Ferritin assay reagent

Abstract

Provided is a measuring reagent and method for accurately measuring high concentrations of ferritin. A ferritin measuring reagent and ferritin measuring method characterized by using ferritin from a liver as a standard solution raw material.

Description

Technical Field

[0001] This invention relates to a test kit for detecting ferritin and a method for detecting ferritin. Background Technology

[0002] Ferritin is an intracellular iron storage complex protein containing 24 polypeptide subunits and approximately 480 kDa. This iron storage complex protein can be found in high concentrations in serum, and within its ferric hydroxide core, it can contain up to 4,500 iron ions (Fe²⁺). 3+ ).

[0003] Since ferritin is present in many organs such as the liver, spleen, and placenta, its quantitative analysis can be used to diagnose iron deficiency anemia, iron excess, etc.

[0004] In addition, serum ferritin levels increase in leukemia and other solid tumors, regardless of the amount of iron stored in the body. Therefore, quantitative ferritin levels can also be used for the diagnosis and treatment monitoring of malignant tumors.

[0005] Currently, radioimmunoassay is the primary method for determining ferritin concentration. However, because this method uses radioactive isotopes, enzyme immunoassay and immunoassays utilizing latex aggregation (such as latex turbidimetric-count immunoassay) have begun to be used in recent years.

[0006] In particular, the immunoassay method utilizing latex agglomeration is widely used because it allows for rapid and convenient testing compared to other methods (see Patent Document 1).

[0007] In the past, it was necessary to be able to measure low concentrations of ferritin, but in recent years the standard for treating transfusion-induced iron overload has been set at "serum ferritin level above 1,000 ng / mL", so it is also necessary to measure high concentrations of ferritin.

[0008] During clinical examinations, approximately 5% of samples had serum ferritin levels exceeding 1,000 ng / mL. However, existing reagents for measuring ferritin using latex agglutination immunoassays cannot detect high concentrations of ferritin exceeding 1,000 ng / mL. Therefore, these samples need to be diluted and retested.

[0009] Existing technical documents

[0010] Patent documents

[0011] Patent Document 1: Japanese Patent Application Publication No. 10-115615 Summary of the Invention

[0012] Existing ferritin assay reagents cannot measure high concentrations of ferritin, therefore the above samples need to be diluted and retested.

[0013] If high concentrations of ferritin can be measured, the sample does not need to be diluted and retested, which is very useful in terms of cost-effectiveness and shortening the TAT (Turn Around Time: the time from sample arrival to obtaining test results).

[0014] However, it is difficult to accurately measure high concentrations of ferritin.

[0015] The present invention aims to provide a reagent and method for accurately determining high concentrations of ferritin.

[0016] In view of the above-mentioned problems, the inventors discovered that by using ferritin derived from the liver as a standard solution material, high concentrations of ferritin can be accurately detected, and the present invention was made based on this insight.

[0017] That is, the present invention is as follows.

[0018] [1] A ferritin assay reagent, characterized in that ferritin from the liver is used as the raw material for the standard solution.

[0019] [2] The ferritin assay reagent according to [1], wherein the reagent is an immunoagglutination reagent.

[0020] [3] The ferritin assay reagent according to [2], wherein the reagent is a latex coagulation method reagent.

[0021] [4] The ferritin assay reagent according to any one of [1] to [3], wherein the reagent comprises insoluble carrier particles sensitized with antiferritin antibody and a standard solution prepared using ferritin from the liver as a raw material.

[0022] [5] The ferritin assay reagent according to any one of [1] to [4], wherein the reagent contains a standard solution having a concentration of at least 1 point between ferritin concentrations of 1001 and 2000 ng / mL.

[0023] [6] A method for determining ferritin, characterized in that ferritin from the liver is used as the raw material for the standard solution.

[0024] [7] The ferritin assay method described in [6] is an immunoagglutination assay.

[0025] [8] The ferritin determination method according to [7], wherein the method is a latex coagulation method.

[0026] [9] According to any one of [6] to [8], the ferritin assay method is to mix the ferritin in the test sample with antiferritin antibody-sensitized insoluble carrier particles in a solution, measure the absorbance based on the aggregation reaction caused by the antigen-antibody reaction, quantify the ferritin by comparing the absorbance with a standard curve, and prepare a standard curve using a standard solution prepared from ferritin from the liver as a raw material.

