JP6210568B2 - Highly accurate immunoassay - Google Patents

Description

  The present invention relates to an immunological measurement method for accurately measuring small molecules in a specimen.

Small molecules in a sample are bound specifically or non-specifically to high molecular weight substances such as proteins and lipids present in the sample, or are in a certain equilibrium state. In order to measure accurately, it was necessary to devise a device.
As one of such devices, there is a method of releasing a small molecule from the protein or the like using a specific binding inhibitor. For example, as a specific binding inhibitor of steroid hormone, a glutamic acid solution (Patent Document 1), 8-anilino-1-naphthalenesulfonic acid or a salt thereof (Patent Document 2), 2-hydroxy-3-naphthoic acid, 2-aminonaphthalenesulfonic acid, aniline-2,5-disulfonic acid, 3-amino-2,7 -Naphthalenedisulfonic acid (Patent Document 3) has been reported. Specific inhibitors of vitamin D metabolites include 8-anilino-1-naphthalenesulfonic acid ammonium salt, 3- (acetonylbenzyl) -4-hydroxycoumarin (Patent Document 4), cyclodextrin, and sodium salicylate. NaOH (Patent Document 5) has been reported.

JP-A-53-101521 Japanese Patent Publication No. 61-12547 Japanese Patent No. 5343825 Japanese Patent No. 3787121 Japanese Patent No. 4130958

  However, in the study of the present inventor, in the measurement of aldosterone, an attempt was made using 8-anilino-1-naphthalenesulfonic acid, which is a specific inhibitor, but good results were obtained as shown in the Examples. It is not done.

  Therefore, as a result of intensive studies to develop an immunoassay for accurately measuring small molecules in a specimen, the inventor has surprisingly found that a macromolecule that is an indirect labeling method. When measuring small molecules in a sample using a small molecule that binds a label that binds to a substance and a factor that specifically binds to a macromolecular substance, these are mixed in advance, and this mixture is combined with an anti-small molecule antibody. It was found that small molecules in a sample can be measured with high accuracy by measuring small molecules in the sample. The present invention is based on such novel findings. Accordingly, the present invention provides the following inventions.

(1)
An immunological measurement method for accurately measuring small molecules in a specimen, wherein a label bonded to a high molecular weight substance and a small molecule bound to a factor that specifically binds to the high molecular weight substance are mixed in advance, A small molecule immunoassay, which comprises measuring a small molecule in a specimen using the mixture and an anti-small molecule antibody.
(2)
The method according to (1) above, wherein the small molecule to be measured is a hormone or a vitamin.
(3)
The method according to (1) above, wherein the small molecule to be measured is a steroid hormone such as aldosterone, estradiol, testosterone, progesterone, cortisol, androstenedione.
(4)
The combination of high-molecular substances and factors that specifically bind to high-molecular substances is avidin-biotin, neutral avidin-biotin, streptavidin-biotin, IgG-protein A, IgG-protein G, IgG-protein L, histidine tag The method according to (1) above, which is any combination of sequence-nickel, sugar chain-lectin, or aptamer-aptamer recognition molecule.
(5)
The method according to (1) above, wherein an anti-small molecule antibody bound directly or indirectly to a carrier is used.
(6)
The method according to (1) above, wherein the label to be bound to the polymer substance is an enzyme such as peroxidase, alkaline phosphatase, β-galactosidase, luciferase.

Reagents used in the immunological measurement of the present invention are a label bound to a polymer substance, a small molecule bound to a factor that specifically binds to the polymer substance, and an anti-small molecule antibody bound to a solid phase. This method and the reagents used are exactly the same. However, the procedure for which the operation procedure has not been reported so far, that is, a label bonded to a high molecular weight substance and a small molecule bound to a factor that specifically binds to the high molecular weight substance are mixed in advance. Small molecules in a sample can be measured with high accuracy only by changing the procedure to measure small molecules in a sample using an anti-small molecule antibody.
As described above, since the small molecules in the sample that could not be measured with high accuracy can be measured with high accuracy by a simple means of changing the procedure, the method of the present invention is extremely useful in the industry.

