JPH0618526A - Immune measuring method - Google Patents

Abstract

PURPOSE:To suppress the quenching of a fluorescent substance by adding a compound expressed by a specific formula to a measurement system using the fluorescent substance as a marker. CONSTITUTION:The compound expressed by formula I (where, X and Y represent the same or different hydrogen atoms, lower alkyl groups, or lower alkoxyl groups and Z represents hydrogen atoms or expression II (where R<1>, R<2>, and M respectively represent hydrogen atoms or lower alkyl groups, hydrogen atoms or hydroxide groups, and alkali metal atoms) except such a case that the X, Y, and Z represent hydrogen atoms at the same time) is a fluorescence quenching preventing agent which prevents the quenching of a fluorescent substance in a liquid to be inspected, such as the blood serum, etc. The compound expressed by formula I includes N-ethyl-N-sulfopropyl-m- anisidine, m-xylene, etc. One, two, or more kinds of these fluorescence quenching preventing agents are added to a measuring system at a concentration of 10mM. For example, when an object to be inspected is made to react to a liposome containing a fluorescent marker, the preventing agents can be added to a reaction liquid before, during, and after reaction and an antigen or antibody contained in the object to be inspected is measured by detecting the fluorescent substance flowing out of the liposome.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunoassay method, and more particularly to a highly sensitive and simple immunoassay method capable of suppressing the quenching of a fluorescent substance.

[0002]

2. Description of the Related Art In recent years,
Immunoassay methods utilizing the antigen-antibody reaction have become increasingly important in clinical diagnosis of various endocrine diseases.

The above-mentioned immunoassay methods are roughly classified into a labeling method and a non-labeling method. Among them, the labeling method is widely used because of its high measurement sensitivity.

In the labeling method, various labeling substances such as radioisotopes, fluorescent substances, enzymes and enzyme-related substances are used. Among them, the method utilizing fluorescent substances has been attracting attention as a simple method.

On the other hand, the reaction system in the immunoassay is a heterogeneous system which separates (B / F separation) after labeling the reaction substances which are involved in the reaction and those which are not involved in the reaction, and the B
/ F is separated into homogeneous systems that do not require separation. The homogeneous system enables simple and quick measurement because it does not require a washing operation for B / F separation. In particular, when a fluorescent substance is used as a labeling substance, it is highly sensitive and can process a large amount of sample. It should be possible to construct a measurement method capable of

However, in general, when a fluorescent substance is added to serum as a sample, it causes a quenching phenomenon mainly due to adsorption to albumin, so that there is a problem that accurate measurement is difficult, and a solution to this point is desired. Was there.

[0007]

Means for Solving the Problems The inventors of the present invention have made extensive studies in view of such circumstances, and as a result, if compound (1) described below and a fluorescent substance are coexistent in a measurement system, immunoassay can be performed with high sensitivity. Moreover, they have found that they can be carried out easily and have completed the present invention.

That is, the present invention provides an immunoassay method in which a fluorescent substance is present as a labeling substance in the assay system,
In the measurement system, the following general formula (1)

[0009]

[Chemical 2]

The present invention provides an immunoassay method characterized by allowing the compound represented by

The compound represented by the general formula (1) used in the present invention is a so-called fluorescence quenching inhibitor having a function of preventing quenching of a fluorescent substance in a test solution such as serum.
In the general formula (1), as the lower alkyl group, an alkyl group having 1 to 6 carbon atoms, for example, a methyl group, an ethyl group, n-
Examples thereof include propyl group, isopropyl group, n-butyl group and the like. Examples of the lower alkoxy group include an alkoxy group having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group and an isopropoxy group. Further, examples of the alkali metal atom include sodium and potassium.

Specific examples of the compound represented by the general formula (1) used in the present invention include N-ethyl-N-sulfopropyl-m-anisidine (ADPS) and N-ethyl-N.
-Sulfopropylaniline (ALPS), N-ethyl-
N-sulfopropyl-3,5-dimethoxyaniline (D
APS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N-sulfopropyl-m-toluidine ( TOP
S), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N-. Ethyl-N- (2-hydroxy-
3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS),
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl)-
Examples thereof include m-toluidine (TOOS), N-sulfopropylaniline (HALPS) and m-xylene.

These fluorescence quenching inhibitors may be used in combination with other optional components, buffers, surfactants, antioxidants, etc., if necessary, and may be in liquid or powder form. Can also be

Further, one or two of these fluorescence quenching inhibitors are used.
The seeds or more are added to the measurement system so that the concentration is 10 mM or less.