[0027]

[10] The method for determining ferritin according to any one of [6] to [9], wherein the standard solution contains at least one concentration of ferritin between 1001 and 2000 ng / mL.

[0028] This specification includes the disclosure of Japanese Patent Application No. 2020-161136, which forms the basis of the priority claim of this application.

[0029] By using the ferritin assay reagent of the present invention, high concentrations of ferritin can be accurately measured.

[0030] In addition, the ferritin assay reagent of the present invention does not require sample dilution and retesting, which is useful in terms of cost-effectiveness and shortening TAT (Turn Around Time: the time from sample arrival to obtaining test results). Attached Figure Description

[0031] Figure 1 This is a graph showing the results obtained by measuring samples in low-concentration regions using a standard solution prepared from liver ferritin.

[0032] Figure 2 This is a graph showing the results obtained by measuring samples in low-concentration regions using a standard solution prepared from ferritin derived from the spleen.

[0033] Figure 3 This is a graph showing the results obtained by measuring samples in the low-concentration region using a standard solution prepared using ferritin from the placenta.

[0034] Figure 4 This is a graph showing the results obtained by measuring samples in high-concentration regions using a standard solution prepared using ferritin from the liver.

[0035] Figure 5 This is a graph showing the results obtained by measuring samples in high-concentration regions using a standard solution prepared from spleen ferritin.

[0036] Figure 6 This is a graph showing the results obtained by measuring samples in high-concentration regions using a standard solution prepared from placental ferritin. Detailed Implementation

[0037] The method of the present invention will now be described. It should be noted that, unless otherwise specified, "%" in this specification represents the quality standard (w / v%).

[0038] The present invention is characterized in that, when performing the assay using ferritin immunoassay, ferritin derived from the liver is used as the raw material for the standard solution.

[0039] Immunoassay is well known. The immunoassay method using the method of the present invention is an immunoagglutination method using insoluble carrier particles. In the present invention, the immunoagglutination method is known as follows: an antigen-antibody reaction occurs between sensitized particles sensitized (bound) with antibodies in solution and an antigen in a biological sample, forming agglutination, and the agglutination is detected optically. Turbidimetric or colorimetric methods are preferably used for detection. Immunogglutination is also called immunoturbidimetric method. For example, light in the visible to near-infrared region, typically 300–1000 nm, preferably 500–900 nm, is irradiated from outside the container, and changes in absorbance or intensity of scattered light are detected, thereby determining the degree of agglutination of the sensitized particles. In immunoagglutination, methods for detecting the agglutination of sensitized particles are well known; in the present invention, well-known methods such as methods for detecting absorbance or light scattering caused by the agglutination of sensitized particles can also be used.

[0040] The insoluble carrier particles used in immunoaggregation are not particularly limited and can be any well-known material commonly used in immunoassay reagents. Examples include latex particles such as polyethylene and polystyrene, alumina particles, silica particles, colloidal gold, and magnetic particles. Among these insoluble carriers, latex particles are preferred, and polystyrene latex particles are particularly preferred. Immunoggregation using latex particles is also called latex coagulation. The size of the insoluble carrier particles, such as latex particles, is not particularly limited, but a particle size of 30–600 nm is preferred.

[0041] Antiferritin antibodies (antiferritin antibodies) or their antigen-binding fragments are immobilized on the aforementioned latex particles. Antibodies against ferritin can be prepared using known methods, or commercially available products can be used. Examples of antigen-binding fragments include immunoglobulin fragments such as Fab and F(ab')2, or recombinant antibodies such as scFv, dsFv, diabody, and minibody expressed as recombinants. Methods for preparing these fragments are well known in the art. Immobilization methods are also well known, performed by methods such as physical adsorption or covalent bonding. The amount of immobilized antibody is not particularly limited, but preferably 0.01–2.0 mg / mL in the latex suspension. After the resulting suspension of sensitized particles is mixed with the test sample, the analyte (antigen) contained in the test sample causes the sensitized particles to aggregate, changing the absorbance of the sensitized particle suspension. At this time, the concentration of sensitized particles in the suspension is not particularly limited, but preferably 0.01–0.5%. For example, the change in absorbance of a 5–20 μL sample solution can be measured (endpoint determination method) or the rate of change (rate method). Multiple standard samples containing the antigen to be tested at various known concentrations are prepared, and the change in absorbance or the rate of change is measured using the above method. A standard curve is plotted with the concentration of the antigen to be tested in the standard samples on the x-axis and the measured change in absorbance or the rate of change on the y-axis. For unknown test samples, the change in absorbance or the rate of change is measured using the same method, and the results are compared with a standard curve prepared using ferritin standard solution. This allows for the quantification of the antigen in the test sample.

