JPH06109733A - Immunological detecting method using electron carier as labeling material - Google Patents

Abstract

PURPOSE:To perform immunological detection with a stable labeling material without requiring a special device by labeling a target material or a material peculiarly bound to it with an electron carrier other than enzyme, and detecting the signal derived from the labeling material. CONSTITUTION:A target material or a material peculiarly bound to it is labeled by an electron carrier other than enzyme, the signal derived from a labeling material is detected, and the target material in a specimen is detected. The specimen is a biological sample having the possibility of containing the target material, e.g. a biological liquid such as blood and blood serum, cells, and a tissue extract. The target material is an inspection subject material contained in the specimen, practically protein and enzyme, e.g. an antigen, an antibody, and a receptor. The electron carrier is reduced when accepting electrons from an electron donor, then it is oxidized when delivering electrons to an electron acceptor. Any material other than enzyme can be used for the labeling material, and a polycyclic compound containing azine or oxazine is preferably used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically detecting a target substance in a test sample using an electron carrier other than an enzyme as a labeling substance. Further, it also relates to a substance obtained by labeling the electron carrier.

[0002]

2. Description of the Related Art It has been a long time since an immunological measuring method has been used for measuring a target substance in a test sample, especially a trace amount component in a biological sample. The content of most of these components in the test sample is generally on the order of μg / ml or less, and for example, a complex produced by an antigen-antibody reaction can be directly analyzed by an immunodiffusion method or laser nephelometry. Since there is a limit to the detection sensitivity of the means for measurement, a method of labeling either the antigen or the antibody with some substance and detecting the signal based on it, a so-called labeled immunoassay method is often used. As the labeling substance, radioactive isotopes ( ¹²⁵ I, ³² P, etc.), fluorescent substances (fluorescein isothiocyanate, etc.), enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), metals (gold colloids, etc.), or luminescent substances (Acridinium ester, luminol, etc.) are used.

[0003]

However, among these labeling substances, the radioisotope is not only problematic in terms of stability, but also its use is limited in terms of equipment.
Also. The stability of enzyme labels and metal labels was also problematic. Therefore, an object of the present invention is to provide a labeling substance that is stable and has no facility restrictions, and to provide an immunological detection method using the labeling substance.

[0004]

According to the present invention, in the method for immunologically detecting a target substance in a test sample, either the target substance or a substance that specifically binds to the target substance is used. This can be achieved by an immunological detection method characterized by detecting a target substance in a test sample by labeling with an electron carrier other than an enzyme and detecting a signal derived from the labeling substance. Further, the present invention is a label characterized in that an electron carrier other than an enzyme is bound to a thiol group or an artificially introduced thiol group of a target substance or a substance that specifically binds to the target substance in a test sample. It also relates to chemical substances.

In the present specification, the test sample is a sample, especially a biological sample, which may contain a target substance, and is, for example, a biological fluid such as blood, serum, plasma, urine, saliva, spinal fluid or cells. And tissue extract and the like. The target substance is a substance to be tested contained in the above-mentioned test sample and refers to any physiologically active substance that can be detected by an immunological reaction. Specifically, proteins, enzymes, sugars, lipids,
Examples of the nucleic acid include various antigens, antibodies, receptors and the like. Furthermore, fibrinogen, albumin, C-reactive protein, anti-streptolysin O, rheumatoid factor, alpha-fetoprotein (AFP),
Treponema pallidum antibody, HBs antibody, HBc antibody, HB
e antibody, an antibody against HTLV-I, an antibody against HIV and the like can also be mentioned. Further, haptens which are low molecular weight compounds, for example, various drugs such as hormones and antiepileptic drugs, and antibodies against the haptens can be mentioned. Therefore, the substance that specifically binds to the target substance means an immunological partner for the target substance, such as an antibody whose antigen is various physiologically active substances or viruses, or an antigen for various antibodies. .

The electron carrier used as a label in the present invention means a substance capable of receiving an electron from an electron donor to be reduced, and then transferring the electron to the electron acceptor to be oxidized. In other words, it means a substance having a catalytic function for the redox reaction. In a broad sense, an enzyme is also included in this concept, but in the present invention, an electron carrier other than an enzyme is used as a labeling substance in immunological detection. The substance that can be used as the labeling substance may be any substance other than the enzyme having the above-mentioned action, and a polycyclic compound containing azine or oxazine is preferable.

