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 reference substance for ferritin measurement, a method for producing the same, and a method for measuring ferritin using the above reference substance. Further, the present invention also relates to a method for reducing the difference in ferritin measurement results.

Background Art

[0002] Ferritin is a globular protein complex containing 24 polypeptide subunits and having a molecular weight of about 480 kDa. As the polypeptide subunits, two types of H chains (21 kDa) and L chains (19 kDa) with different molecular weights are known. The inside of the ferritin molecule is hollow, and a large number of iron ions can be stored in this space. Each ferritin molecule can store 4500 ferric ions (Fe 3+ ).

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

[0004] Also, it has been found that the ferritin concentration in serum is closely related to the iron storage amount in the body, and it has become an important measurement item in clinical tests such as iron deficiency anemia (for example, Non-Patent Document 1). In the measurement of ferritin, various immunological measurement methods are known, but in recent years, an immunometric method by latex agglutination method has become a method that can be measured more simply and quickly than other methods, and thus is widely used. And in order to improve the detection sensitivity of the latex agglutination method, a method of containing one selected from the group consisting of polyvinylpyrrolidone, pullulan, and polyethylene glycol having a predetermined molecular weight in the reaction solution (Patent Document 2), and a method of containing a quaternary ammonium salt in the reaction solution in order to suppress the influence of the freshness of the serum sample (Patent Document 3) have been proposed.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in the above Non-Patent Document 1, ferritin as a measurement item is classified as "Category 3) Items for which standard measurement operation methods and reference materials have not been established and for which there are large differences between facilities". Thus, in the immunological measurement of ferritin, differences may occur in the measurement results when different measurement facilities are used, etc., and accuracy management has been an important issue.

[0008] The above Patent Document 2 has been proposed as a highly sensitive, rapid, and simple ferritin quantification method, and the above Patent Document 3 has been proposed as a ferritin measurement method with little influence of the freshness of ferritin specimens. However, even in these methods, they still could not be said to be sufficient from the perspective of accuracy management.

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

Means for Solving the Problems

[0010] As a result of research to solve the above problems, the inventors of the present invention defined ferritin monomers as ferritin containing 24 polypeptide subunits and having a molecular weight of about 480 kDa. They found that ferritin monomers associate to form ferritin dimers, ferritin oligomers, etc., and the reactivity in immunological measurements differs among them, and the differences in reactivity of these in the standard substances contribute to the differences in measurement values. That is, in measuring ferritin in a specimen, when preparing a standard substance (calibrator) for an immunological measurement method, ferritin derived from a biological tissue is often used as the standard substance. However, when using ferritin from different lots, the ratios of ferritin monomers, dimers, oligomers, etc. may differ, so the standard substances may also have different compositional ratios. And since ferritin monomers, dimers, oligomers, etc. have different reactivities in immunological measurements, the fact that the compositional ratios of ferritin monomers, dimers, oligomers, etc. differ among multiple standard substance lots is the cause of the calibration curve fluctuating, and ultimately contributes to the change in the measured value of the specimen. Then, the inventors found that the above problems can be solved by using ferritin in which the ratios of ferritin monomers, dimers, oligomers, etc. are within a predetermined range in the standard substance, and thus completed the present invention. Specifically, the present invention is as follows.