FIG. 1 shows the measurement results and the effect of the inhibitor in the two-step method without premixing. FIG. 2 shows the measurement results by the one-step method using a reagent in which aldosterone is directly labeled. FIG. 3 shows the measurement results obtained by the method of the present invention using premixed reagents.

  As described above, the feature of the present invention is that, as described above, a label bound to a polymer substance and a small molecule to which a factor that specifically binds to the polymer substance is bound are mixed in advance, and this mixture is combined with an anti-small molecule antibody. The present invention relates to an immunoassay method for small molecules, characterized in that it is used to measure small molecules in a specimen.

The sample to be measured in the present invention is a blood sample such as blood, serum or plasma, or a urine sample.
Small molecules to be measured include hormones or vitamins, and hormones include steroid hormones such as aldosterone, estradiol, testosterone, progesterone, cortisol, androstenedione, ACTH, arginine pasopressin, PTH, insulin, oxytocin, etc. Examples include peptide hormones.
Examples of vitamins include vitamin D and vitamin B12 .

Examples of the label used for the measurement include enzymes, fluorescent dyes, chemical coloring dyes, chemiluminescent substrates, and the like. Specific examples of the enzyme include peroxidase, alkaline phosphatase, β-galactosidase, β- glucosidase, luciferase and the like.

  As a labeling method for such a label, an indirect labeling method is used. The indirect labeling method is already known, and specific combinations include avidin-biotin, neutral avidin-biotin, streptavidin-biotin, IgG-protein A, IgG-protein G, IgG-protein L, histidine tag sequence -Any combination of nickel, sugar chain-lectin, aptamer-aptamer recognition molecule may be used.

Next, the antibody used for the measurement may be an antibody against the small molecule to be measured, and it may be a monoclonal antibody or a polyclonal antibody (antiserum) or a fragment thereof ( (Fab ′) 2 , Fab, etc.). It doesn't matter.

Such an antibody is preferably used in which an antibody is directly or indirectly bound to a carrier for BF separation.
As the carrier, any form such as a tube shape, a well shape, and a particle shape (beads, latex particles, magnetic particles, etc.) can be used, and the material may be appropriately selected according to the measurement.
The binding method between the antibody and the carrier is known, and may be appropriately selected from conventional methods (physical binding method, chemical binding method, complete binding method using a linker, etc.). Furthermore, an indirect binding method using an anti-immunoglobulin antibody or the like can also be used.

In the present invention, using such a conventionally known reagent, (1) first, a label bound to a polymer substance and a small molecule bound to a factor that specifically binds to the polymer substance are mixed, 2) It consists of a two-step method of measuring small molecules in a specimen using this mixture and an anti-small molecule antibody.
The reaction of mixing the label bound to the first stage macromolecular substance and the small molecule bound to the factor that specifically binds to the macromolecular substance is 0 to 50 ° C., preferably 5 to 30 ° C. Well, it is not necessary to incubate for a certain period of time.

The measurement of small molecules in the sample using the mixture and anti-small molecule antibody in the second stage can be performed using the reaction conditions performed on the target small molecules as they are. Can be determined.
The small molecule can be quantified by a method usually used for the label used (for example, an absorbance method for an enzyme).

  Hereinafter, the present invention will be described in detail by showing examples of aldosterone, but it is clear that the present invention is not limited thereto.

Preparation of reagents (preparation of diluted solutions of each constituent reagent)
50 mM containing 1% bovine serum albumin (Gibco), 0.2% skim milk (Becton Dickinson), 0.02% Tween 20 (Nacalai Tesque), 0.1% ProClin 950 (Sigma) Sodium phosphate buffer (pH 6.5) was used.

