JPH0427867A - Fluorescence labelled substance - Google Patents

Abstract

PURPOSE:To measure the amount of a target substance by detecting the fluorescence from the binding substance with the target substance by using a fluorescent rare earth metal chelate having fluorescence decay life longer than the longest fluorescence decay life of a completing unlabelled circumferential substance. CONSTITUTION:A specimen containing DNA is subjected to the contact reaction with a disc or cellulose pulp having DNA complementary to the target DNA sequence present in the specimen immobilized thereon. This reaction product is washed about twice under a condition not liberating DNA subjected to hybridization. Next, a DNA probe standardized by a fluorescent rare earth metal chelate having complementarity to immobilized DNA is reacted under a proper hybridization condition. The reaction body is washed under a condition liberating the hybridized reaction product. The fluorescence of the fluorescent reaction body thus obtained is detected by a technique of time resolution fluorometry.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention is applicable to the detection of signature components present in blood, body fluids, urine, or other biological components, as well as genes in genetic recombination technology and DNA diagnosis. Regarding new technology.

The present invention also relates to a time-resolved fluorescence measurement method using a new measurement reagent.

(Prior art and problems to be solved by the invention) Currently, measurement methods based on various biological affinities are:
It has become an indispensable tool for microanalysis, diagnosis of diseases, detection of substances in genetic engineering, etc.

In such measurement methods, radioassays using radioactive isotopes such as 125, ff2p, and ff53 have been used. This radioassay has the advantages of relatively little change in the properties of the substance during labeling, high measurement sensitivity, and wide applicability. However, because the label itself is a radioactive decay product, the reagent itself is unstable and cannot be stored for a long time, and special care must be taken when using it. There are major problems, such as the fact that even females cannot use it with confidence, and the total cost increases because special equipment such as an isotope processing facility is required. In order to overcome these drawbacks, enzyme assays using enzymes as labeling agents and fluorescence assays using fluorogenic substances have been devised.

Recently, the method of labeling with enzymes has been widely used, but since these enzymes are inherently unstable, they are easily subjected to various treatments during labeling, storage, and use. In addition to problems such as denaturation, enzyme-labeled reagents are easily affected by temperature, pH 1 reaction time, etc., and there are also problems that measurement results are not always obtained consistently or that measurement requires skill. . Furthermore, since the enzyme itself is expensive, there is also the problem of high cost.

In contrast, the method of labeling with a fluorescent substance has good stability both in the fluorescent substance itself and the stability of the fluorescent substance-labeled reagent itself, and furthermore, the measurement itself using the reagent is simple and inexpensive. It also has the advantage of being inexpensive.

However, for the following reasons, this method has a problem in that "measurement sensitivity", which is the most important condition in a trace substance measurement system, is inferior to the first two methods.

Fluorescence is a phenomenon in which specific radiation light is emitted based on the electronic level of a compound when irradiated with a certain excitation light. However, because it requires excitation light, the generation of background noise due to Rayleigh scattering is unavoidable. In addition, much fluorescence derived from various substances present in serum and proteins [11
In order to solve these problems, it is generally necessary to increase the Stokes shift of the fluorescent substance, or to distinguish the labeled fluorescence from other background fluorescence. Examples of such methods include imparting special properties to fluorescent substances.

As a method that has these properties to some extent, a fluorescence measurement method using a technique called time-resolved fluorescence measurement has been proposed.As a method based on the zero-line measurement principle, US Patent No. 4.
No. 058,732 and U.S. Patent No. 4. 341. 957
Examples include those listed in the issue. However, since the reagents shown in these documents are antibodies immobilized with fluorescent substances, they can only be used to measure specific antigenic substances. There is a problem of having to prepare. In general, in the literature, there are recent frequent reports on HSS-redization assay methods or DNA cleavage in genetic recombination.
There is no description of the nucleic acid probe used as the analytical reagent.

(Means for Solving the Problem) As a result of intensive research in order to solve the problems of such conventional methods, we have found that a biological reactant selected from an antigen or a nucleic acid and a fluorescence bonded to the biological reactant. a fluorescent rare earth metal chelate, and the fluorescent rare earth metal chelate has no competing
! A fluorescent biological reactant whose H signature is that it has a long fluorescence decay lifetime compared to the longest fluorescent decay lifetime of surrounding substances, and the range of applications of the fluorescent biological reactant is wide. They discovered this and completed the present invention.