[0011] [1] A method for reducing the difference in the ferritin measurement results in a specimen among at least two or more lots of standard substances for ferritin measurement, the method comprising using ferritin that satisfies the following (1) or (2) in the standard substance: (1) The ratio of ferritin monomers in the ferritin in the standard substance is 90% or more; (2) The difference in the ratio of ferritin oligomers of trimer or higher in the ferritin in the standard substance is 8 points or less among the standard substances. [2] The method according to [1], wherein in [2], the difference in the ratio of ferritin dimer in ferritin in the standard substance is 10 points or less between the standard substances. [3] The method according to [1] or [2], wherein in [1] or [2], the ratio is adjusted with respect to ferritin before being prepared as a standard substance. [4] A method for producing a standard substance for ferritin measurement, The method, wherein ferritin satisfying the following (1) is used in the standard substance: (1) The ratio of ferritin monomer in the ferritin is 90% or more. [5] A method for producing a second lot of standard substance for ferritin measurement with reference to a first lot of standard substance for ferritin measurement, The method, wherein ferritin satisfying the following (2) is used in the second standard substance: (2) The difference in the ratio of ferritin oligomers of trimer or more in ferritin in the standard substance is 8 points or less between the first standard substance and the second standard substance. [6] The method for producing a standard substance for ferritin measurement according to [5], wherein in [2], the difference in the ratio of ferritin dimer in 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 ferritin measurement according to [4] to [6], wherein in [1] or [2], the ratio is adjusted with respect to ferritin before being prepared as a standard substance. [8] A standard substance for ferritin measurement containing ferritin, A standard substance for ferritin measurement, characterized in that the ratio of ferritin monomer in ferritin in the standard substance is 90% or more. [9] The standard substance for ferritin measurement according to [8], wherein the ratio of ferritin oligomers of trimer or more in ferritin in the standard substance is 5% or less. A method for measuring ferritin, characterized by using a reference substance produced by the method described in

[10] , [4] to [7], or the reference substance described in [8] or [9].

Advantages of the Invention

[0012] According to the present invention, the ferritin reactivity in the reference substance can be stabilized, and the measured value of the ferritin concentration in the sample can be stabilized.

Embodiments for Carrying Out the Invention

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

[0014] 1. Ferritin Ferritin is a globular protein complex containing 24 polypeptide subunits and having a molecular weight of about 480 kDa. The ferritin used in this embodiment can be purified from the liver, placenta, spleen, etc., and recombinant proteins can also be used. However, from the viewpoint of easy availability, etc., the ferritin used in this embodiment may not be a recombinant of the L chain, and it is preferably derived from the liver or placenta, and particularly preferably derived from the liver.

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

[0016] As shown in the examples described later, as ferritin multimerizes into monomers, dimers, oligomers, and multimers, its reactivity in immunological measurements decreases. Therefore, if the composition ratios of these components vary significantly in the reference substance, the calibration curve prepared from the reference substance will vary even with the same measuring device, and differences will occur in the measured values of the ferritin concentration in the sample. On the other hand, by adjusting the ferritin contained in the reference substance so that the above composition ratios do not vary significantly, the calibration curve can be stabilized without significant variation, and the measured values of the ferritin concentration in the sample can also be stabilized.

[0017] Although it was known that monomers, dimers, and oligomers exist in ferritin, it was not known at all that the reactivity in immunological measurements differs among them, which is a new finding of the present inventors. Also, as shown in the examples described later, when measuring with different measuring devices using the same ferritin measurement reagent, differences in reactivity in immunological measurements are observed among monomers, dimers, and oligomers.

[0018] 2. Ratios of Monomer, Dimer, and Oligomer In the present embodiment, the ratios of monomer, dimer, and oligomer in ferritin can be expressed, for example, by absorbance conversion. Specifically, ferritin is fractionated by gel filtration chromatography or the like, and the ratios as the protein amounts of monomer, dimer, and oligomer relative to total ferritin can be expressed by absorbance conversion. Monomers, dimers, and oligomers can be fractionated by molecular weight according to conventional methods, and specific conditions for gel filtration chromatography can be exemplified by the conditions shown in the examples described later as an example. The absorbance can be measured, for example, by the absorbance at a wavelength of 280 nm. The ratios as the amounts of protein of the monomer, dimer, and oligomer can be specified by, for example, the area ratio of the peaks of gel filtration chromatography measured at the said wavelength.

[0019] In the case where the absorbance of each fraction cannot be regarded as the absorbance of ferritin for reasons such as that the composition for which the ratio of the monomer etc. in ferritin is to be determined contains components other than ferritin, the above ratio can be determined, for example, by the method of (a) or (b) below.

