wherein the proportion of ferritin oligomers of trimers or higher in ferritin in the standard substance is 5% or less.

[10] A method for measuring ferritin, comprising using a standard substance produced by the method according to any one of [4] to [7], or the standard substance according to [8] or [9]. Effect of the Invention

[0012] According to the present invention, the reactivity of ferritin in a standard substance can be stabilized, and the measured value of the ferritin concentration in a sample can be stabilized. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described. A method according to one embodiment of the present invention is a method for reducing the difference in ferritin measurement results in a sample between at least two or more lots of standard substances for ferritin measurement, in which ferritin in the standard substance is used in which the proportions of monomers, dimers, oligomers, etc. in the ferritin in the standard substance are within a specified range.

[0014] 1. Ferritin Ferritin is a globular protein complex containing 24 polypeptide subunits and weighing approximately 480 kDa. The ferritin used in this embodiment can be obtained by purification from the liver, placenta, spleen, etc., and recombinant proteins, etc. can also be used. However, from the viewpoint of availability, etc., the ferritin used in this embodiment does not have to be an L-chain recombinant, and is preferably derived from the liver or placenta, and is particularly preferably derived from the liver.

[0015] In this specification, ferritin (a protein complex) containing 24 polypeptide subunits and having a molecular weight of approximately 480 kDa is referred to as "ferritin monomer" (or simply as "monomer"). Furthermore, the complex of approximately 960 kDa formed by further association of the two monomer molecules is called a "ferritin dimer" (or sometimes simply called a "dimer"). Furthermore, a large complex formed by the association of three or more molecules of the above monomers is called a "ferritin oligomer of trimer or more" (sometimes simply called a "ferritin oligomer" or "oligomer").

[0016] As shown in the examples below, as ferritin polymerizes into monomers, dimers, and oligomers, its reactivity in immunological measurement decreases. Therefore, if the composition ratio of these components in the standard substance varies significantly, the calibration curve created from the standard substance will vary even in the same measurement device, and the measured value of the ferritin concentration in the sample will also differ. In contrast, by adjusting the above-mentioned constituent ratio in the ferritin contained in the standard substance so that it does not fluctuate significantly, the calibration curve will be stable and will not fluctuate significantly, and the measured value of the ferritin concentration in the sample can also be stabilized.

[0017] It was known that ferritin exists as a monomer, dimer, and oligomer; however, it was not known at all that there is a difference in reactivity between these in immunological assays, and this is a new discovery by the present inventors. Furthermore, as shown in the Examples below, when the same ferritin measurement reagent is used in a different measurement device, differences in reactivity in immunological measurement are observed among monomers, dimers, and oligomers.

[0018] 2. Ratio of monomers, dimers and oligomers In this embodiment, the proportions of monomers, dimers, and oligomers in ferritin can be expressed, for example, by absorbance conversion. Specifically, ferritin is fractionated by gel filtration chromatography or the like, and the proportions of the protein amounts of monomers, dimers, and oligomers relative to the total ferritin can be expressed by absorbance conversion. Monomers, dimers and oligomers can be fractionated based on molecular weight in a conventional manner. Specific conditions for gel filtration chromatography can be exemplified by the conditions shown in the Examples below. The absorbance can be measured, for example, at a wavelength of 280 nm. The proportions of monomers, dimers, and oligomers in terms of protein amount can be determined by the peak area ratios in gel filtration chromatography measured at that wavelength.

[0019] In addition, when the absorbance of each fraction cannot be regarded as the absorbance of ferritin because the composition in which the proportion of monomers, etc. in ferritin is to be determined contains components other than ferritin, the above proportion can be determined, for example, by the following method (a) or (b).

[0020] (a) Ferritin is purified by affinity purification, crystallization, etc. Then, as described above, it is fractionated by gel filtration chromatography, etc., and the protein amount is calculated by measuring absorbance, and the ratio of monomers, etc. (protein amount ratio) is determined.