Specifically, for example, the general formula (1)

[Chemical 1] The phenazinium compound represented by is preferable. Especially 1
-Methoxy-5-methylphenazinium methylsulfate can be preferably used.

Further, the general formula (2)

[Chemical 2] A phenazoxonium compound represented by is also preferable.

In particular, equation (3)

[Chemical 3] 9-dimethylaminobenzo-α-phenazoxonium chloride (trade name: Meldola blue) represented by can be preferably used.

The above-mentioned electron carrier labeling substance is used as an inspection target.
Either the target substance or the substance that specifically binds to the target substance can be labeled and used. For example,
When the target substance is an antigen that may be contained in the serum sample, the electron carrier labeling substance is bound to either the same antigen or an antibody against the antigen.

To prepare the labeled product of the electron carrier, the functional group originally possessed by the labeled product can be used for direct bonding, but preferably, the labeled product has two or more functional groups. The electron carrier can be bound using a linker agent. Many means are known in the art for linking enzymes to antigens or antibodies. For example, the glutaraldehyde method utilizing the amino group of the enzyme and the amino group of the antibody,
Periodate method utilizing the sugar residue of the enzyme and amino group of the antibody, maleimide method utilizing the amino group of the enzyme and thiol group of the antibody, maleimide method utilizing the thiol group of enzyme and the thiol group of antibody , There is a pyridyl disulfide method that utilizes the amino group of the enzyme and the thiol group of the antibody,
Various reagents have also been developed. However, it has not hitherto been known that an electron carrier other than an enzyme, which is the labeling substance of the present invention, is bound to a physiologically active substance as a label, and quinones simply bind to a thiol group to form an SS bond or a thiol derivative. Of the formation of [Chem.Pharm.Bull., 16
(12), 2334, 1968].

The present inventors have found that the number of electron carriers (labeling substances of the present invention) that can be bound per molecule of a target substance or a substance that specifically binds to the target substance is It varies considerably in the range of 0.01 to 100 depending on the properties of the chemical substance and the properties of the electron carrier. Furthermore, the electron carrier of the present invention and reduced glutathione and antibodies,
It was also observed that the reaction occurred stoichiometrically with oxygen with the thiol (-SH) group of the cysteine moiety present in proteins such as albumin. Therefore, in the present invention, it is convenient to use a thiol group for binding the labeling substance. When the functional group used for binding is a thiol group, there is a limit to the number of functional groups that can be used within the range that does not impair the physiological activity of the substance to be labeled. It is preferably introduced. For example, when a peptide or protein is used as the substance to be labeled, S-acetylmercaptosuccinic anhydride, N-succinimidyl-3-
(2′-pyridyldithio) propionate, methyl-3
A thiol group can be introduced by a linker agent such as-(4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrimidate, or iminothiolene. Particularly preferably, 2-iminothiolene hydrochloric acid is used to introduce a thiol group by reacting with a primary amine such as an amino terminal or lysine under weakly acidic to weakly alkaline condition, and excess 2-iminothiolene hydrochloric acid is removed by desalting. Then, the target electron carrier labeling substance can be obtained by mixing with the electron carrier which is the labeling substance of the present invention.

The labeled substance of the present invention prepared by binding an electron carrier other than an enzyme to a target substance or a substance that specifically binds to the target substance as described above can be used in a conventionally known labeled immunodetection method. . The reaction system can be applied to either a homogeneous system which does not require B / F separation or a heterogeneous system which requires B / F separation. Further, the reaction atmosphere can be carried out not only in the liquid but also in a so-called dry state in which the immune reaction is carried out on the filter paper (test piece) or the like.
Specifically, a test sample containing a target substance is added to a polystyrene solid phase on which an antibody against the target substance is preliminarily fixed as a primary antibody, and the mixture is reacted, followed by washing to perform B / F separation.
The target substance bound to the primary antibody and immobilized in solid phase is reacted with an electron carrier-labeled antibody prepared by previously binding the labeling substance to an antibody against the target substance as a secondary antibody. It is possible to quantify the target substance by measuring the amount of electron carrier immobilized after washing to remove unreacted substances. B / F by reacting with the target substance
The target substance can be similarly quantified by continuously measuring the amount of the electron carrier in the reaction system without performing the separation. On the other hand, in the case of simple measurement with a test piece such as a urinalysis, the electron carrier-labeled antibody and components necessary for signal measurement from the labeling substance are previously dried and held in separate compartments of the test piece. The target substance can be easily measured by arranging them so that they are mixed when the sample is applied. Further, if necessary, the measurement can also be carried out by keeping the labeled substance in which the electron carrier is bound to the target substance or its analog and the antibody against the target substance similarly in a dry state.