The present invention uses a fluorescent rare earth metal chelate suitable for time-resolved fluorescence measurement as a fluorescent labeling agent, and also
Antigen or DN as a biological reactant to be msd with ms agent
A. Use a nucleic acid selected from RNA and other nucleic acids.

Accordingly, it is an object of the present invention to provide an antigen, a biological reactant selected from a nucleic acid, and a fluorescent rare earth metal chelate bound to the fluorescent biological reactant, wherein the fluorescent rare earth metal chelate is The objective is to provide a fluorescent biological reactant that has a long fluorescent decay lifetime compared to the longest fluorescent decay lifetime of the surrounding materials of the label. Furthermore, the present invention comprises a biological reactant selected from an antigen or a nucleic acid, and a fluorescent rare earth metal chelate bound to the biological reactant, wherein the fluorescent rare earth metal chelate binds to a competing unlabeled surrounding area. Target substances with fluorescent biological reactants that have a long fluorescence decay lifetime compared to the longest fluorescence decay lifetime of the substance! ! Then, the amount of the target substance is measured by detecting the fluorescence from the combination of the thus obtained fluorescent biological reactant and the target substance to be measured.

Antigens that can be used in the present invention include complete antigens, habten, and their analogs, such as cortisol, cortisol amine, thyroxine, digoxin, biotin, α-
Fetoprotein, human chorionic gonadotropin, ferritin, thyrotropin, folithrobin, lutrobin,
Thyroxine-binding globulin, growth hormone, prolactin, avidin, streptavidin, hemocyanin,
Various hormones such as myosin and catalase, blood coagulation factors, enzyme inhibitors, enzymes, cell surface antigens, proteins with specific binding properties, their subunits or fragments, their variants, various allergy-causing substances, etc. can give.

Nucleic acids that can be used in the present invention include DNA, R
This includes NA and its analogues, which may be extracted from chromosomal genes or synthesized from Metzenger RNA using genetic engineering methods, and may also be derived from the amino acid sequence of the protein. It may be chemically synthesized based on.

These nucleic acids may contain, in addition to normal nucleic acid components, aminomethylated or formylated radicals.

Examples of fluorescent rare earth metal chelates that can be used in the present invention include organometallic chelates in which an organic compound molecule is coordinated with a rare earth metal ion. In addition to not exhibiting a high quantum yield, a high quantum yield can be obtained. Furthermore, it exhibits a long lifetime and a characteristic fluorescence spectrum.

Such fluorescent rare earth gold dust chelates include S,
1. Weismann, et al., Jour
nal of Chemical Physics
, vol, 10. p214-217 (1942),
RoA, Gudmundsen, et al., Jou.
rnal of Chemical Physics v
ol, 39 p, 272-274 (1963), and O.
pt.

Soc, Amer, vol. 54. p1211 (196
Examples include benzoylacetonate, benzoyl trifluoroacetonate, hexafluoroacetylacetonate, acetylacetonate, thenoyl trifluoroacetonate, etc. of europium and terbium described in 4). Examples include fluorescent rare earth metal chelates with ligands described in JP-A-61-200988.

In addition to the above rare earth metals, dysprosium,
Examples include elements selected from elements belonging to the lanthanides such as samarium.

Methods for binding fluorescent rare earth metal chelates to biological reactants selected from antigens or nucleic acids include ionic bonding, adsorption reactions, and covalent bonding. Such binding is not particularly limited as long as the fluorescent rare earth metal chelate can stably label a biological reactant selected from antigens and nucleic acids, but such binding methods include covalent bonding. As a bonding method using a covalent bond, which is particularly preferable from the viewpoint of stability, a wide variety of conventionally known methods can be applied, such as the glutaraldehyde method, the periodic acid method, the maleimide method, and the isothiocyanate method. method, p-nitrobenzoyl chloride method, triazine method, pyridine disulfide method, diazo method,
Examples include methods such as a carbodiimide method, a mixed acid anhydride method, and methods that suitably combine these methods. In applying such a binding method, the biological reactant of the above invention can be modified in advance by applying various modification methods. Methods that can be used here include Journal of the Society for Organic Synthesis and Chemistry, Vol. 42, No. 4, p. 283-29.
For example, if the biological reactant has a free amine group, a fluorescent rare earth metal chelate modified to have a sulfonyl chloride group can be reacted, or the amino group can be reacted with a fluorescent rare earth metal chelate modified to have a sulfonyl chloride group. There is a method in which the amine group of the chelating agent is further substituted with an isothiocyanate group, and the chelating agent substituted with the isothiocyanate group is reacted with a biological reactant.