Preparation of biotinylated aldosterone
Aldosterone (manufactured by Sigma) and Biotin- (AC5) 2-hydrazide (manufactured by Dojindo) were stirred overnight at room temperature in a mixed solvent of 0.5 M acetic acid aqueous solution and ethanol. The progress of the reaction was followed by thin layer chromatography. After confirming the disappearance of the raw material aldosterone, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain biotinylated aldosterone.

[Preparation of horseradish peroxidase directly labeled with aldosterone]
Aldosterone (manufactured by Sigma) and carboxymethoxylamine hemihydrochloride (manufactured by Nacalai Tesque) were stirred in ethanol in the presence of anhydrous sodium acetate for 4 days. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography to obtain a derivative having a carboxyl group introduced at the 3-position of aldosterone. This 3-position derivative, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojindo), N-hydroxysuccinimide (manufactured by Dojindo), and horseradish peroxidase (manufactured by Toyobo) ) In 0.1M 2-morpholinoethanesulfonic acid (MES) -NaOH buffer (pH 5.5) and allowed to react overnight at 4 ° C. The reaction solution was dialyzed against PBS to obtain horseradish peroxidase directly labeled with aldosterone.

[Streptavidin-horseradish peroxidase conjugate]
Streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 0.1 M sodium phosphate buffer (pH 8.0) containing 5 mM ethylenediaminetetraacetic acid (EDTA), and 2-iminothiolane hydrochloride is added thereto, and 30 The reaction was carried out at 2 ° C. for 2 hours. Further, an aqueous solution of tri (carboxyethyl) phosphine hydrochloride was added, and the mixture was reacted at 37 ° C. for 1 hour, and then the buffer solution was changed to 5 mM ethylenediaminetetraacetic acid (EDTA) with an ultrafiltration device Amicon (manufactured by Merck Millipore). The reagent residue was removed at the same time as replacement with 0.1 M sodium phosphate buffer (pH 6.0), and streptavidin into which a mercapto (SH) group was introduced was obtained. On the other hand, horseradish peroxidase (manufactured by Toyobo Co., Ltd.) was dissolved in 0.1 M sodium phosphate buffer (pH 7.5), and N- (6-maleimidocaproyloxy) dissolved in N, N-dimethylformamide was dissolved therein. ) After succinimide (EMCS) was added and reacted at 30 ° C. for 30 minutes, the buffer solution was 0.1 M containing 5 mM ethylenediaminetetraacetic acid (EDTA) using an ultrafiltration device Amicon (manufactured by Merck Millipore). The reagent debris was removed simultaneously with the replacement with sodium phosphate buffer (pH 6.0) to obtain horseradish peroxidase into which a maleimide group was introduced. The mercapto group-introduced streptavidin and maleimide group-introduced horseradish peroxidase prepared as described above were mixed at a molar ratio of 1: 5 and reacted at 4 ° C. for 24 hours. The reaction solution was purified by gel filtration column chromatography (Sephacryl S200HR, manufactured by GE Healthcare) to obtain a streptavidin-horseradish peroxidase conjugate.

[Preparation of aldosterone standard solution]
1.2 mL of aldosterone (manufactured by Sigma) was dissolved in 1 mL of ethanol (manufactured by Nacalai Tesque) to prepare an ethanol solution of aldosterone. This was further appropriately diluted with ethanol, and the absorbance at 240 nm was measured. The exact concentration was calculated using the molecular weight of aldosterone of 360.44 and a molar extinction coefficient of 15,000 at 240 nm. This ethanol solution was diluted with the above-mentioned diluent to prepare a 2,000 pg / mL solution. Furthermore, a 2,000 pg / mL aldosterone solution was appropriately diluted to prepare 1,000, 500, 250, 100, 50, and 25 pg / mL aldosterone solutions. These seven types of aldosterone solutions and a total of eight types of solutions in which a diluted solution not containing aldosterone was 0 pg / mL were used as aldosterone standard solutions.

