JPH10115615A - Assay of frerritin and immunological kit for measuring frerritin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To determine the amount of ferritin in a sample highly accurately, quickly and simply. SOLUTION: By this method, a sample and an insoluble carrier, wherein antiferritin antibody is immunized, are mixed in solution, and aggregation reaction by antigen-antibody reaction is generated. The assay of the amount of the ferritin comprising the measurement of the changing amount of absorbency based on the above descried aggregation reaction and the collating with a working curve is performed. In this case, the above described insoluble carrier is made to be latex particles. In the above described solution, one kind of 0.1-5wt.%, which is selected from the group comprising polyvinyl pyrrolidone having the average molecular weigh of 1,000-2 million molecules, pullulan having the average molecular weight of 1,000-1 million molecules and polyethylene glycol having the average molecular weight of 1,000-2 million molecules, is contained. The above described aggregate reaction is performed for 5 seconds - 15 minutes at a constant temperature. Here, the above described chaining amount of the absorbency is made to be the increasing amount of the absorbency accompanied by the aggregation reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying ferritin in a sample with high sensitivity, speed, and convenience, and an immunological kit for measuring ferritin for carrying out the method for quantifying ferritin.

[0002]

2. Description of the Related Art Ferritin is an iron storage protein having a molecular weight of about 450,000, is composed of 20 to 24 subunits, and contains 4000 iron atoms per molecule. Ferritin is used in the liver,
Since it is present in many organs such as spleen and placenta, quantifying this ferritin is useful for diagnosis of iron deficiency anemia, iron overload and the like.

In the case of malignant tumors such as leukemia and other solid cancers, serum ferritin increases irrespective of the amount of iron stored in the body. Therefore, quantification of ferritin is necessary for diagnosis and treatment of malignant tumors. It is also useful for monitoring.

[0004] Ferritin can be quantified by reverse passive hemagglutination (RPHA) or enzyme immunoassay (EI).
A), a radioimmunoassay (RIA) and the like. However, the RPHA method is complicated in operation, takes a long time for measurement, and has relatively low sensitivity. Also, EIA
The RIA method and the RIA method can perform highly sensitive detection, but the reagent price is high. Furthermore, since the RIA method uses a radioactive element, it is called "nuclear medicine technology" (Kanao et al., Vol.
2, 1992) had the disadvantage of requiring special facilities for measurement. Also,
Due to the clinical importance of ferritin, automation of a method for quantifying ferritin has been desired.

[0005]

SUMMARY OF THE INVENTION In view of the above, the present invention provides a method for quantifying ferritin which is highly sensitive, rapid and simple, and an immunological kit for measuring ferritin for carrying out the method for quantifying ferritin. The purpose is to do.

[0006]

According to the present invention, when quantifying ferritin in a sample, the sample is mixed with an insoluble carrier sensitized with an anti-ferritin antibody in a solution to perform an agglutination reaction by an antigen-antibody reaction. And a method for quantifying ferritin by measuring the amount of change in absorbance based on the agglutination reaction and comparing it with a calibration curve, wherein the insoluble carrier is
Latex particles, having an average molecular weight of 10
Polyvinylpyrrolidone of 200 to 2,000,000, average molecular weight 1
000 to 1 million pullulan and average molecular weight of 100
0.1 to 5% by weight selected from the group consisting of 0 to 2,000,000 polyethylene glycols, and the agglutination reaction is carried out at a constant temperature for 5 seconds to 15 minutes, and the amount of change in absorbance is This is a method for quantifying ferritin, which is an increase in absorbance due to an agglutination reaction. Hereinafter, the present invention will be described in detail.

In the present invention, in quantifying ferritin in a sample, first, the sample and an insoluble carrier sensitized with an anti-ferritin antibody are mixed in a solution to cause an agglutination reaction by an antigen-antibody reaction.

The sample is not particularly restricted but includes, for example, serum, plasma and the like. Further, the method for quantifying ferritin of the present invention can be applied to various other biological samples and non-biological samples.

In the present invention, latex particles having a relatively uniform particle size and capable of industrially producing products of a constant quality and performance in large quantities are used as the insoluble carrier. The latex particles are not particularly limited, and include, for example, styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates, methacrylates, and other vinyl-based monomers such as homopolymers and copolymers; styrene-butadiene copolymers, Fine particles such as a butadiene-based copolymer such as a methyl methacrylate-butadiene copolymer are included. Among these, polystyrene-based latex particles are preferably used because they have excellent antibody-adsorbing properties and can stably maintain biological activity for a long period of time.

The particle size of the latex particles as the insoluble carrier is preferably 0.05 to 1 μm. If it is too small, dispersion becomes difficult when freeze-drying, and if it is too large, self-aggregation proceeds and dispersibility decreases. More preferably, it is 0.1 to 0.5 μm.

