JP2005321411A - Transferrin-transferrin receptor complex measuring reagent and measuring method - Google Patents

Abstract

Provided are a reagent capable of measuring transferrin-transferrin receptor complex (Tf-TfR complex) by a simple operation, and a measuring method.
A Tf-TfR complex measuring reagent composed of insoluble particles to which a substance having affinity for TfR is immobilized, and a Tf-TfR complex using this reagent are measured. As the affinity substance, an antibody against TfR or Tf is advantageous.
[Effect] By providing a Tf-TfR complex measurement technique based on particle agglomeration reaction with excellent operability, the TfR measurement range can be expanded, and a wide concentration range can be measured without diluting the sample. Expansion of the measurement range is particularly effective when measuring TfR as a diagnostic marker for hematopoietic function.
[Selection figure] None

Description

The present invention relates to a measuring reagent for use in diagnosis of various diseases based on the transferrin receptor (hereinafter abbreviated as TfR) concentration in human body fluid, and a method for measuring TfR using this reagent. TfR is present on the surface layer of various cells and plays a role of binding to iron transport protein transferrin (hereinafter abbreviated as Tf). The affinity between TfR and Tf is about 10 ^−9, which is comparable to that of an antigen-antibody reaction. Furthermore, since Tf in blood is generally 10 ³ times larger than TfR, most TfR is present in blood. It exists in a form combined with Tf. The blood TfR concentration is known to be useful as an indicator of cancer, hematopoietic function and the like.

As a method for measuring TfR in body fluid, there has been a report (Patent Document 1) in which a labeled immunoassay is applied for the purpose of cancer diagnosis. An immunoassay method using a labeling substance typified by an enzyme immunoassay method (hereinafter abbreviated as EIA) employed in this report is a method generally used for increasing sensitivity. Examples of frequently used labeling substances include radioisotopes such as ¹²⁵ I, and enzymes such as peroxidase (hereinafter abbreviated as POD), β-galactosidase, and alkaline phosphatase. An immunoassay using a radioisotope as a labeling substance is called an RIA method, and an immunoassay using an enzyme as a labeling substance is called an EIA method. The RIA method is difficult to say because it requires a special facility or apparatus for using a radioisotope as a labeling substance. In addition to the immune reaction, the EIA method has an enzyme reaction step in which an enzyme, which is a labeling substance, reacts with a specific substrate, so that the reaction mode and reagent configuration are complicated. In any method, as long as the heterogeneous reaction principle is adopted, separation of the bonded phase and the free phase (B / F separation) is required, which tends to be an obstacle to automation and speedup.

  Furthermore, since both the RIA method and the EIA method have been developed in the direction of increasing the sensitivity of the measurement, if the substance to be measured exists in a large amount exceeding the upper limit of the measurable range in the body fluid that is the sample, The step of diluting is required. Since TfR is present in blood at a relatively high concentration, a measurement technique using a labeling method such as the EIA method often requires dilution of the specimen.

By the way, it has been pointed out that TfR becomes a marker of hematopoietic function in addition to the use as a cancer diagnostic marker introduced earlier (Non-patent Document 1). When measuring TfR as a marker of hematopoietic function, a higher level of TfR value is often measured than when measuring as a cancer marker. Therefore, in a measurement system in which high sensitivity has been pursued so far, a sample exceeding the measurement range often appears, and a dilution operation is required. That is, the measured concentration range of the TfR measurement technique reported so far cannot cover the actually required concentration range. Therefore, it is desired to provide a simple TfR measurement technique that can be used for functional tests of patients suspected of hematopoietic dysfunction. Moreover, in the measurement technique using the labeling method, it is often the case that a long reaction time is used so that high sensitivity can be easily obtained. Against this background, provision of a TfR measurement technique that realizes quick measurement with a simpler operation is awaited.
Japanese Patent Laid-Open No. 62-6169 Clin. Chem. 41 / 7,1053-1064; 1995

  The present inventor considered that the significance of measuring the TfR concentration in a body fluid that is a receptor for the iron transport protein Tf was significant from the relationship between iron and the hematopoietic function. For this purpose, a measurement system that can be measured more easily and easily automated, and a reagent having a more appropriate measurement concentration range are required. That is, the present invention is a new TfR measurement technique that realizes a wide measurement range that can be measured under the same conditions as the low concentration range even in the high concentration range where the dilution of the sample is required in the conventional TfR measurement technology. Providing is an issue.