[Preparation of antibody solid phase plate]
To a 96-well microplate (manufactured by Thermo Scientific), 100 μL / well of goat anti-rabbit IgG Fc polyclonal antibody (manufactured by Thermo Scientific) adjusted to a concentration of 5 μg / mL with PBS was dispensed at room temperature. Left overnight. After removing the liquid in the well by suction, the operation of dispensing and aspirating 300 μL / well of PBS containing 0.05% Tween 20 was performed three times in total to wash the well. Thereafter, PBS containing 0.5% skim milk, 5% sucrose, and 0.1% ProClin 300 was dispensed at 300 μL / well, and blocking was performed for 2 hours. Finally, the blocking solution was extracted and stored at 4 ° C. until use.

[Preparation of control sample]
EDTA-2Na pooled plasma with aldosterone added to about 800, 400, 200, 100 pg / mL was used as a control sample, dispensed, and stored frozen at −80 ° C. until use.

Measurement in the two-step method without pre-mixing (conventional method) and the effect of the inhibitor (1) Whether the target aldosterone and biotinylated aldosterone bind to proteins in plasma and the measurement system is affected, and specific binding inhibition These molecules are released from the proteins in the plasma using an agent, and it is confirmed whether measurement is possible. This is evaluated by a so-called addition recovery test for determining whether or not the amount of aldosterone added can be recovered from these measured values using plasma added with aldosterone.

(2) Method The personal plasma samples to which standard solution is added are aldosterone standard solution 1 (0 pg / mL), 6 (500 pg / mL), 7 (1,000 pg / mL), and 8 (2,000 pg / mL). Plasma specimens were prepared by mixing at a 1: 9 ratio.
Rabbit anti-aldosterone polyclonal prepared by diluting 100 μL / well of biotinylated aldosterone prepared by diluting aldosterone standard solution or standard solution added to antibody solid phase plate to 25 μL / well, 10 fmol / mL, 40,000 times An antibody (Applied Biological Materials) was added in an amount of 50 μL / well sequentially, and reacted at room temperature for 2 hours while stirring on a plate shaker. At this time, a biotinylated aldosterone solution containing 1 mg / mL 8-anilino-1-naphthalenesulfonic acid (hereinafter referred to as ANS) and not containing it was prepared. After the reaction solution in the well was removed by suction, an operation of dispensing and aspirating 300 μL / well of PBS containing 0.05% Tween 20 was performed three times in total to wash the well. After washing the wells, 100 μL / well of streptavidin-horseradish peroxidase conjugate diluted to 0.5 μg / mL was dispensed into all wells and allowed to react for 30 minutes. After the reaction solution in the well was removed by suction, an operation of dispensing and aspirating 300 μL / well of PBS containing 0.05% Tween 20 was performed three times in total to wash the well. A coloring solution containing 3,3,5,5-tetramethylbenzidine is dispensed at 100 μL / well and allowed to develop color at room temperature for 20 minutes, and then 0.5 N sulfuric acid is dispensed at 100 μL / well to stop the coloring reaction. The absorbance at 450 nm of each well was measured with a plate reader (manufactured by Tecan).

(3) Results The results are shown in the following Tables 1 and 2 and FIG.

Discussion First, when the absorbance in the measurement of the standard solution is observed, sufficient absorbance is seen in the standard solution 1 regardless of the presence or absence of ANS. Therefore, solid phase antibody (goat anti-rabbit IgG Fc polyclonal antibody): rabbit anti-aldosterone It can be seen that an immune complex composed of three components of polyclonal antibody: biotinylated aldosterone is appropriately formed. In addition, since the absorbance decreases as the aldosterone concentration increases, it is considered that the formation of immune complexes is inhibited by aldosterone added in the system. This is the antigen measurement itself in the competitive method, and means that aldosterone can be measured in the composition of the standard solution (Table 1 and FIG. 1).