[0011] The insoluble carrier is one to which an anti-ferritin antibody has been sensitized. The anti-ferritin antibody is not particularly limited, and any of a polyclonal antibody and a monoclonal antibody can be used. The origin of ferritin, which is an immunogen for the liver, spleen, placenta and the like, and the animal species derived from humans, rabbits, goats and the like are not particularly limited. When the anti-ferritin antibody is the polyclonal antibody, the purity of the globulin fraction, the affinity-purified fraction, etc., the method of treating Fab ′, F (ab) 2, and the like are not particularly limited.

The sensitization of the anti-ferritin antibody to the insoluble carrier can be performed by a method of physically or chemically adsorbing the antibody. In this case, the anti-ferritin antibody is used in a solution that causes an agglutination reaction.
The insoluble carrier is sensitized to have a concentration of 0.1 to 100 μg / ml. If it is too low, sensitivity is low and accurate measurement at a low concentration cannot be performed. If it is too high, aggregation due to nonspecific reaction occurs, and accurate measurement cannot be performed.

In the present invention, the sample and the insoluble carrier are mixed in a solution. The above solution is not particularly limited, and examples thereof include a phosphate buffer, a glycine buffer, a Tris-HCl buffer, and a good buffer.

In the present invention, the concentration of the insoluble carrier in the solution is preferably 0.01 to 0.5% by weight.
If it is too low, the formation of aggregates is insufficient, and the required sensitivity cannot be obtained. If it is too high, the absorbance as the background is too high, and accurate quantification cannot be performed.

In the present invention, the solution contains one selected from the group consisting of polyvinylpyrrolidone, pullulan and polyethylene glycol. When these are contained in the above solution, the sensitivity can be improved and the reaction can be promoted.

The average molecular weight of the polyvinylpyrrolidone is 1,000 to 2,000,000. If it is too small, the effect of improving sensitivity or accelerating the reaction is insufficient, while if it is too large, the solubility in the solution becomes extremely low, and a problem arises in solubility, so that it is limited to the above range. Preferably, it is 5000 to 2,000,000.

The above-mentioned pullulan is a linear α-glucan obtained by repeatedly polymerizing maltotriose having α-1,4 bonds at both ends thereof with α-1,6 bonds. The above pullulan has an average molecular weight of 1,000 to 1,000,000. If too small, the effect of improving sensitivity or promoting the reaction is insufficient,
If it is too large, the solubility in the above solution becomes extremely low,
Since a problem arises in solubility, it is limited to the above range. Preferably, it is 2000-1,000,000.

The polyethylene glycol has an average molecular weight of 1,000 to 2,000,000. If it is too small, the effect of improving sensitivity or accelerating the reaction is insufficient, while if it is too large, the solubility in the solution becomes extremely low, and a problem arises in solubility, so that it is limited to the above range. Preferably, it is 5000 to 2,000,000.

The polyvinylpyrrolidone in the solution,
One concentration selected from the group consisting of pullulan and polyethylene glycol is 0.1 to 5% by weight. If it is too low, the sensitivity is low, and accurate measurement in a low concentration range cannot be performed. If it is too high, aggregation due to nonspecific reaction occurs, and accurate measurement cannot be performed. Therefore, the range is limited to the above range. Preferably, it is 0.4 to 2.0% by weight.

One selected from the group consisting of polyvinylpyrrolidone, pullulan and polyethylene glycol is preferably used after being dispersed or dissolved in an appropriate medium. In this case, the insoluble carrier and one selected from the group consisting of polyvinylpyrrolidone, pullulan and polyethylene glycol are dispersed or dissolved in the same medium to be used as a one-part latex reagent. Alternatively, by dispersing or dissolving them in separate media, respectively, they may be used as a two-part reagent of a latex reagent and a liquid for diluting a sample, which is a reagent in the form of a solution.

The medium is not particularly restricted but includes, for example, phosphate buffer, glycine buffer, Tris-HCl buffer, Good buffer and the like. The pH of the medium is
5.5 to 8.5 are preferred. More preferably, 6.5 to 6.5
8.0.

In the one-pack type latex reagent, bovine serum albumin, sucrose, sodium chloride or the like may be appropriately dissolved for adjusting the salt concentration. In addition, the above 2
In the liquid type reagent, bovine serum albumin, sucrose, sodium chloride or the like may be appropriately dissolved for adjusting the salt concentration.

In the present invention, the agglutination reaction by the antigen-antibody reaction is performed at a constant temperature. Preferably, 2
5-37 ° C. The agglutination reaction is performed for 5 seconds to 15 seconds.
Do for a minute. If the length is too short, accurate measurement cannot be performed. If the length is too long, aggregation due to nonspecific reaction occurs, and accurate measurement cannot be performed.

