JPH03112496A - Labeling agent for quantitative analysis of substance - Google Patents

Abstract

PURPOSE:To eliminate problems during use of radioisotope of conventional labeling agent and enzyme and to determine a target substance accurately and in high sensitivity in analysis of drug or useful substances in organisms by using a labeling agent for determining a substance comprising polyadenylic acid. CONSTITUTION:Polyadenylic acid or a labeling material of polyadenylic acid is determined by the following method. Namely, a process wherein polyadenylic acid or the labeling material of polyadenylic acid is hydrolyzed to liberate ADP, a process wherein phosphoric acid is bonded to ADP and the ADP is converted into ATP and a process using organism luminous method utilizing luciferase derived from a firefly are simultaneously or successively carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a labeling agent for quantitative analysis of a substance made of polyadenylic acid, a method for quantitative analysis of a substance, and a method for quantifying polyadenylic acid or polyadenylic acid labeled substances.

[Conventional technology]

When attempting to quantify an analyte with high sensitivity in a sample that contains many contaminants with similar properties to the analyte to be measured, a substance that specifically binds to the analyte (e.g. Antibodies whose antigen is the target substance, nucleic acid fragments containing specific sequences in nucleic acids, etc.) or the target substance itself is labeled with a radioactive substance or enzyme, and the labeled substance does not participate in the amount or binding of the specific binder. There is a known method of measuring the concentration of an analyte in a sample by measuring the amount of radioactivity or enzyme activity.

[Problem to be solved by the invention]

However, when a substance to be analyzed is labeled with a radioactive substance, strict control is required for its handling, and even when labeling with an enzyme, the enzyme may be There are problems in that the activity may decrease and it is impossible to use the optimal enzyme for measurement.

[Means to solve the problem]

Therefore, the present inventors first developed a new method for accurately quantifying polyadenylic acid in a sample.

According to this quantitative method, polyadenylic acid can be used very suitably as a labeling agent for quantitative analysis of substances,
The above problems can be solved.

This knowledge led to the completion of the present invention.

That is, the present invention is first characterized by a labeling agent for quantitative analysis of a substance made of polyadenylic acid.

The present invention also includes a step of hydrolyzing polyadenylic acid or a polyadenylic acid label in a sample to liberate ADP;
Polyadenylic acid in a sample can be converted to polyadenylic acid in a sample by simultaneously or sequentially performing a step of binding phosphoric acid to the ADP and converting it into ATP, and a step of measuring the ATP by luminescence intensity using a bioluminescence method using firefly-derived luciferase. Alternatively, the present invention is characterized by a method for quantifying polyadenylic acid or a polyadenylic acid label, which is characterized in that the polyadenylic acid label is quantified.

The present invention will be explained in more detail below.

The substance to be quantified using the labeling agent of the present invention may be any substance, and there are no particular limitations on the analysis sample containing these substances to be quantified.

The labeling agent of the present invention may be used to directly label an analyte to quantify the analyte, but it is also possible to use the labeling agent to label a substance that can specifically bind directly or indirectly to the analyte. is labeled, this polyadenylic acid label is used to react with the analyte in the sample, and after removing the unreacted label, the amount of the polyadenylate label bound to the analyte is determined. Accordingly, the amount of the target substance in the sample can be calculated. In this case, the substances that can specifically and directly bind to the target substance are:
Examples include m-RNA, DNA probes, antibodies, etc. Furthermore, if biotin is used, avidin and antibodies can be divided into equal parts to indirectly bind specifically to any substance to be analyzed.

Moreover, the molecular weight of polyadenylic acid may be any molecular weight, and furthermore, the content of adenylic acid may be any rate.

Furthermore, the polyadenylic acid used for preparing the polyadenylic acid label is also the same as described above.

The labeling method may be any method, for example, Ayu et al., Collected Abstracts of the 109th Annual Meeting of the Pharmaceutical Society of Japan, etc., p. 164, 19
An example is the method described in 1989.