[0020] (a) Purify ferritin by affinity purification, crystallization, etc. Thereafter, fractionate by gel filtration chromatography or the like in the same manner as described above, measure the absorbance to obtain the amount of protein, and determine the ratio (protein amount ratio) of the monomer etc.

[0021] (b) Fractionate generally available ferritin into monomer, dimer, and oligomer. For each fraction, measure the absorbance and measure the signal intensity with a specific combination of ferritin measurement reagent and measurement device, and specify the relational expression between the signal intensity and the absorbance in ferritin monomer etc. Thereafter, for the composition for which the ratio of the monomer etc. in ferritin is to be determined, fractionate by gel filtration chromatography or the like, and measure the signal intensity of ferritin for each fraction with the above specific combination of ferritin measurement reagent and measurement device. From the signal intensity of each fraction and the above relational expression, convert the signal intensity in each fraction to absorbance, and calculate the ratios of the monomer, dimer, and oligomer.

[0022] Here, the "absorbance" of ferritin refers to the absorption intensity of light at a wavelength of 280 nm when a ferritin antigen monomer is measured with a spectrophotometer. In addition, the "signal intensity" refers to the amount of change in the signal detected by immunological measurement. The above-mentioned signal includes turbidity, absorbance, fluorescence, luminescence, RI, etc. according to the method of immunological measurement. The signal intensity increases and decreases in correlation with the amount of ferritin. However, since the reactivity in immunological measurement differs among ferritin monomer, dimer, and oligomer, the degree of the above increase and decrease differs among ferritin monomer, dimer, and oligomer.

[0023] 3. Method for reducing the difference in ferritin measurement results in a sample In the present embodiment, as described above, in the ferritin contained in the standard substance, by adjusting so that the composition ratios of monomer, dimer, and oligomer do not fluctuate greatly, the calibration curve is stabilized without large fluctuations, and the measured value of the ferritin concentration in the sample can be stabilized. Here, as a method for adjusting so that the composition ratios of monomers and the like in ferritin do not fluctuate greatly, specifically, a method of using ferritin that satisfies the following (1) as ferritin in the standard substance can be mentioned. (1) The ratio of ferritin monomer in the ferritin contained in the standard substance is 90% or more.

[0024] By doing as in the above (1), fluctuations in the ratios of dimers and oligomers with low reactivity can be suppressed, the reactivity of ferritin in the standard substance can be stabilized, the calibration curve can be stabilized, and the measured value of the ferritin concentration in the sample can be stabilized. That is, by using ferritin that satisfies the above (1), the difference in the ferritin measurement results in the sample can be reduced among standard substances with different lots. In the above (1), the ratio of ferritin monomer is more preferably 95% or more, and particularly preferably 98% or more.

[0025] Also, as shown in the examples described below, when measurements are performed using the same ferritin measurement reagent with different measurement devices, differences in reactivity are observed among monomers, dimers, and oligomers. Due to this, when the proportion of ferritin in the standard substance that is an oligomer (or dimer) is high, even when measuring the same sample using the same lot of standard substance, differences in measurement results may occur between measurement devices. On the other hand, in the ferritin used for the standard substance, by increasing the proportion of ferritin monomers, for example, to 90% or more, 95% or more, or 98% or more, not only can the difference in measured values between different lots of standard substances be reduced, but also the difference in measured values between different immunological measurement devices using the same lot of standard substance can be reduced.

[0026] Here, the "lot" in this specification refers to a product unit manufactured under the same conditions using the same raw materials. For example, if the same raw materials are used for ferritin or the like and the manufacturing conditions are the same, it can be referred to as the same lot of standard substance. Regarding lots, it can be defined not only for standard substances but also for ferritin. For example, ferritin manufactured under the same conditions using the same raw materials is the same lot of ferritin. If the lots of ferritin are the same, the compositional ratios of monomers, dimers, and oligomers in the ferritin can be regarded as the same. In addition, when the lots of ferritin used as raw materials are different in at least two or more standard substances, since the raw materials used are not the same, the lots of the standard substances are also different. Also, when the manufacturing processes, locations, times, etc. are different in at least two or more standard substances (or ferritin), since the manufacturing conditions are not the same, the lots of the standard substances (or ferritin) are also different.