The fluorescent substance used as the labeling substance in the present invention is particularly preferably carboxyfluorescein (CF), fluorescein isothiocyanate (FITC) or the like.

In the immunoassay method of the present invention, as long as the above-mentioned fluorescence quenching inhibitor and fluorescent substance are used in the assay system,
The method is not particularly limited, and examples thereof include a method using a fluorescent substance-bound antigen or antibody, a method of encapsulating a fluorescent substance as a marker in liposomes, and the like.

Specifically, in the method using the above-mentioned liposome, the sample containing the antigen or the antibody is reacted with the antibody corresponding to the antigen or the antibody or the fluorescent marker-encapsulated liposome bound with the antigen to destroy the liposome. The fluorescent substance flowing out from the sample is detected, and the antigen or antibody in the sample is measured. At this time, the fluorescence quenching inhibitor may be added to the reaction solution before or during the reaction between the sample and the liposome, or may be added to the reaction solution after the liposome is destroyed.

The fluorescence quenching inhibitor used in the present invention can be prepared in various forms depending on the type of the immunoassay method. For example, in the method using a fluorescent substance-bound antigen or antibody, compound (1) can be added to the assay system alone or in combination with other appropriate reagents, while in the method using liposomes, the complement reaction is stopped. It can be added to the measurement system after being combined with a buffer solution capable of being mixed and lyophilized if necessary. The addition may be done at any stage of the reaction.

[0019]

INDUSTRIAL APPLICABILITY The immunoassay method of the present invention efficiently suppresses the quenching action of a fluorescent substance by albumin contained in human serum, so that immunoassay can be carried out with high sensitivity and easily.

[0020]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Reference Example The inhibitory effect of each compound shown below on the fluorescence quenching action by human serum was examined. 10 ^-7 M carboxyfluorescein (CF) solution in a test tube (gelatin - veronal buffer (GVB ^-) those that have been dissolved in) were placed 2 ml, and then fluorescent anti-quenching agent adjusted to each concentration (FIGS. 2-9) containing for GVB ^- 2 ml and pooled human serum 50μ
1 was dispensed. After stirring with a vortex mixer, the fluorescence intensity was measured with a fluorimeter (excitation; 490 nm, fluorescence; 530 n
m, "Hitachi F-4000" manufactured by Hitachi, Ltd.). GVB ^- 2.0 in 2 ml of 10 ^-7 MCF aqueous solution
Figure 2 shows the fluorescence intensity of 5 ml dispensed as 100%.
As shown in 9, the relative fluorescence intensity (%) at each concentration of each compound was determined.

As is clear from the results shown in FIGS.
When the fluorescence quenching inhibitor is not contained, quenching due to the action of human pool serum is observed (about -20%), but quenching is suppressed by addition of each fluorescence quenching inhibitor, and 100%.
You can see that it recovers to the front and back.

Example CF is used as a fluorescent substance, and MAOS is used as a fluorescence quenching inhibitor.
Was used to perform the immunoassay shown below.

(Preparation of Liposomes) 1 μmol dipalmitoylphosphatidylcholine (DPPC), 1 μmol cholesterol and dithiopyridylated dipalmitoylphosphatidylethanolamine (DTP-DPPE) 0.0
5 μmol was placed in a pear-shaped flask, and chloroform in which the lipid was dissolved was distilled off with an evaporator. After drying in a vacuum desiccator for 1 hour, add 0.2 MCF to a pear-shaped flask.
200 μl was added and shaken vigorously, and the lipid thin film on the glass wall of the pear-shaped flask was peeled off to prepare a CF-encapsulated liposome.

The unencapsulated CF was separated by centrifugation with 0.01 M Hepes buffer (containing 0.15 M NaCl; pH 7.5) three times.

(Sensitization of anti-HBs antibody to liposome) Anti-HBs monoclonal antibody (manufactured by Biokit) 2 mg /
IgG was fractionated into ml to give N-hydroxysuccinimidyl-3- (2-pyridyldithio) propionate (SP
DP) 5 μl of a 30 mM ethanol solution was added, and the mixture was reacted at room temperature for 30 minutes. To remove excess SPDP,
0.1M acetate buffer (containing 0.15M NaCl, pH
Gel filtration was performed on Sephadex G-25 equilibrated in 4.5). 12 mg of dithiothreitol (DTT) was added to the obtained protein fraction (1.5 ml) and the mixture was reacted at room temperature for 30 minutes, and then 0.01 M Hepes buffer (0.15 M) was added in advance.
The excess DTT and the protein fraction were separated by gel filtration with Sephadex G-25 equilibrated with NaCl, pH 7.5). The liposome pellet obtained by preliminarily washing by centrifugation in 1 ml of this protein fraction was suspended to give 6-1.
The reaction was allowed to proceed at 0 ° C. for 18-20 hours with slow stirring.