Examples of chelating agents having an amino group that can be used here include, for example, U.S. Pat. Kotoji is published in issue 341957.

According to the present invention, a biological reactant having a plurality of labeling sites is desirable, such as having a plurality of free amino groups. It has been found that this reagent has good fluorescence properties and provides good measurement sensitivity.

The thus obtained biological reactant selected from the antigen and nucleic acid labeled with the fluorescent rare earth metal chelate is used for detection or analysis of the target substance. Examples of such detection analysis methods include immunoassay and hybridization analysis, which can be applied based on conventionally known principles as they are or with appropriate modifications.
Typical methods include Sand-Germany method, double antibody method, and
Examples include antibody solid phase method.

The target substances targeted by the present invention include, first of all, antibodies. The amount of antibodies in a living body reflects various diseases or the state of the living body, and it is of great significance to detect antibodies, including their presence or absence.The second target substance targeted by the present invention is: Enzymes and their specific inhibitors, biotin and avidin or streptavidin, cholera toxin and its receptor, and other highly biologically compatible combinations. Third, target substances targeted by the present invention include oncogenes, viral nucleic acids, genes that cause specific diseases, and RNA and DNA used in genetic recombination.

In detecting a target substance according to the present invention, first, a sample containing the target substance is immobilized on a well, disk, microbead, etc., and then reacted with a biological reactant reactive with the target substance. The thus immobilized target substance is washed under conditions under which it remains immobilized, and then reacted with the fluorescently labeled biological reactant of the present invention. If necessary, instead of reacting the fluorescently recognized biological reactant directly with the target substance, for example, the target substance can be reacted with biotin or other substances and an antibody specific to the target substance. , and then, after a suitable washing treatment, a fluorescent biological reactant according to the invention capable of specifically recognizing biotin or other substances may be reacted, so that the complex thus formed is not liberated. After washing under these conditions, the amount of target substance is detected by measuring the fluorescence of the produced Humbrex in a time-resolved manner.

In addition, when the target substance is a nucleic acid such as DNA or RNA, the biological reactants ffm-formed with the fluorescent rare earth total chelate of the present invention include the fluorescent rare earth total chelate).
It is preferable to use a nucleic acid that has been labeled with Keyaki.The processing conditions used here can be those in which a normal nucleic acid is used as a probe, and the nucleic acid used is used in a state where it is immobilized on a filter etc. You can also do that.

Hereinafter, the measurement method generally described above will be explained using a specific example.

(1) A method for measuring IgE antibodies as an allergy-causing substance will be explained. First, allergens are immobilized on microbeads by a known method. Then, serum containing allergen-specific IgE is reacted. This reaction is
This is done by incubating for several minutes in an appropriate buffer.

Then, the immobilized allergen and allergen-specific Ig
Wash 2 to 3 times with an appropriate buffer solution under conditions that do not dissociate the bound product with antibody E.Next, react with goat anti-human IgE antibody labeled with fluorescent rare earth gold chelate suspended in buffer solution. and let it react for a few minutes.

Next, the obtained microbeads are washed 2 to 3 times with an appropriate buffer solution to remove excess labeled antibody, and the fluorescence of the thus obtained fluorescent reactant is measured using a conventional time-resolved fluorescence measuring device. do. Calculate the amount by comparing with a separately prepared standard curve.

(2) Next, a method for detecting DNA will be explained. A sample containing DNA is brought into contact with a disk or cellulose on which DNA complementary to the target DNA sequence present in the sample is immobilized. This reaction is
Wash twice under conditions that do not release hybridized DNA. First, use a fluorescent rare earth metal chelate that is complementary to the immobilized DNA! The weakened DNA probe is reacted. During this reaction, zero reactants carried out under appropriate hybridization conditions are washed under conditions that do not release hybridized reactants. The fluorescence of the fluorescent reactant thus obtained is detected using a time-resolved fluorescence measurement technique. In addition, in the above method, detection may be performed using a DNA probe or the like in which a secondary probe is labeled with a fluorescent rare earth metal chelate.

According to the present invention, since relatively stable substances such as various allergens are labeled as biological reactants, the reagent can be stored for a long period of time, and the stability of the reagent itself of the present invention is radioactive. Since this is higher than that of an isotope-labeled reagent, the reproducibility and sensitivity of measurement results when using the reagent according to the present invention are good.