[0027] Also, for at least two or more lots of standard substances, in addition to the above (1), ferritin that satisfies the following (2) can be used as the ferritin in the standard substance. (2) The difference in the ratio of ferritin oligomers of trimer or higher in ferritin in the reference material is 8 percentage points or less between reference materials. Note that the "point" in this specification is a percentage point.

[0028] By doing as described in the above (2), the variation in the ratio of the least reactive oligomer can be suppressed, and even when using reference materials of different lots, the ferritin reactivity in the reference material can be stabilized, the calibration curve can be stabilized, and the measured value of the ferritin concentration in the sample can be stabilized. That is, by using ferritin that satisfies the above (2), the difference in the ferritin measurement results in the sample can be reduced among at least two or more lots of ferritin measurement reference materials of different lots. In the above (2), it is more preferable that the difference in the ratio of oligomers in ferritin in the reference material is 5 percentage points or less between reference materials, and particularly preferably 3 percentage points or less.

[0029] Also, when manufacturing a reference material, by using ferritin as described in the above (1) or (2), even if a plurality of lots of reference materials are manufactured using ferritin of any lot, a reference material with a reduced difference in ferritin measurement results can be obtained.

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

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

[0032] Furthermore, in the above (2), as ferritin in the reference substance, it is preferable to use ferritin in which the ratio 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, as a method for adjusting so that the composition ratio of monomers and the like in ferritin does not vary greatly (for example, the above (1) to (4), etc.), for example, a method for confirming the composition ratio of monomers and the like in ferritin can be mentioned. In this case, when using ferritin from different lots, the above composition ratio should be confirmed for each lot. The method for confirming the composition ratio of monomers and the like in ferritin is as described above, and ferritin can be fractionated by gel filtration chromatography or the like, and the composition ratio can be expressed in terms of absorbance conversion. When the composition ratio of monomers and the like in ferritin satisfies the desired conditions (for example, the above (1) or (2), etc.), since it has already been adjusted to the desired composition ratio, the ferritin can be used as it is for the reference substance. On the other hand, when the desired conditions are not satisfied, for example, each fraction obtained by fractionation by gel filtration chromatography or the like can be appropriately combined to adjust to the desired composition ratio. In the present embodiment, it is preferable that the composition ratio of monomers and the like in the ferritin before being prepared as the reference substance is adjusted.

[0034] 4. Reference Substance The reference material for ferritin measurement according to an embodiment of the present invention contains ferritin in which the composition ratio of monomers or the like is within the above range. In addition to containing the above ferritin, the reference material according to this embodiment may contain other components allowed in the reference material. As such components, for example, water; salts; buffers; stabilizers; serum, etc. can be appropriately added.

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

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

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

[0038] Furthermore, serum may be used to dilute the reference material. Such serum is preferably defatted serum and preferably serum that does not contain ferritin. The fact that it does not contain ferritin can be confirmed, for example, in the immunological measurement described later. Specifically, it can be determined that the serum does not contain ferritin when no significant difference is observed between the results of measuring the serum multiple times and a control sample that does not contain ferritin.

[0039] Furthermore, the reference substance according to this embodiment may be appropriately blended with other additives, such as preservatives such as sodium azide; preservatives such as benzoic acids and sorbic acids; and fungicides such as orthophenylphenols, diphenyl, thiabendazole, etc., as long as the effects of this embodiment are not impaired.

[0040] Note that the pH of the reference substance can specifically be pH 5 to 10, and more preferably pH 6 to 8.

[0041] 5. Method for producing reference substance The method for producing a reference substance for ferritin measurement according to an embodiment of the present invention can be produced according to a conventional method, in addition to using ferritin in which the composition ratio of monomers and the like is in the above range. For example, the reference substance according to this embodiment can be produced by mixing ferritin and, if desired, other components.

[0042] More specifically, when producing the reference substance, ferritin satisfying the following (1) is used: (1) The proportion of ferritin monomers in the ferritin is 90% or more.