[0042] The preferred reaction temperature for the coagulation reaction is around 30–40°C, with 37°C being the most preferred. The reaction time can be approximately 2–10 minutes.

[0043] It should be noted that there are various commercially available automated devices for performing this immunoagglutination method, which can be used to easily and conveniently perform the method.

[0044] The substance to be tested in this invention is ferritin.

[0045] The test sample is not particularly limited as long as it contains ferritin. Examples include: blood, serum, plasma, urine, feces, saliva, tissue fluid, cerebrospinal fluid, swab fluid, and other bodily fluids or their dilutions. Blood, serum, plasma, urine, feces, cerebrospinal fluid, or their dilutions are preferred.

[0046] In the method of the present invention, it is preferable to dilute the sample 5 to 20 times, more preferably 10 to 20 times, before measurement. Dilution can be performed using physiological saline, buffer solution, pure water, etc. For example, 5 to 10 μL, preferably 7 μL, of sample can be diluted with 100 μL of buffer solution before use. The sample can be measured after mixing 20 to 100 μL, preferably 25 to 75 μL, more preferably 50 μL of latex suspension into the diluted sample.

[0047] As described above, in this invention, ferritin derived from human liver is used as the standard solution raw material for ferritin determination. The standard solution can be prepared as a dilution series between 0 ng / mL and 2000 ng / mL, for example, at 100 ng / mL or 200 ng / mL. The standard solution has at least one concentration point between 1001 and 2000 ng / mL of ferritin. Preferably, the standard solution is determined simultaneously with the determination of ferritin in the sample, but alternatively, the standard solution can be pre-determined using immunoagglutination to create a standard curve (calibration curve), then the ferritin in the sample can be determined, and the ferritin concentration in the sample can be calculated based on the obtained absorbance according to the standard curve.

[0048] The concentration range of 0 ng / mL to 1000 ng / mL is called the low concentration range, and the concentration range exceeding 1000 ng / mL is called the high concentration range. According to the method of the present invention, which uses liver ferritin as a standard sample, ferritin in the sample can be accurately quantified even in the high concentration range.

[0049] As described above, in the method of the present invention, the sample is diluted 5 to 20 times before measurement. This sample dilution is typically performed within an automated analytical device. In existing methods, samples with serum ferritin values ​​exceeding 1,000 ng / mL cannot be measured even after dilution and require further redilution before measurement. However, the method of the present invention uses liver ferritin as the raw material for the standard solution, which allows for measurement of samples with serum ferritin values ​​exceeding 1,000 ng / mL without redilution.

[0050] Blank samples used for immunoassay can be any substance that does not contain the analyte ferritin, and are not particularly limited. Pure water, physiological saline, buffer solution, negative sample or dilution thereof are preferred.

[0051] As described in the following examples, non-specific reactions are suppressed when a polyoxyethylene alkyl ether surfactant with 8 to 14 carbon atoms in the alkyl group is present in the reaction and / or assay system. For example, the liquid mixed with the sample may contain a polyoxyethylene alkyl ether surfactant with 8 to 14 carbon atoms in the alkyl group. Alternatively, it may be included in a latex particle suspension. Furthermore, the specificity is significantly improved compared to when a polyoxyethylene alkyl ether surfactant with 8 to 14 carbon atoms in the alkyl group is absent. Therefore, by using the method of the present invention, reagent performance can be improved, particularly in highly sensitizing reagents that readily produce non-specific reactions, compared to the past. The concentration of the polyoxyethylene alkyl ether surfactant in the reaction and / or assay system is 0.0001 to 2%, preferably 0.005 to 1%.

[0052] This invention comprises a ferritin assay reagent, characterized in that it uses ferritin derived from the liver as the standard solution raw material. The assay reagent comprises at least insoluble carrier particles sensitized with antiferritin antibody and a standard solution prepared using ferritin derived from the liver as the raw material. This assay reagent is also referred to as an assay kit.

[0053] Furthermore, whether the standard solution raw material comes from liver ferritin or other organs can be confirmed by analyzing the organ-specific substances mixed in. As an analytical method for organ-specific substances, proteomics and metabolomics can be used for analysis.