[0021] (b) Generally available ferritin is fractionated into monomers, dimers, and oligomers, and the absorbance of each fraction is measured, and the signal strength is measured using a combination of a specific ferritin measurement reagent and a measurement device, and a relational formula between the signal strength and absorbance in ferritin monomers, etc. is identified. Thereafter, the composition in which the proportion of monomers, etc. in ferritin is to be determined is fractionated by gel filtration chromatography, etc., and the signal strength of ferritin is measured for each fraction using the combination of the specific ferritin measurement reagent and a measurement device. From the signal strength of each fraction and the above relational formula, the signal strength in each fraction is converted to absorbance, and the proportions of monomers, dimers, and oligomers are calculated.

[0022] Here, the "absorbance" of ferritin refers to the absorption intensity of light with a wavelength of 280 nm when the ferritin antigen alone is measured using a spectrophotometer. Moreover, "signal intensity" refers to the amount of change in a signal detected by immunological measurement. The above-mentioned signal may be turbidity, absorbance, fluorescence, luminescence, RI, etc., depending on the method of immunological measurement. The signal intensity increases or decreases in correlation with the amount of ferritin, and since the reactivity in immunological measurement differs between ferritin monomers, dimers, and oligomers, the degree of the above-mentioned increase or decrease differs between ferritin monomers, dimers, and oligomers.

[0023] 3. Methods for reducing differences in ferritin measurement results in samples In this embodiment, as described above, by adjusting the composition ratio of monomer, dimer and oligomer in the ferritin contained in the standard substance so that it does not fluctuate significantly, the calibration curve is stable and does not fluctuate significantly, and the measured value of the ferritin concentration in the sample can be stabilized. Here, a specific example of a method for adjusting the constituent ratio of monomers, etc. in ferritin so that it does not vary significantly is to use ferritin that satisfies the following (1) as the ferritin in the standard substance. (1) The proportion of ferritin monomers in the ferritin contained in the standard material is 90% or more.

[0024] By doing as described above in (1), it is possible to suppress the fluctuation of the ratio of low-reactivity dimers, oligomers, etc., stabilize the reactivity of ferritin in the standard material, stabilize the calibration curve, and stabilize the measured value of the ferritin concentration in the sample. In other words, by using ferritin that satisfies the above in (1), it is possible to reduce the difference in the measurement result of ferritin in the sample between standard materials of different lots. In the above (1), the proportion of ferritin monomers is more preferably 95% or more, and particularly preferably 98% or more.

[0025] In addition, as shown in the examples below, when the same ferritin measurement reagent is used and different measurement devices are used, differences in reactivity are observed between monomers, dimers, and oligomers. Due to this, if the proportion of oligomers (or dimers) in ferritin in the standard material is high, differences in measurement results may occur between measurement devices even when the same sample is measured using the same lot of standard material. In contrast, by increasing the proportion of ferritin monomers in the ferritin used as the standard substance, for example to 90% or more, 95% or more, or 98% or more, not only can the measurement value difference between different lots of standard substances be reduced, but also the measurement value difference between different immunological measurement devices using the same lot of standard substance can be reduced.

[0026] Here, the term "lot" in this specification refers to a product unit manufactured using the same raw materials under the same conditions. For example, if the same raw materials such as ferritin are used and the manufacturing conditions are the same, it can be said that the reference material is from the same lot. The lot can be defined not only for the standard substance but also for ferritin, for example, if the ferritin is produced using the same raw material under the same conditions, it is considered to be the same lot of ferritin. If the lot of ferritin is the same, the composition ratio of monomers, dimers, and oligomers in the ferritin can be considered to be the same. In addition, when at least two or more reference materials are made from different lots of ferritin, the raw materials used are not the same, and the lots of the reference materials are also different. In addition, when at least two or more reference materials (or ferritin) are manufactured in different processes, places, times, etc., the manufacturing conditions are not the same, and the lots of the reference materials (or ferritin) are also different.