[0043] In addition, when producing reference substances of different lots, that is, when producing a reference substance of a different lot (second lot) with reference to the reference substance of the first lot, ferritin satisfying the following (2) is used for the second reference substance: (2) The difference in the proportion of ferritin oligomers of trimer or higher in ferritin in the reference substance is 8 points or less between the first reference substance and the second reference substance. Here, the case of producing the reference substance of the second lot with reference to the reference substance of the first lot may be that the reference substances of the first and second lots are produced simultaneously while referring to each other, or a new reference substance (second lot of reference substance) may be produced with reference to the already obtained reference substance (first lot of reference substance).

[0044] Regarding ferritin, it is preferable that the more favorable conditions for the composition ratio of ferritin monomers and the like, and other components allowed in the reference substance, are as described above.

[0045] 6. Method for Measuring Ferritin The method for measuring ferritin according to an embodiment of the present invention uses the reference substance for ferritin measurement obtained as described above. By using such a reference substance, the calibration curve can be stabilized, and the measured value of the ferritin concentration in the sample can be stabilized. Specifically, for the measurement of ferritin, first, using the reference substance for ferritin measurement, the signal intensity of the reference substance for ferritin measurement with various known concentrations is measured with a ferritin measurement reagent to create a calibration curve. Next, the signal intensity of the sample is measured with a ferritin measurement reagent, and the concentration is calculated by applying it to the calibration curve prepared previously.

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

[0047] As the immunological measurement method, for example, it can be by an immunoprecipitation method such as a latex agglutination method or a gold colloid agglutination method, a sandwich method such as an enzyme immunoassay (ELISA method, etc.) or a chemiluminescence measurement method, a radioimmunoassay, an immunochromatography method, etc., and is not particularly limited. Examples of the measurement method include a method of measuring the absorbance, scattered light, luminescence, fluorescence, etc. of the immunoreaction solution by an optical technique, a method of measuring the radioactivity of a labeled radioisotope, etc. As the optical technique, for example, a general-purpose optical measurement device may be used, and for example, it can be measured using a 7180 type Hitachi automatic analyzer (manufactured by Hitachi High-Technologies Corporation).

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

[0049] According to the ferritin measurement standard substance according to the above-described embodiment, the ferritin reactivity in the standard substance can be stabilized, the calibration curve can be stabilized, and thus the measured value of the ferritin concentration in the specimen can be stabilized.

[0050] The embodiments described above are described for ease of understanding of the present invention and are not described for limiting the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design changes, equivalents, and equivalent methods belonging to the technical scope of the present invention.

Example

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

[0052] 〔Test Example 1〕Measurement using calibrators (standard substances) with different lots of ferritin Using ferritin (manufactured by BBI, derived from human liver, lot 1 and lot 2, purity by SDS-PAGE: 95% or more), calibrators (standard substances) for immunological measurement of ferritin were prepared. 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 1000 ng / mL by diluting with defatted serum based on the manufacturer's indicated value, and calibrators with concentrations of 25, 250, 500, and 750 ng / mL were also prepared. The defatted serum used for dilution was a commercially available defatted serum from which ferritin was removed using an affinity gel with an anti-ferritin antibody. The fact that the defatted serum after removal was free of ferritin was confirmed using the LZ test 'Eiken' FER.

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

[0054]

Table 1

[0055] As shown in Table 1, it was revealed that calibration curves differed due to differences in signal intensity for calibrators from different lots using ferritin from different lots, resulting in differences in measurement results. Since other components in the calibrator were prepared in the same way, it was considered that the differences in measurement results were due to differences in the lots of ferritin used in the calibrator.

[0056] 〔Test Example 2〕Analysis by Gel Filtration Chromatography Regarding the ferritin of lot 1 and lot 2, it was dissolved in a buffer solution (0.05 M phosphate buffer: pH 7.0, sodium chloride: 0.3 M) (ferritin concentration: 10% by mass in terms of the indicated value), and analysis by gel filtration chromatography was performed under the following conditions. As the molecular weight marker, Gel Filtrate Standard (manufactured by BIO RAD) was used.