  An object of the present invention is to provide a Tf-TfR complex measuring reagent composed of insoluble particles to which a TfR affinity substance is immobilized, or insoluble particles to which a TfR affinity substance and a Tf affinity substance are immobilized individually or mixed, and It is solved by the measurement method using this reagent.

A typical TfR affinity substance in the present invention is an antibody such as an anti-TfR antibody or an anti-Tf-TfR complex antibody. In addition, since the Tf-TfR complex is an object to be measured, anti-Tf antibodies can also be mentioned as affinity substances. This is because it is considered to specifically bind to TfR via Tf. In the case of using an antibody as an affinity substance, it is advantageous to select an antibody having a sufficiently high antibody titer because there is no competing point in terms of affinity in the biological fluid as a specimen. As the antibody, a polyclonal antibody obtained by immunizing mice, rats, rabbits, goats and the like by a usual method can be used. Of course, it goes without saying that monoclonal antibodies can also be used. Several antibodies against TfR have been reported so far. These known antibodies, whether polyclonal antibodies or monoclonal antibodies, are used in the present invention as long as they have affinity and specificity that can be aggregated by reacting with TfR in a state sensitized to insoluble particles. be able to.

  Among them, the polyclonal antibody can be shown as a preferable antibody from the following background. First, considering the reaction principle of the agglutination reaction, it is generally more advantageous that the number of binding sites between the affinity substance immobilized on the particle and the test substance is larger. That is, a polyclonal antibody that recognizes a large number of binding sites is more advantageous than a monoclonal antibody in which binding sites are limited. Furthermore, in order to measure TfR that forms a complex with Tf, the Tf binding site on TfR is covered by Tf. In such a complex, an antibody that recognizes the Tf binding site can no longer bind. For these reasons, polyclonal antibodies are preferred antibodies.

  When higher specificity is expected or when it is desired to reduce the consumption of TfR or Tf as an immunogen, a monoclonal antibody may be used as an antibody. However, when using a monoclonal antibody, a clone having a more advantageous binding affinity should be selected in consideration of the circumstances described above. For example, an antibody that recognizes the Tf binding site of TfR should not be selected because the binding site is blocked by Tf. Therefore, it is preferable to use an antigenic determinant existing other than the Tf binding site of TfR, an antigenic determinant specifically present in the Tf-TfR complex, an antigenic determinant specific to Tf, etc. as the binding site of the monoclonal antibody. . In particular, the combination of a monoclonal antibody that recognizes an antigenic determinant other than the Tf-binding site of TfR and an antigenic determinant specific to Tf can reliably measure TfR forming a complex in blood. A preferred combination. The reagent according to the present invention can be obtained by mixing insoluble particles to which these monoclonal antibodies are immobilized, or by immobilizing the mixture of monoclonal antibodies to insoluble particles. The reagent according to the present invention composed of an anti-Tf monoclonal antibody binds to free Tf but cannot form a cross-linked structure, so it does not lead to an agglutination reaction. That is, since it becomes a reagent with high accuracy that specifically causes aggregation only when the Tf-TfR complex is present, it can be shown as an embodiment in which the use of a monoclonal antibody is advantageous. Monoclonal antibodies can also be used in combination with polyclonal antibodies.

  When using a monoclonal antibody against Tf, it is expected that sufficient particle aggregation cannot be expected because free Tf present in the blood in a large amount compared to Tf-TfR competes with this monoclonal antibody. In such a case, a monoclonal antibody against the same Tf as that immobilized on the particles is added without being immobilized on the particles, and the reaction may be performed in the coexistence thereof.

  The antibody used in the present invention can be made less susceptible to non-specific reactions such as complement and rheumatoid factor by removing the Fc site. As a method for removing the Fc site, a method in which various proteolytic enzymes are allowed to act is known.

  As an affinity substance other than an antibody, α-1 antitrypsin for TfR can be assumed. Since these affinity substances each have a substance that competes with TfR in terms of affinity in biological fluids, it is preferable to carry out the reaction under conditions where the affinity for TfR is as large as possible. Alternatively, a method of blocking a binding site with a competitor is also effective. For example, when α-1 antitrypsin is used, it is preferable to prepare particles to which trypsin is bound and to indirectly bind α-1 antitrypsin to the particles via trypsin. By this method, the trypsin binding site of α-1 antitrypsin is blocked, and the binding activity with TfR is present on the particles.