After the immune reaction is completed, the target substance in the test sample can be qualitatively or quantitatively measured by detecting the signal derived from the labeling substance. That is, since the labeling substance of the present invention is an electron carrier other than an enzyme (a substance that catalyzes a redox reaction), an appropriate electron donor and electron acceptor are required. An electron donor refers to an atom, an ion, or a molecule that donates an electron (hydrogen) to another molecule or ion.
Specifically, aromatic hydrocarbons such as benzene or naphthalene and various substitution products thereof, for example, phenols, anilines, benzoic acid, etc., reducing agents such as Fe ⁺⁺ and metals, and basic substances such as amines. , Succinic acid and reduced nicotinamide adenine dinucleotide (NADH)
It can be appropriately selected and used from so-called nucleophilic reagents such as coenzymes such as and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The electron acceptor refers to an atom, an ion, or a molecule that receives an electron (hydrogen) from an electron donor. The electron acceptor receives an electron from the electron donor, oxidizes the electron donor, and is itself reduced.
Specifically, dye-based compounds such as quinolines, thiazines, azoles, and acridines, various synthetic dyes such as methylene blue and tetrazolium salts, or oxygen, fluorescent substances or luminescent substances, so-called electrophilic reagents It can be appropriately selected and used from among them. The addition concentration of these electron donor and electron acceptor can be appropriately selected and adjusted by those skilled in the art depending on the reaction form and the concentration of the target substance. As an example, NAD (P) H can be used as an electron donor in combination with a tetrazolium salt as an electron acceptor. Alternatively, it can be measured using electrodes.

For example, when α-fetoprotein in serum is measured by the method of the present invention, 1 is used as a labeling substance.
50 μl of a reagent prepared by binding -methoxy-5-methylphenazinium methylsulfate to an anti-α-fetoprotein specific antibody and adjusting the concentration of the labeling substance to about 10 μM
Was mixed with 50 μl of serum to carry out an immunoreaction, and then 5 mM NADH was used as an electron donor and 3,3 ′-[3,3′-dimethoxy- (1,1′-biphenyl) -4, as an electron acceptor. Addition of 500 μl of a reagent containing 0.3 mM of 4′-diyl] -bis [2- (p-nitrophenyl) -5-phenyl-2H tetrazolium chloride (NBT), and detecting the produced coloring substance by a colorimetric method. Can be used to quantitatively measure α-fetoprotein. When albumin in serum is measured by the method of the present invention in a homogeneous system, 9-dimethylaminobenzo-α-phenazoxonium chloride is bound to albumin as a labeling substance, and the concentration of the labeling substance is adjusted to about 20 μM. 50 μl of the adjusted reagent is mixed with 50 μl of serum and 50 μl of anti-albumin antibody to carry out an immune reaction, and then albumin can be quantitatively measured by the same operation as described above.

[0016]

In the present invention, an electron carrier is attached as a label to either one of the target substance and the substance that specifically binds to it, for example, one of the pair of immunological partners. Therefore, when the present invention is applied to a conventionally known labeled immunodetection method, as a result of an immune reaction, the electron carrier immobilized via a target substance uniquely targets the immobilized amount. Since it can correspond to the amount of substance, it can be detected in the presence of an appropriate electron donor and electron acceptor. On the other hand, in the case of a homogeneous system that does not require B / F separation, a complex is formed by the immune reaction between the pair of immunological partners, and at the same time, the electron carrier is contained in the complex. The present inventor has found that, unlike the case of the above-described solid phase immobilization, the activity of the electron carrier in the free solution is always lower than that before the immune reaction. That is, a difference occurs in the activity of the electron carrier before and after the immune reaction. Moreover, this decrease in activity is proportional to the amount of electron carrier contained in the immune complex. Therefore, in the assay system of the present invention, it is not necessary to separate the immune reaction complex and the unreacted substance after the immune reaction, and detection can be performed in a homogeneous system. The reason for this is unknown at present, but for example, inhibition of binding (approx.) To a macromolecule having an activity-inhibiting action due to steric hindrance caused by formation of immune complex (Japanese Patent Publication No. 63-1544). Gazette) and inhibition of the binding between the labeled substrate and the enzyme (RC Boguslaski
Others, Immunoassays Clinical Laboratory Techniques fo
r the 1980s, ed.RM Nakamura et al., p. 45, 1980, Alan R. Li
(ss, Inc., New York), because it uses a small molecule electron donor and electron acceptor, and that the label is attached to one of the immunological partners, It can be distinguished.