[0057] =Gel Filtration Chromatography Conditions= Column: Yarra SEC3000 (manufactured by Shimadzu GLC) Mobile phase: 0.05 M phosphate buffer, 0.3 M NaCl, pH 7.0 Flow rate: 0.5 mL / min Detection: UV (280 nm)

[0058] In both Lot 1 and Lot 2, three peaks were observed, and these were recognized as peaks corresponding to the monomer (molecular weight approximately 480 kDa), dimer (approximately 960 kDa), and oligomer (approximately 1400 kDa or more) of ferritin, respectively. The compositional ratios determined from the ratio of peak areas were as shown in Table 2, and since they differed between the two lots, it was considered possible that the compositional ratio of monomers, etc. in ferritin was the cause of the difference in the measurement results.

[0059]

Table 2

[0060] 〔Test Example 3〕Measurement using a calibrator with monomers, etc. Regarding the ferritin in 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 ferritin WHO international standard (recombinant, 3rd IS, 94 / 572), and the extinction coefficient of ferritin was determined from the absorbance of the monomer fraction. Here, although the ferritin standard should conform to the above WHO international standard, the WHO international standard is a dilution of recombinant ferritin protein 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, and the concentration of the monomer fraction was determined based on the indicated 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., ferritin extinction coefficient), the concentration of the monomer fraction was adjusted to 1000 ng / mL using the defatted serum. Further, the concentrations were adjusted to 25, 250, 500, and 750 ng / mL to prepare a series of calibrators. Also, based on the above ferritin extinction coefficient, the concentrations of the dimer and oligomer fractions were adjusted to 1000 ng / mL, and a series of calibrators were prepared in the same manner as the monomer fraction. Using the obtained calibrators, a calibration curve was created in the same manner as in Test Example 1, and the ferritin concentration in human specimens (No. 1 to 6) was measured. However, for the results where the measured value exceeded 1000 ng / mL, they were considered incomparable as being outside the range of the calibration curve (0 to 1000 ng / mL). The results are shown in Table 3.

[0062]

Table 3

[0063] As shown in Table 3, as the monomer, dimer, oligomer, and multimerization proceeded, the reactivity decreased significantly, and the calibration curves obtained from each calibrator became lower. As a result, it became clear that when using dimers or oligomers as calibrators, even for the same specimen, the measured values would be calculated higher. Therefore, it was shown that the composition ratio of monomers, dimers, oligomers, etc. in ferritin is very important for the ferritin standard. Also, it was recognized that by increasing the ratio of monomers, high reactivity can be obtained even with a small amount of ferritin, which is useful for improving productivity.

[0064] 〔Test Example 4〕Measurement using a calibrator with an adjusted composition ratio of monomers, etc. For each fraction of monomer, dimer, and oligomer, based on the concentrations determined in Test Example 3, they were mixed at various ratios shown in Table 4. 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 obtained 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, for the results where the measured values exceeded 1000 ng / mL, they were considered incomparable as being outside the range of the calibration curve (0 to 1000 ng / mL). The results are shown in Table 4.

[0065]

Table 4

[0066] As shown in Table 4, as the monomer ratio in the calibrator decreased, the measured values tended to increase even for the same specimen. When using ferritin from different lots as the reference substance, it was found useful to increase the proportion of monomer (for example, to 90% or more) in order to reduce the difference in measured values for the same specimen. Also, even when using ferritin with a low monomer ratio, by adjusting the composition ratio in ferritin as consistently as possible in the next lot (for example, making the difference in the proportion of oligomers with low reactivity 8 points or less, and further making the difference in the proportion of dimers 10 points or less, etc.), it was found that the difference in ferritin measured values between lots could be reduced.