[0027] Furthermore, in at least two or more lots of standard substances, in addition to the above (1), ferritin satisfying the following (2) may be used as the ferritin in the standard substance. (2) The difference in the proportion of ferritin oligomers of trimers or higher in the ferritin in the standard materials is 8 percentage points or less between the standard materials. In this specification, "points" refer to percentage points.

[0028] By doing as described above in (2), it is possible to suppress the variation in the ratio of the least reactive oligomer, and even when using a standard substance of a different lot, it is possible to stabilize the ferritin reactivity in the standard substance, stabilize the calibration curve, and stabilize the measured value of the ferritin concentration in the sample. In other words, by using ferritin that satisfies the above in (2), it is possible to reduce the difference in the measurement result of ferritin in the sample between at least two or more lots of standard substances for ferritin measurement that are different in lot. In the above (2), the difference in the proportion of oligomers in ferritin in the standard substances is more preferably 5 points or less, and particularly preferably 3 points or less, between the standard substances.

[0029] Furthermore, when producing a standard substance, by using ferritin such as (1) or (2) above, it is possible to obtain a standard substance with reduced differences in ferritin measurement results, even if multiple lots of different standard substances are produced using any lot of ferritin.

[0030] In the above (1) or (2), it is preferable that the ferritin used as the standard substance further satisfies the following (3): (3) The percentage of ferritin oligomers of trimers or higher in the ferritin in the standard material is 5% or less. Furthermore, the proportion of the above oligomer is more preferably 4% or less, and particularly preferably 3% or less. By reducing the proportion of the least reactive oligomer, the ferritin reactivity in the standard can be further stabilized, and the measured value of the ferritin concentration in the sample can be stabilized.

[0031] Furthermore, between at least two or more lots of standard substances, it is preferable that the ferritin used as the standard substance satisfies the following (4) in addition to the above (1) or (2). (4) The difference in the proportion of ferritin dimer in the ferritin in the above standard materials is 10 points or less between the standard materials. Furthermore, it is more preferable that the difference in the proportion of dimers in ferritin in the standard substances is 7 points or less, and particularly preferably 5 points or less, between the standard substances.

[0032] Furthermore, in the above (2), it is preferable to use ferritin in the standard substance in which the proportion of ferritin monomers in the ferritin is 50% or more, 60% or more, 70% or more, 80% or more, and most preferably 90% or more.

[0033] Here, an example of a method for adjusting the constituent ratio of monomers, etc. in ferritin so that it does not vary significantly (e.g., the above (1) to (4), etc.) is to confirm the constituent ratio of monomers, etc. in ferritin. In this case, when using ferritin from different lots, the above constituent ratio is confirmed for each lot. The method for confirming the constituent ratio of monomers, etc. in ferritin is as described above, and ferritin is fractionated by gel filtration chromatography or the like, and the constituent ratio can be expressed in terms of absorbance. When the composition ratio of monomers in ferritin satisfies the desired conditions (e.g., (1) or (2) above), the ferritin has already been adjusted to the desired composition ratio, and therefore the ferritin can be used as is as a standard substance. On the other hand, when the desired conditions are not satisfied, the composition ratio can be adjusted to the desired ratio by, for example, appropriately combining each fraction obtained by fractionation by gel filtration chromatography or the like. In this embodiment, it is preferable to adjust the constituent ratios of monomers, etc. in ferritin before it is prepared as a standard substance.

[0034] 4. Standard material A standard substance for ferritin measurement according to one embodiment of the present invention contains ferritin in which the constituent ratios of monomers, etc. are within the above-mentioned ranges. The reference material according to the present embodiment may contain, in addition to the above-mentioned ferritin, other components that are acceptable for reference materials. For example, water, salts, buffers, stabilizers, serum, etc. may be appropriately added as such components.