  The insoluble particles used in the present invention may be those commonly used in the field of immunology such as animal erythrocytes, metal sols, latex, gelatin, or pigments, but are not limited thereto. Among them, latex particles made of synthetic resin such as polystyrene are already widely used and thus are easily available and inexpensive, and are advantageous in terms of qualitative uniformity compared to animal erythrocytes. The polystyrene latex may have an average particle size of 0.02-1.0 μm, preferably 0.04-0.6 μm.

  The dispersion solution for dispersing the affinity particles of the present invention is used by adding a dispersant, a surfactant, a substance that prevents non-specific reactions, a small amount of a preservative, and the like to a buffer that provides a pH necessary for an immune reaction. The selection of these additive substances and concentrations is appropriately adjusted according to the affinity substance to be immobilized, the amount thereof, the type of insoluble particles, and the like. For example, inactive proteins such as gelatin and bovine serum albumin are effective in stabilizing the aggregation reaction and preventing nonspecific reactions. Specifically, when dispersing polystyrene latex immobilized with an antibody as an affinity substance, phosphate buffer (hereinafter abbreviated as PBS) or buffer that gives a neutral region such as HEPES, BSA, normal animal serum, etc. Add

  The present invention also provides a method for measuring a Tf-TfR complex using the Tf-TfR complex measurement reagent. The reaction between the reagent of the present invention and the Tf-TfR complex can be followed by observing the degree of aggregation. As a method for observing the agglutination reaction, there are a method using the naked eye and a method of measuring the change in the amount of scattered light or transmitted light using an optical device. In general, the method of observing with the naked eye can be quantitatively measured only by diluting the specimen to several levels and observing each of them. The optical method is more preferable as an embodiment of the present invention because an appropriate calibration curve can be prepared in advance or at the time of measurement, so that quantitative measurement can be performed without taking several concentrations of the sample.

  At this time, a Tf-TfR complex derived from a blood material may be used as a standard substance. Since TfR released into the blood from cells has a different structure from TfR extracted from tissue, there is a possibility that an accurate result cannot be expected by the method using purified TfR as a standard due to a difference in reactivity. In fact, TfR released in the blood lacks 100 amino acids on the N-terminal side compared to complete TfR present in the tissue. Considering that most of TfR in blood is complex with Tf, in order to obtain the value of TfR in blood, it should be measured as Tf-TfR complex. If free TfR that does not form a complex with Tf in blood is measured, free TfR is used as a standard.

  The TfR measurement reagent of the present invention realizes measurement in a wide concentration range that has not been conventionally available. Due to this feature, the present invention provides a Tf-TfR complex measurement method in which most serum samples are measured without dilution. In addition, the measurement which does not require dilution operation said here means that a low concentration sample and a high concentration sample can be measured on the same reaction conditions. That is, in the conventional measurement method in which the measurable concentration range is narrow, dilution conditions different from those for the low concentration sample are necessary for the measurement of the high concentration sample. On the other hand, in the present invention, even a high concentration sample can be measured under the same conditions as a low concentration sample.

  The measured value of the Tf-TfR complex obtained by the present invention can be converted into a TfR value. Since TfR and Tf are bonded at a ratio of 1: 1, the molar ratio between the two becomes 1: 1. Since most of TfR in the blood exists in a state of being bound to Tf, the TfR value is obtained by multiplying the ratio of the amount of TfR protein to the measured value of the Tf-TfR complex. This value is 47% as shown in the examples, and it can be converted to a TfR value by applying 47% to the measured value according to the present invention.

  The standard substance used in the present invention is desirably a standard substance for measuring a Tf-TfR complex including a Tf-TfR complex. The Tf-TfR complex can be purified using serum with a high TfR value as a raw material. When measuring the Tf-TfR complex in the blood, it is advantageous to use a standard substance purified from the blood in order to make the reactivity with the reagent common. Purification of the Tf-TfR complex from serum can be easily performed by purification steps such as serum ammonium sulfate fractionation, anion exchange chromatography, and immunoadsorption chromatography. The purified Tf-TfR complex can be diluted in an appropriate buffer solution or added to TfR-free serum to test the concentration and use it as a standard substance. As this standard substance, a substance having a high concentration of about 14,000 to 70,000 ng / mL is useful so that it can be used as a standard substance over a wide concentration range that enables diagnosis of hematopoietic function.

