JP7709818B2 - Ferritin measurement standard substance and ferritin measurement method - Google Patents

Description

The present invention relates to a standard substance for measuring ferritin, a method for producing the same, 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.

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 many iron ions can be stored in this space. Each ferritin molecule can store 4,500 ferric ions (Fe ³⁺ ).

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).

It is also 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 other diseases (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).

Special Publication No. 2011-516419 Japanese Patent Application Publication No. 10-115615 JP 2000-88849 A

Guidelines for quantitative test evaluation methods and samples for clinical laboratory quality control surveys, Medical Testing, 2008, 57: 109-117

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

The above-mentioned Patent Document 2 proposes a highly sensitive, rapid, and simple method for quantifying ferritin, and the above-mentioned Patent Document 3 proposes a method for measuring ferritin that is less affected by the freshness of the ferritin sample. However, even these methods are still not sufficient from the standpoint of quality control.

Therefore, the present invention aims to provide a method for stabilizing the measured value of ferritin concentration in a sample.

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 difference in reactivity of these in standard substances is one of the causes of the difference in measurement values.
That is, in the measurement of ferritin in a sample, when preparing a standard substance (calibrator) for immunoassay, ferritin derived from biological tissue is often used as the standard substance, but when different lots of ferritin are used, the ratio of ferritin monomer, dimer, oligomer, etc. may differ, and therefore the composition ratio of these may also differ in the standard substance. Furthermore, since ferritin monomer, dimer, oligomer, etc. have different reactivity in immunoassay, it has been found that the composition ratio of ferritin monomer, dimer, oligomer, etc. differs 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 measurement value 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.

[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 in which ferritin that satisfies the following (1) or (2) is used as the standard substance:
(1) The proportion of ferritin monomers in the ferritin in the standard substance 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 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 in which ferritin that satisfies the following (1) is used 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 standard material is 8 points or less between the first standard material and the second standard material.
[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 the ratio is adjusted for ferritin before it is prepared as a standard substance in the method (1) or (2).
[8] A standard substance for measuring ferritin, comprising ferritin,
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 the 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].

According to the present invention, it is possible to stabilize the ferritin reactivity in the standard substance, and to stabilize the measured value of the ferritin concentration in the sample.

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.

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.

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 monomer is called a "ferritin oligomer of trimer or more" (sometimes simply called a "ferritin oligomer" or "oligomer").

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 becomes stable without fluctuating significantly, and the measured value of the ferritin concentration in the sample can also be stabilized.

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.

2. Ratio of Monomer, Dimer, and Oligomer In this embodiment, the ratio of each of the 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 ratio of the protein amount of the monomer, dimer, and oligomer 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 the same wavelength.

If 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).

(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 the absorbance, and the ratio of monomers, etc. (protein amount ratio) is determined.

(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 measurement device, and the relationship between the signal strength and absorbance of 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 measurement device. From the signal strength of each fraction and the above relationship, the signal strength in each fraction is converted to absorbance, and the proportions of monomers, dimers, and oligomers are calculated.

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 increase or decrease differs between ferritin monomers, dimers, and oligomers.

3. Method for reducing differences in ferritin measurement results in a sample In this embodiment, as described above, by adjusting the composition ratio of monomers, dimers, and oligomers in the ferritin contained in the standard substance so that it does not vary significantly, the calibration curve is stable and does not vary 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 fluctuate significantly is a method of using 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.

By doing as described above in (1), it is possible to suppress the fluctuation of the ratio of dimers and oligomers with low reactivity, 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.

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 the ferritin in the standard material is high, even when the same sample is measured using the same lot of standard material, differences in the measurement results may occur between measurement devices.
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 difference in measurement values between different lots of standard substances be reduced, but also the difference in measurement values between different immunological measurement devices using the same lot of standard substance can be reduced.

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 made from different processes, places, times, etc., the manufacturing conditions are not the same, and the lots of the reference materials (or ferritin) are also different.

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 ferritin in the standard substances is 8 percentage points or less between the standard substances.
In this specification, "points" refer to percentage points.