[0054] For example, proteomics analysis involves using proteases to break down trace amounts of proteome mixed in a standard solution into peptide fragments, and then analyzing the amino acid sequences of these peptides using mass spectrometry to obtain a comprehensive proteome analysis. Similarly, metabolomics analysis uses mass spectrometry to comprehensively identify trace amounts of metabolites mixed in a standard solution. If the identified proteome and metabolites contain liver-specific expressed proteomes or metabolites, the ferritin is considered to originate from the liver.

[0055] Example

[0056] The present invention will be specifically described through the following embodiments, but the present invention is not limited to these embodiments.

[0057] (1) Preparation of reagents

[0058] Using antibodies against ferritin, the assay reagents for immunoagglutination were prepared as follows.

[0059] Sensitized particles were suspended in a buffer solution (Tris, pH 8.5) to achieve a concentration of 0.09%, thereby preparing a latex suspension. The sensitized particles were prepared by loading 0.04 mg of antiferritin antibody into 1 mL of polystyrene latex suspension.

[0060] (2) Preparation of standard solution

[0061] Ferritin was used to prepare a standard solution for creating a standard curve.

[0062] Standard solution A (Example 1) was prepared by adding liver ferritin (commercially available from SCRIPPS LABORATORIES) to a buffer solution (Hepes, pH 7.5). As comparative examples 1 and 2, standard solutions B and C were prepared by adding ferritin from different organs.

[0063] [Table 1]

[0064] Example 1 A liver Comparative Example 1 B spleen Comparative Example 2 C placenta

[0065] (3) Measurement is performed using an automatic analysis device.

[0066] The automatic analysis device uses Hitachi's 7180 automatic analysis device to perform automatic measurements using the endpoint determination method.

[0067] Using the aforementioned reagents, 48 ​​serum samples were analyzed. 100 μL of buffer (Hepes, pH 7.4) was added to 7.0 μL of sample solution, and the mixture was stirred at 37°C. After standing for 5 minutes, 50 μL of latex suspension was added, and the mixture was stirred again at 37°C. The change in absorbance was measured after approximately 5 minutes of agglomeration, and the ferritin concentration of each sample was calculated using a standard curve prepared using standard solutions A–C.

[0068] (4) Comparison of different parameters at the metering point

[0069] The samples were measured using two different parameters at the photometer point to compare their reactivity.

[0070] In the low concentration range (0–1000 ng / mL), the results of Example 1 will be shown. Figure 1 And showing the results of comparative examples 1-2 Figures 2-3 After comparison, the correlation coefficient was good under all conditions, reaching 0.999, and no difference was found due to organ origin of ferritin.

[0071] On the other hand, in the high concentration range (1000–2000 ng / mL), the results of Example 1 will be shown. Figure 4 And showing the results of comparative examples 1-2 Figures 5-6 After comparison, the correlation coefficient reached 0.997 when using liver ferritin as the standard solution raw material, which was better than other conditions. The good correlation at different measurement points indicates that the measured values ​​did not change even when the measurement points were changed, suggesting that the time curves of the standard solution and the sample were similar. This demonstrates that the standard solution using liver ferritin as the raw material exhibits reactivity similar to the sample in the high concentration region.

[0072] Industry applicability

[0073] The method of this invention enables the determination of ferritin in a sample.

[0074] All publications, patents and patent applications cited in this specification are incorporated herein by reference.

Claims

1. A reagent for ferritin assay, characterized in that, Ferritin derived from the liver was used as the raw material for the standard solution. The reagent comprises a standard solution with a ferritin concentration between 1600 and 2000 ng / mL. The reagent is a latex coagulation reagent. The reagent comprises insoluble carrier particles sensitized with antiferritin antibody and a standard solution prepared using ferritin derived from the liver as a raw material.

2. A method for determining ferritin, characterized in that, Ferritin derived from the liver was used as the raw material for the standard solution. It contains standard solutions with ferritin concentrations between 1600 and 2000 ng / mL. The method described is latex coagulation. Ferritin in the test sample was mixed with antiferritin antibody-sensitized insoluble carrier particles in solution, and the absorbance based on the aggregation reaction caused by the antigen-antibody reaction was measured. Ferritin was quantified by comparing the absorbance with a standard curve. The standard curve was prepared using a standard solution made from liver ferritin as a raw material.

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