In the present invention, the amount of change in absorbance based on the agglutination reaction is measured and compared with a calibration curve to quantify ferritin. The amount of change in absorbance is an increase in absorbance due to the agglutination reaction.

The measurement wavelength at the time of measuring the change in absorbance is usually 500 to 1000 nm, preferably 500 to 1000 nm.
An appropriate wavelength is selected from the range of ８００800 nm. The measuring device used for measuring the change in absorbance is not particularly limited as long as it can measure the absorbance of the solution over time, and examples thereof include a general-purpose biochemical automatic analyzer. The measurement wavelength, sample amount, reagent amount, and the like can be appropriately selected according to the measurement device.

As a specific method of comparing with the above calibration curve, for example, a change in absorbance is measured for a ferritin sample having a known concentration such as a dilution series of ferritin, and a calibration curve is obtained from the measured value and the amount of ferritin. The method can be carried out by preparing a ferritin sample of unknown concentration and determining the corresponding ferritin amount in the above calibration curve from the measured value of the change in absorbance measured under the same conditions.

[0027] The present invention 2 comprises a latex particle sensitized with an anti-ferritin antibody, polyvinylpyrrolidone having an average molecular weight of 1,000 to 2,000,000, pullulan having an average molecular weight of 1,000 to 1,000,000, and polyethylene glycol having an average molecular weight of 1,000 to 2,000,000. A ferritin assay immunological kit for carrying out the method for quantifying ferritin according to the first aspect of the present invention, which comprises a sample diluent containing one selected from the group consisting of:

As a specific method for quantifying ferritin using the immunological kit for measuring ferritin of the present invention 2, for example, a sample to be quantified for ferritin,
The method can be carried out by dispensing the above-mentioned liquid for diluting a sample into a reaction container, incubating, and then adding and mixing a latex reagent comprising latex particles sensitized with the anti-ferritin antibody.

[0029]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of anti-human placental ferritin antibody-sensitized latex solution A solution prepared by dissolving anti-human placental ferritin antibody at a concentration of 2.0 mg / ml in 50 mM phosphate buffer (pH 7.4) 7 0.5 ml of polystyrene latex having an average particle size of 0.3 μm (solid content: 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) and a phosphate-salt buffer (0.036 M phosphate buffer ( pH 6.6) and 0.1 M NaCl) and stirred at 25 ° C. for 60 minutes. Next, a phosphate-salt buffer (0.05 M phosphate buffer (pH 7.0), 0.1 M NaCl) containing 1% by weight of bovine serum albumin (hereinafter, referred to as “BSA”) in the solution (hereinafter, referred to as 0.1 M NaCl) Then, the mixture was stirred at 25 ° C. for 60 minutes, and washed by centrifugation at 4 ° C. for 20 minutes at 18,000 rpm. The washing operation was performed three times. PBS 10m containing 1% by weight of BSA in the obtained precipitate
After suspending the latex, dispersion treatment was performed using an ultrasonic crusher to prepare an anti-human placental ferritin antibody-sensitized latex solution having a solid content of 0.08% (W / V).

(2) Preparation of Sample Dilution Solution Polyvinylpyrrolidone having an average molecular weight of 1.2 million (Luvi
skol K-90, manufactured by BASF) (hereinafter "PVP")
Is added to PBS containing 1% by weight of BSA.
It was dissolved to a concentration of 0% by weight.

(3) Ferritin Assay Reagent The ferritin assay reagent of this example comprises the first reagent comprising the anti-human placental ferritin antibody-sensitized latex solution of the above item (1) and the PVP solution of the above item (2). This is a two-part reagent composed of a second reagent.

(4) Standard ferritin solution Ferritin was added to 0, 25, 50, 100, 300, 600
Human serum at a concentration of ng / ml was used as a standard.

(5) Measuring method: 20 μl of the sample and 150 μl of the sample diluent described in the above item (2)
And the mixture is kept at 37 ° C. as appropriate, and then the anti-human placental ferritin antibody-sensitized latex solution (150 μl) described in the above (1)
Was added and stirred. After this, the wavelength 75 after 1 minute and 5 minutes
The absorbance at 0 nm is measured, and this difference is determined by the change in absorbance (Δ
Abs). The measurement used Hitachi automatic analyzer 7150 type. The standard ferritin solution was measured, a calibration curve was prepared in advance, and the amount of change in absorbance of the sample was extrapolated to the calibration curve to calculate the amount of ferritin in the sample.

(6) Measurement of Sample Using human serum as a sample, the amount of ferritin was measured according to the above-mentioned measurement method (hereinafter referred to as "latex method").

Example 2 Preparation of (2) Sample Diluent Solution in Example 1
(5) The amount of ferritin was calculated in the same manner as in Example 1, except that the measurement was performed as follows.

(2) Preparation of Sample Dilution Solution Pullulan having an average molecular weight of 200,000 (manufactured by Hayashibara Corporation)
Was dissolved in PBS containing 1% by weight of BSA to a concentration of 1.1% by weight.