By doing as described above in (2), it is possible to suppress the variation of the ratio of the least reactive oligomer, and even when using different lots of standard substances, 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 specimen. That is, by using ferritin that satisfies the above in (2), it is possible to reduce the difference in the ferritin measurement result in the specimen between at least two or more lots of ferritin measurement standard substances of different lots.
In the above (2), it is more preferable that the difference in the proportion of oligomers in ferritin in the standard substances is 5 points or less, and particularly preferably 3 points or less, between the standard substances.

In addition, when producing a reference material, by using ferritin as described above in (1) or (2), it is possible to obtain a reference material with reduced differences in ferritin measurement results, even if multiple lots of reference material are produced using any lot of ferritin.

In the above (1) or (2), it is preferable that the ferritin used as the standard substance further satisfies the following (3):
(3) The proportion 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.

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-mentioned standard substances is 10 points or less between the standard substances.
Furthermore, it is more preferable that the difference in the dimer ratio in ferritin in the standard substances is 7 points or less, and particularly preferably 5 points or less, between the standard substances.

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.

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 different lots of ferritin are used, 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 composition ratio has already been adjusted to the desired composition ratio, and therefore the ferritin can be used as it 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 composition 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 ratio of monomers, etc. in ferritin before it is prepared as a standard substance.

4. Standard Substance A standard substance for measuring ferritin according to one embodiment of the present invention contains ferritin in which the constituent ratio of monomers, etc. falls within the above-mentioned range.
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.

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

Examples of buffers include Good's buffers such as HEPES and PIPES, phosphate buffers, Tris buffers, glycine buffers, and glycylglycine buffers, 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.

The stabilizer is used to stabilize ferritin, and specific examples include bovine serum albumin, skim milk, gelatin, etc., and one type can be used alone or two or more types can be used in combination. The concentration of the stabilizer can be, for example, 0.01 to 20% by mass, or 0.1 to 10% by mass.

Furthermore, serum may be used to dilute the standard substance. 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 measurements, 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 a control sample that does not contain ferritin.

Furthermore, the standard 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, and thiabenzol, within the scope that does not impair the effects of this embodiment.

The pH of the standard substance can be specifically set to pH 5 to 10, or even pH 6 to 8.

5. Method for Producing Standard Material The method for producing a standard material 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 and other constituent ratios in the above-mentioned ranges. For example, the standard material according to this embodiment can be produced by mixing ferritin with other components as desired.

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

In addition, when a standard substance of a different lot is produced, that is, when a standard substance of a different lot (a second lot) is produced with reference to a standard substance of a first lot, ferritin that satisfies the following (2) is used in the second standard substance:
(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 first standard substance and the second standard substance.
Here, when a second lot of standard material is produced by referring to a first lot of standard material, the first and second lots of standard materials may be produced simultaneously while referring to each other, or a new standard material (a second lot of standard material) may be produced by referring to an already obtained standard material (a first lot of standard material).

The more preferable conditions for the composition ratio of ferritin monomers in ferritin and other components acceptable for the standard substance are preferably as described above.

6. Ferritin Measurement Method The ferritin measurement method according to one embodiment of the present invention uses the standard substance for ferritin measurement 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.

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.

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

Examples of specimens include whole blood, serum, plasma, etc. The specimen may be used as is, or may be diluted to prepare a sample for measurement.

According to the above-described embodiment of the standard substance for measuring ferritin, the reactivity of ferritin in the standard substance can be stabilized, and the calibration curve can be stabilized, so that the measured value of the ferritin concentration in the sample can be stabilized.

The above-described embodiments are described to facilitate understanding of the present invention, and are not described to limit the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design modifications, equivalents, and equivalent methods that fall within the technical scope of the present invention.

The present invention will be explained in more detail below by showing test examples, but the present invention is not limited to the test examples below.