[0035] As the salt, sodium chloride, potassium chloride, lithium chloride, cesium chloride, phosphates, etc. can be appropriately used, and one type can be used alone or two or more types can be used in combination. The concentration of the salt can be, for example, 5 to 3000 mM, and can be 100 to 2000 mM.

[0036] Examples of the buffer include Good's buffers such as HEPES and PIPES, phosphate buffers, Tris buffers, glycine buffers, glycylglycine buffers, etc., and one type may be used alone or two or more types may be used in combination. The concentration of the buffer may be, for example, 1 to 200 mM, and may be 5 to 50 mM.

[0037] The stabilizer is used to stabilize ferritin, and specifically includes bovine serum albumin, skim milk, gelatin, etc., and can be used alone or in combination of two or more. The concentration of the stabilizer can be, for example, 0.01 to 20% by mass, or 0.1 to 10% by mass.

[0038] Furthermore, serum may be used to dilute the standard. Such serum is preferably delipidated serum, and is preferably serum that does not contain ferritin. The absence of ferritin can be confirmed, for example, by immunological measurement, which will be described later. Specifically, the serum can be determined to be free of ferritin if the results of multiple measurements of the serum are not significantly different from those of a control sample that does not contain ferritin.

[0039] Furthermore, the reference substance according to this embodiment may contain other additives, such as preservatives such as sodium azide; preservatives such as benzoic acids and sorbic acids; and fungicides such as orthophenylphenols, diphenyl, and thiabenzol, as appropriate, within the scope that does not impair the effects of this embodiment.

[0040] The pH of the standard substance can be specifically set to pH 5-10, or further to pH 6-8.

[0041] 5. Manufacturing method of reference material The method for producing a standard substance for ferritin measurement according to one embodiment of the present invention can be produced according to a conventional method, in addition to using ferritin having a monomer content within the above-mentioned range. For example, the standard substance according to this embodiment can be produced by mixing ferritin with other components as desired.

[0042] More specifically, in producing the standard material, ferritin that satisfies the following (1) is used: (1) The proportion of ferritin monomers in the ferritin is 90% or more.

[0043] In addition, when a reference material of a different lot is produced, that is, when a reference material of a different lot (second lot) is produced by referring to a reference material of a first lot, ferritin that satisfies the following (2) is used in the second reference material: (2) The difference in the proportion of ferritin oligomers of trimers or higher in ferritin in the standard material is 8 percentage points or less between the first standard material and the second standard material. Here, when a second lot of standard material is produced by referencing a first lot of standard material, the first and second lots of standard material may be produced simultaneously while referencing each other, or a new standard material (a second lot of standard material) may be produced by referencing an already obtained standard material (a first lot of standard material).

[0044] More preferable conditions for the composition ratio of ferritin monomers, etc. in ferritin and other components acceptable for the standard substance are preferably as described above.

[0045] 6. How to measure ferritin The method for measuring ferritin according to one embodiment of the present invention uses the standard substance for measuring ferritin obtained as described above. By using such a standard substance, the calibration curve can be stabilized, and the measured value of the ferritin concentration in the sample can be stabilized. Specifically, the measurement of ferritin is carried out by first using the above-mentioned ferritin measurement standard substance, measuring the signal intensity of various known concentrations of ferritin measurement standard substance using a ferritin measurement reagent, and creating a calibration curve. Next, measuring the signal intensity of a sample using the ferritin measurement reagent, and calculating the concentration by applying it to the previously created calibration curve.

[0046] The measurement method according to the present embodiment immunologically measures ferritin. That is, it utilizes a specific antigen-antibody reaction between ferritin as an antigen and an anti-ferritin antibody. The anti-ferritin antibody may be either a polyclonal antibody or a monoclonal antibody, or a recombinant antibody, or may be an antibody fragment such as Fab, F(ab')2, Fab', or Fv.