However, when looking at the absorbance of the individual plasma sample to which the standard solution was added, although the absorbance decreased with the increase in the amount of aldosterone added, the absorbance was significantly lower than that of the standard solution. Since the absorbance in the measurement of standard solution 8 (2,000 pg / mL) is about 0.5, all of the plasma samples measured this time have a measured value of 2,000 pg / mL or more, (Table 2).

In addition, since the absorbance in plasma samples is low regardless of the presence or absence of ANS in the reaction system, ANS does not contribute to the inhibition of binding between aldosterone or biotinylated aldosterone and plasma proteins that was initially expected. it is conceivable that. Since the absorbance in the plasma sample is remarkably reduced, it is assumed that biotinylated aldosterone binds to the protein in plasma and cannot react with the rabbit anti-aldosterone polyclonal antibody added in the system (Table 2). .

3) Purpose of measurement by a one-step method using a reagent directly labeled with aldosterone If a very low molecular weight substance such as biotinylated aldosterone binds to a protein in plasma and the rabbit anti-aldosterone antibody does not react, a tracer molecule is used. Modification to a higher molecular weight should improve the reactivity with the antibody. Therefore, it was investigated whether measurement was possible under the condition that aldosterone was directly chemically bound to horseradish peroxidase, a labeling enzyme, as a tracer.

Method Rabbit anti-aldosterone polyclonal antibody (Applied) prepared by diluting aldosterone standard solution or control sample at 25 μL / well, 1 μmol / mL diluted horseradish peroxidase-labeled aldosterone at 100 μL / well, 40,000 times on an antibody solid phase plate. Biological Materials (manufactured by Biological Materials) was added in an order of 50 μL / well, and the mixture was allowed to react at room temperature for 2 hours with stirring on a plate shaker. After the reaction solution in the well was removed by suction, an operation of dispensing and aspirating 300 μL / well of PBS containing 0.05% Tween 20 was performed three times in total to wash the well. After washing the wells, a coloring solution containing 3,3,5,5-tetramethylbenzidine was dispensed at 100 μL / well, and the color was developed at room temperature for 20 minutes, and then 0.5 N sulfuric acid was dispensed at 100 μL / well. The color reaction was stopped, and the absorbance at 450 nm of each well was measured with a plate reader (manufactured by Tecan).

Results The results are shown in Tables 3 and 4 below and FIG.

(4) Consideration Since the absorbance in the measurement of the standard solution 1 is about 2, an immune complex comprising three components of solid phase antibody (goat anti-rabbit IgG Fc polyclonal antibody): rabbit anti-aldosterone polyclonal antibody: horseradish peroxidase labeled aldosterone It can be seen that is properly formed. However, it can be seen that the tendency of the absorbance to decrease with increasing free aldosterone concentration is quite small, and competitive inhibition by aldosterone contained in the standard solution hardly occurs. That is, although the same antibody as the above-mentioned measurement is used at the same dilution factor, the sensitivity is reduced (Table 3, FIG. 2). Further, in the measurement of the control sample, although the measurement values are calculated for the control samples 1 and 2, there is a considerable difference from the assumed value. For the control samples 3 and 4, since the absorbance value obtained is larger than that of the standard solution 1, no measured value is calculated (Table 4). This is because the reactivity of the rabbit anti-aldosterone polyclonal antibody used was significantly different from the reactivity to horseradish peroxidase-labeled aldosterone and the reactivity to aldosterone. Because of its high value, competitive inhibition by aldosterone is less likely to occur, and it is presumed that the tendency for the absorbance to decrease with increasing aldosterone concentration is reduced. In general, in the production of antibodies against low molecular weight substances such as aldosterone, the low immunogenicity of the antibody itself makes it possible to chemically bind low molecules to proteins such as bovine serum albumin or bovine thyroglobulin. It is obtained by immunizing animals with That is, the reactivity of an antibody tends to be higher in an antigenic substance in a form bound to a polymer than in a small molecule itself. In addition, a relatively large amount of horseradish peroxidase-labeled aldosterone was added to the system in order to obtain an appropriate absorbance. Therefore, free aldosterone inhibited the binding of horseradish peroxidase-labeled aldosterone and rabbit anti-aldosterone polyclonal antibody. It is also expected that this will be difficult to occur.