[Test Example 1] Measurement using calibrators (standard substances) using different lots of ferritin Calibrators (standard substances) for ferritin immunoassay were prepared using ferritin (manufactured by BBI, derived from human liver, lot 1 and lot 2, purity by SDS-PAGE: 95% or more). 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 based on the manufacturer's indicated value by diluting with delipidated serum, and calibrators with concentrations of 25, 250, 500, and 750 ng/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.

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

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, resulting in differences in the measurement results. Since the other components in the calibrators were prepared in the same way, it was thought that the differences in the measurement results were due to differences in the lots of ferritin used in the calibrators.

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

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

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 1,400 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.

[Test Example 3] Measurement using a calibrator using monomers, etc. The extinction coefficient of ferritin of lot 1 fractionated in Test Example 2 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 be in accordance with the 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 type automatic analyzer (H7180) were used to determine the concentration of the monomer fraction 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.

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. In addition, a series of calibrators was prepared by adjusting the concentration 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 and the ferritin concentrations in human specimens (Nos. 1 to 6) were measured in the same manner as in Test Example 1. 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.

As shown in Table 3, as the polymerisation 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. This demonstrated that the constituent ratio of monomer, dimer, oligomer, etc. in ferritin is extremely important for the ferritin standard. Furthermore, by increasing the proportion of monomer, sufficiently high reactivity could be obtained even with a small amount of ferritin, which was found to be useful in improving productivity.

[Test Example 4] Measurement using calibrators with adjusted monomer and other constituent ratios The monomer, dimer, and oligomer fractions were mixed at 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 deemed 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.

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 are used as a standard substance, it has been found to be useful to increase the proportion of monomers (for example, to 90% or more) in order to reduce the difference in measured 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).

[Test Example 5] Measurement using a different measuring device Using each of the monomer, dimer, and oligomer calibrators prepared in Test Example 3, the measuring device was changed to TBA-120 Pearl Edition (Canon Medical Systems) and JCA-BM6070 (JEOL), and the ferritin concentration in human specimens (No. 1 to 6) was measured. As in Test Example 3, a latex agglutination reagent (Eiken Chemical Co., Ltd., "LZ Test 'Eiken'FER") was used as the measuring reagent.
The differences in the measured values between the measuring devices were evaluated based on the obtained values. However, 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.

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.

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 (10)

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 immunological measurement of ferritin, comprising:
A method in which ferritin that satisfies the following (1) or (2) is used as the standard substance:
(1) The proportion of ferritin monomers in the ferritin in the standard material is 95 % or more;
(2) The difference in the proportion of ferritin oligomers of trimers or higher in the ferritin in the standard substances is 8 percentage points or less between the standard substances. The method according to claim 1, 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. The method according to claim 1 or 2, wherein in (1) or (2), the ratio is adjusted for ferritin before it is prepared as a standard substance. A method for producing a standard substance for immunological measurement of ferritin, comprising the steps of:
A method in which ferritin that satisfies the following (1) is used as the standard substance:
(1) The proportion of ferritin monomers in the ferritin is 95 % or more. A method for producing a second lot of a standard substance for immunological measurement of ferritin by referring to a first lot of a standard substance for immunological measurement of ferritin, comprising the steps of:
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 standard material is 8 percentage points or less between the first standard material and the second standard material. The method for producing a standard substance for immunological measurement of ferritin according to claim 5, wherein in (2), the difference in the proportion of ferritin dimer in the ferritin in the standard substance is 10 percentage points or less between the first standard substance and the second standard substance. The method for producing a standard substance for immunological measurement of ferritin according to any one of claims 4 to 6, wherein in (1) or (2), the ratio is adjusted for ferritin before it is prepared as a standard substance. A standard substance for immunological measurement of ferritin, comprising ferritin,
A standard substance for immunological measurement of ferritin, characterized in that the proportion of ferritin monomers in the ferritin in said standard substance is 95 % or more. The standard substance for immunological measurement of ferritin according to claim 8, wherein the proportion of ferritin oligomers of trimers or more in the ferritin in the standard substance is 5% or less. A method for immunoassay of ferritin, comprising using a standard substance produced by the method according to any one of claims 4 to 7, or a standard substance according to claim 8 or 9.