[0047] Examples of immunological measurement methods include, but are not limited to, immunoagglutination methods such as latex agglutination and gold colloid agglutination, sandwich methods such as enzyme immunoassay (ELISA, etc.) and chemiluminescence assay, radioimmunoassay, and immunochromatography. Examples of the measurement method include a method of measuring the absorbance, scattered light, luminescence, fluorescence, etc. of the immune reaction solution by an optical method, a method of measuring the radioactivity of a labeled radioisotope, etc. As the optical method, for example, a general-purpose optical measuring device may be used, and for example, the measurement can be performed using a Hitachi automatic analyzer model 7180 (manufactured by Hitachi High-Technologies Corporation).

[0048] Examples of the specimen include whole blood, serum, plasma, etc. The specimen may be used as is, or may be diluted or otherwise used as a sample for measurement.

[0049] According to the standard substance for measuring ferritin according to the embodiment described above, the reactivity of ferritin in the standard substance can be stabilized, and the calibration curve can be stabilized, thereby stabilizing the measurement value of the ferritin concentration in a sample.

[0050] The above-described embodiments are described for the purpose of facilitating understanding of the present invention, and are not described for the purpose of limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design modifications and equivalents / methods that fall within the technical scope of the present invention. EXAMPLES

[0051] The present invention will be described in more detail below by showing test examples, but the present invention is not limited to the following test examples.

[0052] [Test Example 1] Measurement using a calibrator (standard material) made from different lots of ferritin Calibrators (standard materials) for ferritin immunoassay were prepared using ferritin (BBI, human liver-derived, lot 1 and lot 2, purity by SDS-PAGE: 95% or higher). Two types (2 lots) of calibrators were prepared using two lots of ferritin (lot 1 and lot 2, respectively). The ferritin concentration was adjusted to 1000ng / mL by dilution with delipidated serum based on the manufacturer's indicated value, and calibrators with concentrations of 25, 250, 500, and 750ng / mL were also prepared. The delipidated serum used for dilution was a commercially available delipidated serum, from which ferritin had been removed using an affinity gel containing an anti-ferritin antibody. The absence of ferritin in the delipidated serum after removal was confirmed using the LZ test 'Eiken' FER.

[0053] A calibration curve was created using the obtained calibrator, and the ferritin concentration in human samples (No. 1 to 6) was measured. A latex agglutination reagent (Eiken Chemical Co., Ltd., "LZ Test 'Eiken' FER") was used as the measurement reagent, and a Hitachi 7180 automatic analyzer (Hitachi High-Technologies Corporation) was used as the measurement device. Based on the results obtained, the ratio of the measurement value obtained with the lot 2 calibrator to the measurement value obtained with the lot 1 calibrator was calculated as the difference between the lots. The results are shown in Table 1.

[0054] [Table 1]

[0055] As shown in Table 1, it became clear that different lots of ferritin used in calibrators had different calibration curves due to differences in signal intensity, which resulted in differences in the measurement results. Since the other components in the calibrators were prepared in the same way, it was believed that the differences in the measurement results were due to the differences in the lots of ferritin used in the calibrators.

[0056] [Test Example 2] Analysis by gel filtration chromatography Ferritin from Lot 1 and Lot 2 was dissolved in a buffer (0.05 M phosphate buffer: pH 7.0, sodium chloride: 0.3 M) (ferritin concentration: 10% by mass calculated as the indicated value) and analyzed by gel filtration chromatography under the following conditions. Gel Filtrate Standard (BIO RAD) was used as a molecular weight marker.