The quantitative analysis method of the present invention will be explained in more detail below.

First, a polyadenylic acid-labeled substance labeled with polyadenylic acid is hydrolyzed against the substance to be analyzed in the sample.
Release DP. This polyadenylic acid-labeled substance may be one in which the target substance to be analyzed is directly labeled, or a polyadenylic acid-labeled substance in which a substance that can specifically bind directly or indirectly to the target substance contained in the sample is labeled with polyadenylic acid. It may also be a polyadenylic acid labeled product that is reacted with an analyte in a sample. In the latter case, unreacted polyadenylic acid labels are removed.

(Hereinafter referred to as the first step.) Further, any method may be used for hydrolysis, and for example, enzymatic hydrolysis may be used.

The enzyme used may be any enzyme as long as it generates ADP from polyadenylic acid, such as polynucleotide phosphorylase derived from Escherichia coli.

As for the decomposition conditions, for example, the reaction pH is 5 to 1O1, preferably pH 6 to 9, and the reaction temperature is 10 to 70°C.
, preferably 25 to 40°C, and the reaction time is 5 minutes to 10 hours, preferably 30 minutes to 3 hours,
Further, as a buffer solution, phosphoric acid I (EPES) containing magnesium ions of 1 μH or more is used.

Examples include Tris buffer, and the amount of enzyme may be appropriately selected depending on the amount of polyadenylic acid or polyadenylic acid labeled substance in the sample.

Next, the ADP obtained in the above step is converted to ATP by binding phosphoric acid. (Hereinafter referred to as the second step.) Any method may be used to bind phosphoric acid to ADP and convert it into ATP. For example, by using phosphooninol pyruvate and pyruvate kinase, AT
One example is a method of converting to P.

The reaction conditions are, for example, pH 5-1O1, preferably pH 6-9, and reaction temperature 10-70°C.
1 Preferably, the temperature is 25 to 40°C, and the reaction time is 5
minutes to 10 hours, preferably 30 minutes to 3 hours, and buffers include phosphoric acid, 11EPES, l-lis, triethanolamine buffers, etc., and the amount of phosphooninol pyruvate and enzyme amount may be appropriately selected depending on the ADP content in the sample.

Next, using a bioluminescence method using firefly-derived luciferase, the luminescence intensity corresponding to the amount of ATP obtained as described above is measured, and, for example, using a calibration curve as described in Example 1 and Example 2 below, polyadenyl The amount of the analyte bound in the acid-labeled substance is calculated and quantified. (Hereinafter referred to as the third step.) As firefly-derived luciferase,
Any luciferase may be used as long as it is used for ATP measurement using the luciferin-luciferase system, and examples thereof include luciferases derived from Genji fireflies, Heike fireflies, American fireflies, and the like.

Luciferin is added to the ATP-containing sample produced as described above, firefly-derived luciferase is used, and the reaction conditions are such that the pH is 5 to 9, preferably pH 6 to 8.
and the reaction temperature is 10 to 40°C, preferably 2
0 to 30°C, reaction time is within 1 hour, preferably 1 hour.
Furthermore, as a buffer solution, glycylglycine containing magnesium ions of in+M or more, HEPE
The amount of ATP is measured using S, Tricin buffer, etc.

In addition, the amounts of luciferin and luciferase added are A
It may be selected appropriately depending on the content of TP.

On the other hand, in the present invention, the above steps are performed simultaneously, that is, the first and second steps are performed simultaneously and then the third step is performed, or the first step is performed and then the second and third steps are performed simultaneously. Alternatively, steps 1, 2 and 3 may be performed simultaneously.

For example, the reaction conditions for performing the first and second steps at the same time include, for example, a pH of 5 to 10, preferably a pH of 6 to 9, and a reaction temperature of 10 to 70°C, preferably 25 to 40°C. "C, and the reaction time is 5 minutes to 10
The time is preferably 30 minutes to 3 hours, and the buffer solution is phosphoric acid containing 1 μm or more of magnesium ion, HEPES.