In addition, the reason why the measured value of the control sample was considerably lower than the expected value was that the protein in plasma and aldosterone partially bound and reacted with the rabbit anti-aldosterone polyclonal antibody added in the system. It is presumed to be impossible.

Purpose of measurement using the pre-mixed reagent by the method of the present invention The tracer molecule is prepared using the binding of streptavidin-biotin for the binding of aldosterone and labeling enzyme, and the standard solution and plasma sample can be measured for the system using this tracer Find out. As in the previous item 2), the plasma sample is obtained by adding aldosterone to the plasma, and these measured values are evaluated by a so-called addition recovery test for determining whether the amount of the added aldosterone can be recovered.

Individual plasma samples to which method standard solution is added are aldosterone standard solution 1 (0 pg / mL), 6 (500 pg / mL), 7 (1,000 pg / mL), and 8 (2,000 pg / mL). Prepared by mixing in a 1: 9 ratio.
25 μL / well of an aldosterone standard solution or a standard solution added to an antibody solid phase plate, a pre-mixed tracer solution (0.3 pmol / mL biotinylated aldosterone and 0.5 μg / mL streptavidin) -Horseradish peroxidase conjugate) (100 μL / well), rabbit anti-aldosterone polyclonal antibody diluted by 40,000 times (Applied Biological Materials) was added in an order of 50 μL / well, and the mixture was stirred on a plate shaker for 2 hours at room temperature. It was made to react with. After the reaction solution in the well was removed by suction, an operation of dispensing and aspirating 300 μL / well of PBS containing 0.05% Tween 20 was performed three times in total to wash the well. After washing the wells, a coloring solution containing 3,3,5,5-tetramethylbenzidine was dispensed at 100 μL / well, and the color was developed at room temperature for 20 minutes, and then 0.5 N sulfuric acid was dispensed at 100 μL / well. The color reaction was stopped, and the absorbance at 450 nm of each well was measured with a plate reader (manufactured by Tecan).

Results The results are shown in Tables 5 and 6 below and FIG.

Discussion From the absorbance value in the measurement of the standard solution, an immune complex comprising 4 components of solid phase antibody (goat anti-rabbit IgG Fc polyclonal antibody): rabbit anti-aldosterone polyclonal antibody: biotinylated aldosterone: streptavidin-horseradish peroxidase conjugate Is formed appropriately, and further, competitive inhibition of the immune response by free aldosterone occurs (Table 5, FIG. 3). Further, when the absorbance measurement value of the plasma sample is observed, the abnormal low value or high value tendency of the absorbance is not observed as in the past, and the absorbance decrease with the addition of aldosterone is also observed in the plasma sample. A standard curve is prepared from the absorbance in the standard solution, the measurement value of the plasma sample to which the standard solution is added is calculated, and the recovery rate for the added aldosterone amount is calculated. It shows that aldosterone added to plasma can be measured in the same manner as the standard solution (FIG. 6).

Claims (2)

A immunoassay for accurately measuring the aldosterone in the sample, mixing avidin, a label bound to a polymer material selected from neutral avidin and streptavidin, and aldosterone biotin is bonded in advance and, immunoassay of aldosterone, characterized in that to measure aldosterone in a sample using as those this mixture, an anti-aldosterone antibody engaged indirectly binding to the carrier. Label, peroxidase, alkaline phosphatase is bound to the polymeric material, beta-galactosidase, luciferase is an enzyme, such as, The method of claim 1, wherein.

Images