[0067] 〔Test Example 5〕Measurement by Different Measuring Instruments Using each calibrator of the monomer, dimer, and oligomer prepared in Test Example 3, the measuring device was changed to TBA-120 Pearl Edition (manufactured by Canon Medical Systems Corporation) and JCA-BM6070 (manufactured by JEOL Ltd.), and the ferritin concentration in human specimens (No. 1 to 6) was measured. As the measurement reagent, a latex agglutination reagent (manufactured by Eiken Chemical Co., Ltd., "LZ Test 'Eiken' FER") was used in the same manner as in Test Example 3 and the like. From the obtained measurement values, the difference in the measurement values between the measuring devices was evaluated. However, for the results where the measurement value exceeded 1000 ng / mL, they were considered incomparable as being outside the range of the calibration curve (0 to 1000 ng / mL). The results are shown in Table 5.

[0068]

Table 5

[0069] As shown in Table 5, when using a calibrator with 100% monomer, even if the measuring devices were different, the result was obtained that the difference in the obtained measurement values could be kept small. Here, when measuring with different measuring devices using the same measurement reagent, although the measurement principle is the same, there are quite differences between the measuring devices in terms of the material and size of the reaction cell, the order and amount of reagent addition, the heating conditions, the stirring conditions, the detection conditions, etc. It is considered that these differences cause the difference in results between the measuring devices (and thus the difference in results between facilities). From the results in Table 5, it was recognized that the difference in reactivity such as monomer, dimer, and oligomer also affects the difference in results between the measuring devices. Therefore, it was recognized that by increasing the proportion of the monomer in the ferritin standard substance, the difference in results between the measuring devices (and thus the difference in results between facilities) can be reduced, and stable measurement values can be obtained.

Industrial Applicability

[0070] According to the present invention, the measured value of the ferritin concentration in a sample can be stabilized. In ferritin measurement, accuracy stabilization has been a long-standing problem, and the present invention can help solve such a problem.

Claims

1. A method for reducing the difference in ferritin measurement results in a sample between standard substances for immunological measurement of ferritin of at least two or more lots, comprising: using, as the standard substance, ferritin satisfying the following (1) or (2): (1) The proportion of ferritin monomers in the ferritin in the standard substance is 95% or more; (2) The difference in the proportion of ferritin oligomers of trimer or more in the ferritin in the standard substance is 8 percentage points or less between the standard substances.

2. The method according to claim 1, wherein in the above (2), the difference in the proportion of ferritin dimers in the ferritin in the standard substance is 10 percentage points or less between the standard substances.

3. The method according to claim 1 or 2, wherein in the above (1) or (2), the proportion is adjusted with respect to ferritin before being prepared as a standard substance.

4. A method for producing a standard substance for immunological measurement of ferritin, comprising: using, as the standard substance, ferritin satisfying the following (1): (1) The proportion of ferritin monomers in the ferritin is 95% or more.

5. A method for producing a standard substance for immunological measurement of ferritin of a second lot with reference to a standard substance for immunological measurement of ferritin of a first lot, comprising: using, as the second standard substance, ferritin satisfying the following (2): (2) The difference in the proportion of ferritin oligomers of trimer or more in the ferritin in the standard substance is 8 percentage points or less between the first standard substance and the second standard substance.

6. The method for producing a standard substance for immunological measurement of ferritin according to claim 5, wherein in the above (2), the difference in the proportion of ferritin dimers in the ferritin in the standard substance is 10 percentage points or less between the first standard substance and the second standard substance.

7. The method for producing a standard substance for immunological measurement of ferritin according to any one of claims 4 to 6, wherein in the above (1) or (2), the proportion is adjusted with respect to ferritin before being prepared as a standard substance.

8. A standard substance for immunological measurement of ferritin containing ferritin, characterized in that the proportion of ferritin monomers in the ferritin in the standard substance is 95% or more.

9. The standard substance for immunological measurement of ferritin according to claim 8, wherein the proportion of ferritin oligomers of trimer or higher in ferritin in the standard substance is 5% or less.

10. An immunological measurement method of ferritin, characterized by using a standard substance produced by the method according to any one of claims 4 to 7, or the standard substance according to claim 8 or 9.

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