After the reaction, the liposome suspension is washed by centrifugation,
After removal of the unreacted protein solution, gelatin - veronal buffer (0.1% NaNO ₃ containing, pH 7.4) and resuspended in 1 ml.

(H using anti-HBs antibody-sensitized liposome
Bs antigen measurement) The reaction was performed using a 96-well microplate. 0.15 mM anti-HBs antibody-sensitized liposome
Dilute 500-fold with gelatin-veronal buffer (GVB ²⁺ ) containing CaCl ₂ and 0.5 mM MgCl _2, and add 25
μl was dispensed into each well. A dilution series (12.5, 6.25, 3.13, 1.5) prepared by adding purified HBs antigen to inactivated human HBs negative serum.
6, 0.78, 0.39, 0.2ng / ml) 2 each
5 μl was dispensed. After reacting at 37 ° C for 1 hour, rabbit anti-H was further diluted to the optimum concentration with GVB ²⁺ as a secondary antibody.
25 μl of Bs antibody (IgG fraction) and 25 μl of guinea pig complement (3CH ₅₀ ) diluted with GVB ²⁺ were dispensed, respectively, and reacted at 37 ° C. for 1 hour.

As a stop solution for the above-mentioned complement reaction, 10 mM MA
Veronal buffer containing OS and 10 mM EDTA (pH
7.4) 100 μl was added and 10 mM for comparison
Veronal buffer containing EDTA (pH 7.4) 100μ
1 was added separately, and the CF release rate (%) was obtained for each dilution series. The CF release rate was determined as the relative fluorescence intensity (%) with 100% as the fluorescence intensity when 25 μl of 10% Triton X-100 was added instead of the HBs antigen. As a control, a dilution series prepared by adding only purified HBs antigen to GVB ²⁺ (12.
5, 6.25, 3.13, 1.56, 0.78, 0.3
(9, 0.2 ng / ml), the CF release rate was similarly obtained.

The CF release rate measurement results for each of these dilution series are shown in FIG. As is clear from the results shown in FIG. 1, when the stop solution containing only EDTA was used, the quenching was observed to a considerable extent, but when the stop solution containing MAOS was used, the quenching phenomenon was hardly observed. I can't see it.

From these facts, it is understood that MAOS suppresses the quenching of CF by human serum, and the same sensitivity as the reaction sensitivity in a pure system containing no human serum can be obtained.

[Brief description of drawings]

FIG. 1 is a drawing showing a CF release rate for each dilution series obtained in Examples.

FIG. 2 is a drawing showing relative fluorescence intensity with respect to ADOS concentration obtained in Reference Example.

FIG. 3 is a drawing showing the relative fluorescence intensity with respect to the ADPS concentration obtained in Reference Example.

FIG. 4 is a drawing showing the relative fluorescence intensity with respect to the ALPS concentration obtained in the reference example.

FIG. 5 is a drawing showing the relative fluorescence intensity with respect to the ALOS concentration obtained in Reference Example.

FIG. 6 is a drawing showing the relative fluorescence intensity with respect to the MAOS concentration obtained in Reference Example.

FIG. 7 is a drawing showing the relative fluorescence intensity with respect to the DAPS concentration obtained in Reference Example.

FIG. 8 is a drawing showing the relative fluorescence intensity with respect to the MAPS concentration obtained in Reference Example.

FIG. 9 is a drawing showing the relative fluorescence intensity with respect to the m-xylene concentration obtained in Reference Example.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Mamoru Umeda 1075-2 Kitanan Moro, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Co., Ltd.

Claims (2)

[Claims] 1. In an immunoassay method in which a fluorescent substance is present as a labeling substance in a measurement system, the following general formula (1): An immunoassay method comprising the presence of a compound represented by: 2. The immunoassay method according to claim 1, wherein the fluorescent substance is carboxyfluorescein and / or fluorescein isothiocyanate.