Examples include Tris and triethanolamine buffers, and the amount of phosphoninol pyruvate and the amount of enzyme may be appropriately selected depending on the amount of polyadenylic acid in the sample.

Although the above explanation focused on the method for quantitative analysis of the target substance bound to the polyadenylic acid label, the above method can of course also be used when quantifying polyadenylic acid or polyadenylate label in a sample. Needless to say, it can be applied to

〔Effect of the invention〕

By using the labeling agent for substance quantitative analysis made of polyadenylic acid of the present invention, there is no need for strict control as in the case of using a radioisotope as a conventional labeling agent, and there is no need for labeling as in the case of using an enzyme. There is no problem that enzyme activity decreases during operation (it is possible to quantitatively analyze target substances accurately and with high sensitivity, and it greatly contributes to analytical tests for drugs, substances useful in living organisms, etc.) .

Example 1 (1) Preparation of reagents The following reagents (a) to (d) were prepared.

Reagent (a): Polyadenylic acid solution in redistilled water, polyadenylic acid (manufactured by Boehringer Mannheim, about 1500 ADP polymerized).
A polyadenylic acid solution was prepared by dissolving 1 ng-10 pg of each in 1 In.

Reagent (O): A mixed solution of polynucleotide phosphorylase and pyruvate kinase in 0, 1 M I-lith buffer (pl (8,0), polynucleotide phosphorylase (manufactured by Boehringer Mannheim), pyruvate Kynes (manufactured by Boehringer Mannheim), magnesium sulfate, phosphooninol pyruvate, and disodium hydrogen phosphate at 1 ol/d and 1 mol/d, respectively.
A mixed solution of polynucleotide phosphorylase and pyruvate kinase was prepared by dissolving them at concentrations of 00υ/d, IIIM, 30mM, and 10mM.

Reagent (c): D-
A luciferin solution was prepared by dissolving luciferin and magnesium sulfate to a concentration of 86 μH and 5.4 mM, respectively.

Try! (d): Luciferase solution 0.02 mg/firefly luciferase (manufactured by Sigma) in 50 mM IIEPBS buffer (pH 7.5)
A luciferase solution was prepared by dissolving it to a concentration of rttl.

(2) Measurement Using the above reagents (a) to (d), the relationship between the amount of polyadenylic acid and the luminescence intensity was investigated.

Reagent (a)lOu7! and 20u/! of reagent (II)! was added and reacted for 2 hours at a temperature of 36°C.
)400μ! and 1 μl of reagent (d) were added, and the resulting luminescence was integrated for 15 seconds using a luminescence detector (Alloka Luminescence Reader, manufactured by Aloka).

The correlation between the luminescence intensity thus obtained and polyadenylic acid is shown in FIG.

As is clear from Fig. 1, there is a linear correlation between the amount of polyadenylic acid 5 and the luminescence intensity, the detection range of polyadenylic acid is 10 pg to IQ ng/assay, and the accuracy after 5 repetitions ( Cv%) was good with an average of 5.7%.

Furthermore, a sample containing polyadenylic acid on the calibration ring (5
When X10' and 2X10'' pg/l0ml were measured in the same manner as above, luminescence intensities that closely matched the values of the calibration curve were obtained.

Example 2 (1) Preparation of reagents The following reagents (a) to (main) were prepared.

Reagent (a): TSH solution 50aM phosphate buffer (containing 0.1% bovine serum albumin and 0.9% sodium chloride, pH 7.0)
A TSH solution was prepared by dissolving the following to a concentration of 0 to 200 μU/I11.

Reagent (Il+): Biotin-labeled anti-TSH antibody solution 5
0mM phosphate buffer (0,1% bovine serum albumin and 0
．． A biotin-labeled anti-TSH antibody is dissolved in a solution (containing 9% sodium chloride, pH 7.0) to a concentration of about 15a+g/m.