  The insoluble particles to which the TfR affinity substance in the present invention is immobilized bind to the Tf-TfR complex and enable analysis thereof. The present invention is novel in that a particle agglutination reaction that can be carried out with a simpler operation is applied in place of a measurement technique for realizing high sensitivity that has been conventionally used for measuring TfR. By applying the particle agglutination reaction, it is possible to measure up to a high concentration with sufficient accuracy under the same conditions as those in a low concentration region without diluting the sample. Further, in the particle agglutination reaction that is a homogeneous reaction, the B / F separation required for the measurement by the label becomes unnecessary, so that the automation of the measurement becomes remarkably easy. This also contributes to speeding up of inspection time.

  The present invention enables measurement of a Tf-TfR complex by a particle agglutination reaction having excellent operability. According to the present invention, the measurement range of TfR is expanded, and a wide concentration range can be measured without diluting the sample. Expansion of the measurement range is particularly effective when measuring the Tf-TfR complex as a diagnostic marker for hematopoietic function. Furthermore, according to the present invention, the B / F separation required for the measurement by the label becomes unnecessary, so that the speed and automation can be easily realized as compared with the high sensitivity measurement method using the labeling technique.

  By immobilizing a TfR affinity substance on insoluble particles and measuring using a particle agglutination reaction, the measurement range of the TfR-TfR complex was expanded, and the analysis was accelerated and automated.

  1. Reagent preparation

  Preparation of anti-TfR antibody: Human TfR purified from placenta by a known method (J. Biol. Chem., 263, 8318-8325, 1988) is mixed with Freund's complete adjuvant and thoroughly emulsified. The pupa footpad was immunized for the first time and boosted twice in the back skin every 4 weeks. The increase in antibody titer was confirmed by the octellony method with purified human TfR, and blood was collected 2 weeks after the final immunization to separate the antiserum. Antiserum was purified by DEAE-cellulose ion exchange chromatography, and the IgG fraction was used as an anti-TfR antibody. Preparation Example of Reagent of the Present Invention: Anti-TfR antibody was mixed with polystyrene latex (average particle size 0.104 μm) and reacted at 37 ° C. for 1 hour. Latex particles were separated by centrifugation at 3,000 × G for 15 minutes, and then suspended in a PBS buffer containing 1% BSA to obtain a 0.5% latex emulsion, which was used as the measurement reagent of the present invention.

  Example of preparation of reference reagent: The above-mentioned anti-TfR antibody was adjusted to 10 mg / L with PBS buffer solution, dispensed 0.1 mL each into a well of a 96-well microtiter plate (manufactured by NUNK), and then allowed to stand at 37 ° C. for 1 hour. After washing with physiological saline, a block operation with BSA was applied to obtain a solid phase plate for ELISA. Further, the anti-TfR antibody was POD-labeled by the periodate crosslinking method to obtain a labeled antibody for ELISA.

  Preparation of standard substance: Serum was fractionated with 45-70% saturated ammonium sulfate, dialyzed with a buffer of 20 mM Tris-HCl (pH 8.6), and applied to an anion exchange column (Q-Sepharose, Pharmacia). Elution was performed with a linear gradient using 20 mM Tris-HCl buffer (pH 8.6) containing 0.5 M NaCl. Fractions rich in TfR were pooled by following the TfR concentration by ELISA and applied to an affinity column to which an anti-Tf antibody was immobilized. The column was first washed with 50 mM HEPES buffer (pH 7.5) containing 0.1 M NaCl, and then with 50 mM citrate buffer (pH 4.9) containing 100 mM NaCl and 1 mM deferroxamine mesylate. Iron was released from Tf. After washing with 50 mM HEPES buffer (pH 7.5) containing 0.1 M NaCl again, the binding to Tf was reduced by the release of iron in 50 mM HEPES buffer (pH 7.5) containing 3 M KCl. TfR was eluted. The eluate of the affinity column was reacted with an excess amount of iron-saturated Tf to form a complete Tf-TfR complex, dialyzed against a buffer of 20 mM Tris-HCl (pH 8.6), and further subjected to gel filtration column and anion exchange. A purified Tf-TfR complex was obtained using a column (Resource-Q, Pharmacia). The yield of the complex by this method was about 50% (recovering about 2.0 mg of Tf-TfR complex from 2 L of serum containing 2,000 ng / mL Tf-TfR complex).