(5) Measurement method 20 μl of the standard ferritin solution and 150 μl of the sample diluting solution of the above item (2) are mixed and kept at 37 ° C. as appropriate, and then sensitized with the anti-human placental ferritin antibody of the above item (1). 150 μl of a latex solution was added and stirred. Thereafter, the absorbance at a wavelength of 750 nm after 1 minute and 5 minutes was measured, and this difference was defined as the absorbance change (ΔAbs). The measurement used Hitachi automatic analyzer 7150 type.

Example 3 The ferritin amount was calculated in the same manner as in Example 2 except that (2) the preparation of the sample diluent in Example 2 was performed as follows.

(2) Preparation of Sample Dilution Solution 1.5% by weight of polyethylene glycol having an average molecular weight of 500,000 (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as “PEG”) was added to PBS containing 1% by weight of BSA. Was dissolved to a concentration of

Comparative Example 1 For the sample measured in (6) in Example 1, the ferritin in the sample was measured using a commercially available kit for measuring ferritin by EIA method (Enzygnost Ferritin micro, manufactured by Hoechst). The measurement was performed according to the kit instructions.

Comparative Example 2 The ferritin amount was calculated in the same manner as in Example 2 except that (2) the preparation of the sample diluting solution in Example 2 was performed as follows.

(2) Preparation of Sample Dilution Solution Polyvinyl alcohol having an average molecular weight of 92,000 (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as “PVA”) was mixed with 1 BSA.
It was dissolved in PBS containing 1.8% by weight to a concentration of 1.8% by weight.

Results The calibration curve of Example 1 is shown in Table 1 and FIG. Table 2 shows the sample measurement results of Example 1 and Comparative Example 1. FIG. 2 shows the correlation between the EIA method and the latex method. The calibration curves of Examples 2, 3 and Comparative Example 2 are shown in Table 3 and FIG.
It was shown to. From Table 2 and FIG. 2, the latex method, which is a method for quantifying ferritin of the present invention, has a good correlation with the EIA method (correlation coefficient r = 0.98), and has the same sensitivity as the EIA method. It became clear. Table 3 and FIG.
Thus, it has been clarified that a highly sensitive reagent can be obtained by using PVP, pullulan or PEG as a sensitizer.

[0045]

[Table 1]

[0046]

[Table 2]

[0047]

[Table 3]

[0048]

EFFECTS OF THE INVENTION The method for quantifying ferritin and the immunological kit for measuring ferritin of the present invention have the above-mentioned constitutions, so that ferritin in a sample can be quantified with high sensitivity, quickly and easily.

[Brief description of the drawings]

FIG. 1 is a calibration curve of Example 1. The vertical axis represents the change in absorbance (ΔAbs), and the horizontal axis represents the concentration of the standard ferritin solution.

FIG. 2 is a graph showing the correlation between the EIA method and the latex method. The vertical axis is the measured value by the EIA method, and the horizontal axis is the measured value by the latex method.

FIG. 3 is a calibration curve of Examples 2, 3 and Comparative Example 2. The vertical axis represents the change in absorbance (ΔAbs), and the horizontal axis represents the concentration of the standard ferritin solution.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hirofumi Morino 4-8-35-2-207 Nishikoji, Minoh-shi, Osaka (72) Inventor Tomoko Matsuo 2-1-9-305 Hata, Ikeda-shi, Osaka

Claims (2)

[Claims] When quantifying ferritin in a sample, the sample and an insoluble carrier sensitized with an anti-ferritin antibody are mixed in a solution to cause an agglutination reaction by an antigen-antibody reaction, and the agglutination reaction based on the agglutination reaction is performed. A method for quantifying ferritin, comprising measuring the amount of change in absorbance and comparing with a calibration curve, wherein the insoluble carrier is latex particles,
In the above solution, polyvinylpyrrolidone having an average molecular weight of 1,000 to 2,000,000, pullulan having an average molecular weight of 1,000 to 1,000,000, and one kind selected from the group consisting of polyethylene glycol having an average molecular weight of 1,000 to 2,000,000
5% by weight, and the agglutination reaction is carried out at a constant temperature,
It is performed for 5 seconds to 15 minutes, and the amount of change in absorbance is:
A method for quantifying ferritin, which is an increase in absorbance due to an agglutination reaction. 2. Latex particles sensitized with an anti-ferritin antibody, polyvinylpyrrolidone having an average molecular weight of 1,000 to 2,000,000, pullulan having an average molecular weight of 1,000 to 1,000,000,
2. The ferritin for carrying out the method for quantifying ferritin according to claim 1, comprising a sample diluent containing one selected from the group consisting of polyethylene glycol having an average molecular weight of 1,000 to 2,000,000. Immunological kit for measurement.