[0057] Gel filtration chromatography conditions Column: Yarra SEC3000 (Shimadzu GLC) Mobile phase: 0.05M phosphate buffer, 0.3M NaCl, pH 7.0 Flow rate: 0.5mL / min Detection: UV (280 nm)

[0058] In both Lots 1 and 2, three peaks were observed, which were found to correspond to ferritin monomers (molecular weight approximately 480 kDa), dimers (approximately 960 kDa), and oligomers (approximately 1400 kDa or more), respectively. The constituent ratios calculated from the peak area ratios are shown in Table 2. Since they differed between the two lots, it was considered possible that the constituent ratios of monomers, etc. in ferritin were the cause of the difference in the measurement results.

[0059] [Table 2]

[0060] [Test Example 3] Measurement using a calibrator with monomers, etc. For the ferritin of Lot 1 fractionated in Test Example 2, the extinction coefficient of ferritin was determined as follows. First, the concentration of the monomer fraction was determined based on the WHO international standard for ferritin (recombinant, 3rd IS, 94 / 572), and the extinction coefficient of ferritin was determined from the absorbance of the monomer fraction. Here, the ferritin standard should conform to the above-mentioned WHO international standard, but the WHO international standard is a recombinant ferritin protein diluted with plasma and contains many proteins other than ferritin. Therefore, when determining the concentration of the monomer, a latex agglutination reagent (LZ test 'Eiken' FER) and a Hitachi 7180 automatic analyzer (H7180) were used to determine the concentration of the monomer fraction based on the displayed value of the WHO international standard. From the concentration of the monomer fraction thus obtained and the absorbance of the monomer fraction, the extinction coefficient (wavelength: 280 nm) of the ferritin monomer was determined to be 11.27 at 1 mg / mL.

[0061] Based on the determined concentration of the monomer fraction (i.e., the ferritin extinction coefficient), the concentration of the monomer fraction was adjusted to 1000 ng / mL using the delipidated serum described above. In addition, a series of calibrators was prepared by adjusting the concentrations to 25, 250, 500, and 750 ng / mL. Based on the above ferritin absorption coefficient, the concentration of each of the dimer and oligomer fractions was adjusted to 1000 ng / mL, and a series of calibrators were prepared in the same manner as for the monomer fraction. Using the obtained calibrator, a calibration curve was prepared in the same manner as in Test Example 1, and the ferritin concentrations in human specimens (Nos. 1 to 6) were measured. However, results in which the measured values ​​exceeded 1000 ng / mL were deemed to be outside the range of the calibration curve (0 to 1000 ng / mL) and therefore could not be compared. The results are shown in Table 3.

[0062] [Table 3]

[0063] As shown in Table 3, as the polymerization progressed from monomer to dimer to oligomer, the reactivity decreased significantly, and the calibration curves obtained from each calibrator became lower. As a result, it became clear that when dimers or oligomers were used as calibrators, the measured values ​​calculated were higher even for the same sample. Therefore, it was shown that the composition ratio of monomer, dimer, oligomer, etc. in ferritin is very important for the ferritin standard. In addition, by increasing the proportion of monomer, sufficiently high reactivity can be obtained even with a small amount of ferritin, and it was recognized that this is useful for improving productivity.

[0064] [Test Example 4] Measurement using a calibrator with adjusted monomer composition ratio The monomer, dimer, and oligomer fractions were mixed in various ratios shown in Table 4 based on the concentrations determined in Test Example 3. The resulting mixed ferritin was adjusted to concentrations of 25, 250, 500, 750, and 1000 ng / mL in the same manner as in Test Example 3 to prepare a series of calibrators. Using the resulting calibrators, a calibration curve was created in the same manner as in Test Example 1, and the ferritin concentrations in human specimens (No. 1 to 6) were measured. However, results with measured values ​​exceeding 1000 ng / mL were considered to be outside the range of the calibration curve (0 to 1000 ng / mL) and were not comparable. The results are shown in Table 4.