  In the purified product, two bands with molecular weights of about 85 kDa and 65 kDa were recognized by SDS-PAGE in a non-reducing state. In the reduced state, Tf is dissociated from the intramolecular SS bond and the apparent molecular weight increases, so the two bands are close to each other. When amino acid sequence analysis was performed on each protein after blotting to the PVDF membrane, it was confirmed that 85 kDa coincided with the sequence from the 101st position of TfR, and 65 kDa coincided with the sequence from the N-terminus of Tf. The composition ratio of TfR and Tf was approximately 47% to 53% from the densitogram of the stained SDS-PAGE gel, and the number of amino acid residues coincided with the ratio of 660 for TfR and 719 for Tf. That is, it is considered that the molar ratio was 1: 1. A value obtained by multiplying the amount of protein by the concentration ratio of TfR (47%) was determined as the amount of TfR in the purified complex. The protein amount of the purified Tf-TfR complex was determined by the BCA protein quantification method. This purified Tf-TfR complex was diluted with PBS containing 1% BSA and 0.1% Tf and used as a primary standard. In the ELISA, lyophilized products obtained by appropriately diluting serum were assayed by the primary standard, and in the latex agglutination method, lyophilized products obtained by partially purifying and concentrating the Tf-TfR complex from the serum using an ion exchange column were tested with the primary standard. Used as the next standard. Therefore, the concentration value obtained by measurement using this standard is the concentration of the Tf-TfR complex.

  2. Comparison of calibration curves of measurement reagent of the present invention and reference reagent

  Using the measurement reagent of the present invention prepared in 1 above, 30 μL of a sample solution (standard diluted concentration solution for a calibration curve, sample solution for general measurement) and 300 μL of this reagent emulsion are dispensed and mixed in a measurement cell, Scattered light (wavelength 660 nm) was measured after 25 seconds and 300 seconds to determine the difference in intensity of the scattered light. The standard dilution was measured using each of the five stages of the partially purified human Tf-TfR complex, which were serially diluted 2-fold from 16,000 ng / mL to prepare a calibration curve. For the measurement, a fully automatic immunochemical analyzer LX-3000 (trade name, manufactured by Eiken Chemical and Analytical Instruments) was used.

  As a control, a calibration curve for the ELISA method using the reference reagent prepared in 1 was prepared. For measurement, 100 μL of sample solution per well was dispensed onto a solid phase plate, reacted at 37 ° C. for 1 hour, washed with 0.1% Tween 20-added PBS, added with 100 μL of labeled antibody, reacted at 37 ° C. for 30 minutes, washed, and TMB ( The color was developed with tetramethylbenzidine, and the absorbance at a wavelength of 450 nm was measured. The standard dilution concentration solution was measured using each of the five stages obtained by serially diluting serum having a known Tf-TfR complex value from 78.0 ng / mL, and a calibration curve was prepared. The concentration of the standard substance is set based on a concentration range that can be measured with practical accuracy confirmed in a preliminary experiment. The results are shown in FIG. 1 and FIG. In the measurement reagent of the present invention, the measurement range is 500 to 16,000 ng / mL (FIG. 1), whereas in the reference example ELISA method (FIG. 2), it is 2.44 to 78.0 ng / mL. That is, it was confirmed that it was necessary to dilute the sample 200 times in advance to obtain the same measurement range as the measurement reagent of the present invention by ELISA.

  3. Correlation between latex agglutination method and ELISA method according to the present invention

  80 serum samples having various Tf-TfR complex values at various concentrations were measured with the latex agglutination reagent according to the present invention prepared in 1, and the ELISA reagent as a reference reagent, and the correlation between the measured values was investigated. High-value specimens that cannot be directly measured by the ELISA method were measured after dilution as appropriate. The results are as shown in FIG. It was confirmed that the measurement value according to the present invention and the measurement value by ELISA showed a good correlation and high-precision measurement was possible.

  4). Example of measurement using the measurement reagent of the present invention

  By the measurement reagent of the present invention shown in FIG. 2, 15 healthy subjects, 10 iron deficiency anemia patients, 15 stable dialysis patients, 10 dialysis patients with iron deficiency, 2 polycythemia patients, kidney transplantation Tf-TfR complex values were measured for sera from two patients. The results are shown in Table 1. The TfR value in the serum of patients with iron deficiency anemia, polycythemia, etc. was abnormally high, and it was confirmed that the measurement of TfR according to the present invention is useful as a diagnostic marker for hematopoietic function. Such a high value is beyond the measurable range of the ELISA method.