[0017]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Preparation of labeled albumin 7.2 mg of human-derived albumin was added to 5 mM containing 1 mM EDTA.
It was dissolved in 1.0 ml of 0 mM phosphate buffer (pH 7.0). For this solution, add 0.5M triethanol chloride buffer (pH
50 μl of a 120 mM 2-iminothiolene hydrochloride solution dissolved in 8.0) was added, and the mixture was reacted at 25 ° C. for 30 minutes. Sephadex G-25, which had been equilibrated with 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, was used as the reaction solution.
It was injected into a column (diameter 1.5 cm × height 20 cm) to collect a polymer fraction. A part of this fraction was taken and subjected to the DTNB method (Clin. Chim. Acta, 130, 257-2.
61, 1983), the number of SH groups introduced was measured, and it was confirmed that 7.5 SH groups were introduced per molecule of albumin. Then, this fraction (SH group concentration 0.11
200 mM aqueous solution of 10 mM 1-methoxy-5-methylphenazinium methylsulfate (m-PMS)
μl was added and left overnight at room temperature. The obtained solution was injected into a Sephadex G-25 column equilibrated in the same manner as above, and a polymer fraction was collected.

Example 2: Measurement of albumin by competitive reaction
Constant (1) labeled albumin was prepared by the antigen-antibody reaction in Example 1 was diluted 100-fold with 50mM phosphate buffer containing 1 mM EDTA (pH 7.0), it was the dilution with labeled albumin solution. As the antibody, anti-human albumin IgG fraction (Yatron),
10 mM phosphate buffer containing 0.15 M NaCl (PB
S) (pH 7.0) was diluted 128 times, and this diluted solution was used as an antibody solution. The sample is 4.5% bovine albumin PB.
The S solution was diluted to various concentrations before use. 50 μl of labeled albumin solution, 50 μl of sample, 50 μl of 6% polyethylene glycol dissolved in PBS, and 50 μl of antibody solution
Were mixed and heated to 37 ° C. for 8 minutes. (2) Detection method 0.37% Triton X-100 and 0.37 mM 3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-diyl] -bis [ 2- (p-nitrophenyl) -5-phenyl-2H tetrazolium chloride] (NBT) and 0.1M acetate buffer (pH
5.0) was used as solution (A), 25 mM NADH aqueous solution was solution (B), and 0.1N HCl was solution (C). 8 minutes after the start of the above antigen-antibody reaction, (A)
Solution: (B) Solution mixed at a ratio of 4: 1 0.5 ml
Was added and warmed to 37 ° C. for 30 minutes. After adding 1 ml of (C) liquid to this mixture to stop the reaction, the main wavelength of 60
Colorimetric determination was carried out at 0 nm and subwavelength 750 nm. (3) Results As shown in FIG. 1, a good standard curve was obtained from 400 ng / ml.

Example 3: Preparation of labeled antibody 12.4 mg of F (ab ') ₂ fraction of anti-human fibrinogen-specific antibody prepared by digestion with pepsin was dissolved in 1.9 ml of 50 mM phosphate buffer (pH 7.8). 0.5M to the solution
95 μl of a 60 mM 2-iminothiolene hydrochloride solution dissolved in triethanolamine-hydrochloric acid buffer (pH 8.0) was added, and the mixture was heated at 25 ° C. for 110 minutes. The obtained reaction solution is
50 mM phosphate buffer solution (pH 7.
It was injected into a Sephadex G-25 (diameter 1.5 cm × height 20 cm) column that had been equilibrated in 0). The polymer fraction was pooled (2 ml) and the number of SH groups was measured by the DTNB method. As a result, it was confirmed that 13 SH groups were introduced per F (ab ′) ₂ molecule. This SH group concentration 0.11 mM
10 mM Meldola Blue (MB) aqueous solution for each fraction
4 μl was added and left overnight at room temperature. The precipitate was removed by centrifugation (3000 rpm, 10 minutes), and the whole amount of the obtained supernatant was equilibrated with Sephadex G-.
It was injected into a 25-column, and the polymer fraction was collected.