[0065] [Table 4]

[0066] As shown in Table 4, it was confirmed that the measured values ​​tended to become higher even for the same sample as the monomer ratio in the calibrator decreased. When different lots of ferritin were used as a standard substance, it was found to be useful to increase the proportion of monomers (e.g., 90% or more) in order to reduce the difference in measurement values ​​for the same sample. Furthermore, even when using ferritin that does not have a high proportion of monomers, it was found that the difference in ferritin measurement values ​​between lots can be reduced by adjusting the constituent ratios in ferritin in the next lot as constant as possible (for example, by keeping the difference in the proportion of low-reactive oligomers to 8 points or less, and even by keeping the difference in the proportion of dimers to 10 points or less).

[0067] [Test Example 5] Measurement using different measuring devices The ferritin concentrations in human samples (No. 1 to 6) were measured using the monomer, dimer, and oligomer calibrators prepared in Test Example 3, and the measuring device was changed to TBA-120 Pearl Edition (Canon Medical Systems) and JCA-BM6070 (JEOL). As in Test Example 3, a latex agglutination reagent (Eiken Chemical Co., Ltd., "LZ Test 'Eiken' FER") was used as the measuring reagent. The difference in the measured values ​​between the measuring devices was evaluated from the obtained measured values. However, the results exceeding 1000 ng / mL were deemed to be outside the range of the calibration curve (0 to 1000 ng / mL) and were therefore not comparable. The results are shown in Table 5.

[0068] [Table 5]

[0069] As shown in Table 5, when a 100% monomer calibrator was used, the results showed that the difference in the measured values ​​obtained could be kept small even when different measuring devices were used. Here, when the same measurement reagent is used with different measurement devices, although the measurement principle is the same, there are significant differences between the measurement devices in terms of the material and size of the reaction cell, the order and amount of reagents added, heating conditions, stirring conditions, detection conditions, etc. These differences are thought to cause differences in results between measurement devices (and therefore differences in results between facilities). From the results in Table 5, it was found that the difference in reactivity of monomers, dimers, oligomers, etc. also affects the difference in results between measurement devices. Therefore, it was found that by increasing the proportion of monomers in the ferritin standard substance, the difference in results between measurement devices (and therefore the difference in results between facilities) can be reduced, and stable measurement values ​​can be obtained. [Industrial Applicability]

[0070] According to the present invention, it is possible to stabilize the measured value of the ferritin concentration in a sample. Stabilizing the accuracy of ferritin measurement has been a long-standing issue, and the present invention helps to solve this issue.

Claims

1. A method for reducing a difference in measurement results between different measurement devices when measuring ferritin in a sample using a ferritin immunoassay standard substance and an immunoassay reagent, comprising: A method using ferritin that satisfies the following (1) as the standard substance: (1) The proportion of ferritin monomers in the ferritin is 90% or more.

2. The method according to claim 1, wherein the immunological assay method is an immunoagglutination method.

3. The method of claim 1 or 2, wherein the proportion of ferritin monomers is 95% or more.

4. A method for immunoassay of ferritin using at least two or more lots of standard substances for immunoassay of ferritin, A method using ferritin that satisfies the following (1) or (2) as the standard substance: (1) The proportion of ferritin monomers in the ferritin in the standard material is 90% or more; or (2) The difference in the proportion of ferritin oligomers of trimers or higher in the ferritin in the standard materials is 8 percentage points or less between the standard materials.

5. The immunological method for measuring ferritin according to claim 4, wherein in (2), the difference in the proportion of ferritin dimer in the ferritin in the standard substances is 10 percentage points or less between the standard substances.

6. The immunological assay method for ferritin according to claim 4 or 5, wherein the immunological assay method is an immunoagglutination method.

7. The immunological measurement method for ferritin according to any one of claims 4 to 6, wherein in (1), the proportion of the ferritin monomer is 95% or more.

8. A method for producing a standard substance for immunological measurement of ferritin, comprising: A manufacturing method comprising a step of preparing ferritin having a ferritin monomer ratio of 90% or more from ferritin having a ferritin monomer ratio of 66% or less by adjusting the ratio of the monomers.