  4). Example of measurement using the measurement reagent of the present invention

  The measurement reagent of the present invention was composed of a monoclonal antibody that recognizes other than the Tf binding site of TfR and a monoclonal antibody that recognizes Tf. The monoclonal antibody was prepared according to the method described in Japanese Patent Application Laid-Open No. Sho 62-6169, and each was immobilized on insoluble particles and then mixed. The specific operation is as follows. The same amount of purified TfR used in 1 was mixed with Freund's complete adjuvant and immunized subcutaneously in Balb / c mice. Immunization was repeated every 2 weeks, and 50 μg of TfR was immunized intraperitoneally after 4 immunizations. Three days after the final immunization, it was confirmed that the antibody titer had increased, and the spleen was removed and mouse myeloma was obtained by a conventional method (Harada.N et al. Proc. Natl. Acad. Sci. USA 84: 4581-4585,1987). Cells were fused with cells P3-X63Ag8.653.

  Hybridomas were first screened for antibodies specific for TfR by immunoblotting assay using purified TfR as an antigen. Clones that produce monoclonal antibodies that recognize TfR are further screened by an inhibition assay using TfR and Tf, and those that produce monoclonal antibodies that recognize antigenic determinants present at portions other than the Tf binding site are selected. did. Thus, a monoclonal antibody that recognizes other than the Tf binding site of TfR was obtained. On the other hand, the monoclonal antibody recognizing Tf was obtained by substantially the same operation except that Tf was used as the immunogen and screening was simply performed on Tf.

  The two types of monoclonal antibodies (IgG, 0.11 mg / mL) thus obtained were each physically adsorbed at 37 ° C. for 1 hour on polystyrene latex having a particle size of 0.104 μm. After washing with HEPES buffer, the reagent of the present invention comprising a monoclonal antibody was dispersed in HEPES buffer containing 1% BSA in an equal amount so that the particle concentration would be 0.5%.

It was confirmed that the reagent according to the present invention composed of a monoclonal antibody quantitatively produces an optically measurable aggregate when reacted with the purified Tf-TfR complex by the same procedure as 2. This aggregation was not observable with purified Tf or TfR and was clearly specific for the Tf-TfR complex.

The standard curve obtained by the immunological latex agglutination reaction method using the measuring reagent of this invention. In the figure, the vertical axis represents the amount of change in scattered light intensity (DLSE) at 660 nm, and the horizontal axis represents the Tf-TfR complex concentration (ng / mL). Standard curve obtained by ELISA using reference reagents. In the figure, the vertical axis represents the absorbance at 450 nm, and the horizontal axis represents the Tf-TfR complex concentration (ng / mL). The graph which shows the correlation of the measured value by the latex agglutination reaction reagent (LA method) by this invention, and the measured value by an ELISA reagent.

Claims (8)

  Transferrin-transferrin receptor complex measurement reagent that measures the concentration by reacting transferrin receptor affinity substance immobilized on insoluble particles with transferrin-transferrin receptor complex and observing aggregation of insoluble particles caused by antigen-antibody reaction   The reagent for measuring transferrin-transferrin receptor complex according to claim 1, wherein the substance having affinity for transferrin receptor is an antibody.   The reagent for measuring transferrin-transferrin receptor complex according to claim 2, wherein the antibody is selected from an anti-transferrin receptor antibody and an anti-transferrin-transferrin receptor complex antibody.   The transferrin-transferrin receptor complex measuring reagent according to claim 3, wherein the antibody is a polyclonal antibody.   The reagent for measuring transferrin-transferrin receptor complex according to claim 1, wherein the insoluble particles are latex particles.   The reagent for measuring transferrin-transferrin receptor complex according to claim 1, further comprising insoluble particles having a transferrin affinity substance immobilized thereon.   The reagent for measuring transferrin-transferrin receptor complex according to claim 1, comprising insoluble particles in which the substance having affinity for transferrin is immobilized together with the substance having affinity for transferrin receptor.   The transferrin-transferrin receptor complex measurement reagent according to claim 6 or 7, wherein the transferrin-affinity substance is a monoclonal antibody against transferrin.

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