Note that the biotin-labeled anti-TSH antibody was prepared as follows. 50 μg of anti-TSI 11 gG fraction (rabbit) dissolved in 100 uffi of 0.1 M sodium bicarbonate solution
[Prepared according to the method described in Immunochemistry, Medical Chemistry Experimental Methods Course, 4, Antibody Preparation and Purification Methods, pp. 47-76 (1972). ],] dimethylformamide solution of D-biotin-N-hydroxysuccinimide ester (2
rag/d), 10 ul was added and reacted at 25°C for 4 hours, followed by Sephadex G.
-25 column chromatography.

Reagent (c): Washing solution 0.9% (W/V) Saline solution Reagent (d): Avidin solution 50mM phosphate buffer (contains 0.1% bovine serum albumin and 0.9% sodium chloride, pH 7.0) Then, avidin solution was prepared by dissolving avidin to a concentration of 20 ug/ml.

Reagent (ne): Biotin-labeled polyadenylic acid solution 50wM
Phosphate buffer (0.1% bovine serum albumin and 0.9%
A biotin-labeled polyadenylic acid solution was prepared by dissolving biotin-labeled polyadenylic acid in a solution containing sodium chloride (pH 7.0) to a concentration of 2.5 μg/d.

In addition, biotin-labeled polyadenylic acid is available from Arakawa Co., Ltd.
Methods such as Cheap, Pha
rm.

Bull, 37 (7), No. 1831-1833
(1989)] with the following modifications. That is, poly(poly) A 2.5
Biotin-X-hydrazide (Biot
in-X-hydrazide) (manufactured by Calbiochem) Add 1.25 d of lOIIIg/Id aqueous solution and 1.5 d of geltaraldehyde 0.05% aqueous solution,
After reacting at 7°C for 10 minutes, it was purified by a conventional ethanol precipitation method.

(2) Measurement Using the above reagents (A) to (N), perform TS as follows.
A quantitative analysis test of H was conducted.

Add 50μ of reagent (A) to a microtiter well with anti-TSH antibody immobilized on the wall of the well! , and 50μ of reagent (II)! After adding and reacting overnight at a temperature of 4°C, it was washed three times with reagent (c), and then reagent (d) was added to lOOμ!
and reacted for 30 minutes at a temperature of 25°C.

After washing with reagent (c), 100 μl of reagent (main) was added and reacted at 25°C for 30 minutes.
After washing with c), the amount of polyadenylic acid bound to the wells was measured by the method described in Experimental Example 1.

The correlation between the luminescence intensity and the amount of TSH obtained from this is shown in FIG.

As is clear from this figure, there is a linear correlation between the amount of TSH and the luminescence intensity, and the calibration range of TSH is 3, 12 #
U/af~200 //U/1111! , the detection limit is
0.156μυ/assay, accuracy is 1O on average
It was good at 0.07%.

Furthermore, when samples containing TSH (200 and 3.125 μU/111) from the above calibration composition were measured in the same manner as above, luminescence intensities that closely matched the values of the calibration curve were obtained.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the correlation between luminescence intensity and polyadenylic acid, and FIG. 2 is a diagram showing the correlation between luminescence intensity and TSH amount. Fig. 1yA (Hube) Fig. TSHItU/ml

Claims (1)

[Claims] 1. A labeling agent for quantitative analysis of a substance composed of polyadenylic acid. 2. A step of hydrolyzing polyadenylic acid or a polyadenylic acid label in the sample to liberate ADP, a step of binding phosphoric acid to the ADP and converting it to ATP, and a bioluminescence method using firefly-derived luciferase, The ATP
1. A method for quantifying polyadenylic acid or a polyadenylic acid label, characterized in that polyadenylic acid or a polyadenylic acid label in a sample is quantified by simultaneously or sequentially performing steps of measuring the luminescence intensity.