Example 4: Fibrino by non-competitive reaction
Measurement of Gen (1) Antigen-antibody reaction The labeled antibody prepared in Example 3 was added to 5 mM containing 1 mM EDTA.
It was diluted 5-fold with 0 mM phosphate buffer (pH 7.0) and used as a labeled antibody solution. As a sample, human fibrinogen was diluted to various concentrations with a PBS solution containing 4.5% bovine albumin and used. Labeled antibody 50μl, sample 50μ
1 and 50 μl of 6% polyethylene glycol dissolved in PBS were mixed and heated to 37 ° C. for 8 minutes. (2) Detection method Instead of the NADH-containing (B) solution used in Example 2 (2), a (B) solution containing NADPH was used.
As the solution (A) and the solution (C), the same solutions as in Example 2 (2) above were used. 8 minutes after the start of the antigen-antibody reaction, (A)
Solution: (B) Solution mixed at a ratio of 4: 1 0.5 ml
Was added and warmed to 37 ° C. for 30 minutes. Then, the reaction was stopped by adding 1 ml of (C) liquid, and colorimetric measurement was carried out at a wavelength of 560 nm. (3) Results As shown in FIG. 2, human fibrinogen 100 ng / ml
Gave a good standard curve.

Example 5: Measurement of antibody by non-competitive reaction (1) Preparation of labeled human IgG F (ab ') ₂ F of human IgG previously digested with pepsin in the same manner as in the preparation of labeled antibody of Example 3. (Ab ') ₂ fraction was used. However, the reaction with 2-iminothiolene hydrochloride is 8 at 25 ° C.
It went for 0 minutes. The number of SH groups introduced is F (ab ′) ₂ 1
It was 10 per molecule. This SH group concentration is 0.12 mM
10 mM phenazine methosulfate (P
MS) aqueous solution (200 μl) was added and left at room temperature overnight.
The precipitate was removed by centrifugation (3000 rpm; 10 minutes), and the supernatant was treated with Sephadex G-as in Example 3.
It was injected into a 25-column, and the polymer fraction was collected. As a sample, anti-human IgG rabbit antibody (Yatron) was added to 4.5.
Diluted to various concentrations with PBS containing% bovine albumin. (2) Antigen-antibody reaction 20 μl of labeled human IgG F (ab ′) ₂ diluted 20-fold with 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA
1, 100 μl of the sample, and 100 μl of 6% polyethylene glycol dissolved in PBS were mixed and heated to 37 ° C. for 5 minutes. (3) Detection method 0.37% Triton X-100 and 0.37 mM NB
0.1 M acetate buffer (pH 5.0) containing T was used as solution (A), 25 mM NADH aqueous solution was used as solution (B), and solution (A) and solution (B) were mixed in a ratio of 4: 1 in advance. I'll do it.
5 minutes after the start of the antigen-antibody reaction, the solution (A) and the solution (B)
1 ml of the liquid mixture was added, and the change in absorbance at a wavelength of 560 nm was monitored while heating at 37 ° C. for 5 minutes. (4) Results As shown in Fig. 3, a change in activity depending on the antibody concentration was observed even in the case of short-time reaction.

[0022]

EFFECTS OF THE INVENTION According to the detection method of the present invention, a stable labeling substance can be used to perform immunological detection without the use of a special device, and B / F separation can be performed. It is possible to perform the detection operation in a homogeneous system without the need.

[Brief description of drawings]

FIG. 1 is a graph showing the results of albumin measurement by competitive reaction.

FIG. 2 is a graph showing the results of fibrinogen measurement by non-competitive reaction.

FIG. 3 is a graph showing the results of antibody measurement by non-competitive reaction.

Claims (3)

[Claims] 1. A method for immunologically detecting a target substance in a test sample, wherein either the target substance or a substance that specifically binds to the target substance is labeled with an electron carrier other than an enzyme, and the labeled substance is used. An immunological detection method, which comprises detecting a target substance in a test sample by detecting a signal derived from. 2. The immunological detection method according to claim 1, wherein a substance other than an enzyme that catalyzes a redox reaction is used as the electron carrier. 3. A label characterized by binding an electron carrier other than an enzyme to a thiol group or a thiol group artificially introduced in a target substance or a substance that specifically binds to the target substance in a test sample. Chemical substance.