Further, according to the method of the present invention, a reagent having many labelable sites can be used as a reagent, and as a result, measurement sensitivity can be increased. Furthermore, when a protein that specifically reacts with biotin, such as avidin or streptavidin, is labeled, there is no need to change the biological reactant to be labeled depending on the target substance, and biotin m! Another advantage is that biological reactants can be used. In addition, when labeling carrier substances such as albumin, thyroglobulin, polylysine, and their synthetic, natural, or semi-synthetic polypeptides, many labeling sites can be obtained and combinations with various types of habten can be obtained. Another advantage is that a wide variety of reagents can be prepared with just one.

Further, according to the present invention, a fluorescently labeled nucleic acid probe that is stable, inexpensive, and valuable can be provided, and the detection effect of DNA and RNA can be enhanced.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1: Preparation of europium (m) chelate-labeled habuten Journal of Molecular 5pec
troscopy, Volume 8, pp. 315-327,
0.81 g of sodium p-aminomethylbenzoyl trifluoroacetonate, prepared according to the method disclosed in 1962, was dissolved in 20-ounces of distilled water. This solution was mixed with 7.4B/- europium (In) chloride aqueous solution 50-
was added dropwise and stirred. Collect the deposited precipitate,
Wash with a small amount of cold distilled water, then store under reduced pressure at room temperature for 1
It was dried for 0 hours to obtain tris(p-aminobenzoyltrifluoro7cetonate) europium (m).

Progesterone compound 2oB was dissolved in dioxane 5-1, and N-methylmorpholine 5-1 and isobutyl chloroformate 14-1 were added dropwise under water cooling and stirring.

Next, the aforementioned tris(p-benzoyltrifluoroacetonate) europium ([11>
A dioxane solution 5- containing dioxane was added dropwise, stirred as it was for 30 minutes under water cooling, and continued stirring at room temperature for 1 hour.
After the reaction, the solvent was removed under reduced pressure and redissolved in a small amount of acetonitrile/water (55/45) solution.
Preparative HP using column (φ2.2 x 25 cm)
Europium (In) chelated progesterone was detected by LC. After brewing, remove the solvent under reduced pressure,
50mM Tris-HCl buffer containing 0.1% sodium chloride and 0.05% sodium azide (pH 7,
75) and stored in a cool, dark place.

Example 2: Measurement of progesterone by competitive method An anti-progesterone monoclonal antibody was immobilized on a plastic cuvette. That is, 100 J of the antibody solution was added to a cuvette and incubated overnight at 4°C.

After washing the cuvette with deionized water, 0.5%
50mM phosphate buffer containing bovine serum albumin (pH 7)
, 2) Blocking was performed by adding 500 μm and incubating overnight at 4°C. After washing the cuvette, the europium chelate-labeled progesterone described in Example 1 and the standard antigen (progesterone temperature: O, l,
10.100゜1000 ng/aQ) 50J, and 0.5% BSA, 0.01χ) Vienna 20.0
50mM phosphate buffer containing 9χ sodium chloride (pH 7)
, 2) 250 J was added to the cuvette and allowed to react at room temperature for 1 hour. 500 J of ethanol was added to the cuvette from which europium chelate-labeled progesterone unbound to the solid phase and progesterone had been removed by washing, and the cuvette was analyzed using a 4-hour resolution fluorometer (Horiba, HAES-1100,
Fluorescence intensity was measured using Hamamatsu Photonics Universal Photon Counting System (Hamamatsu Photonics Universal Photon Counting System). As standard antigen perfusion increased, the fluorescence intensity decreased.

Example 3; Preparation of europium (III) chelate 5 oligonucleotide probe Science, Vol. 227, 484-
Page 492, based on the nucleobase sequence of gag1595-1635 of HIV Idako disclosed in 1985,
An oligonucleotide for an HIV probe was synthesized using DNA synthesizer model 381A# (Applied Biosystems). At that time, the oligonucleotide
The synthesized oligonucleotide, in which an amino group had been introduced at the 5-terminus using Am1nolink 2@ (Applied Biosystems) for the purpose of binding to the oligonucleotide, was purified using an oPC cartridge.

2 containing 5° of the oligonucleotide prepared in this way.
0.18 sodium carbonate buffer (p
H8,0) 2.6 mg/- of dissuccinimidyl suberate (25- prepared with dimethyl sulfoxide)
DSS) I solution 50- was added. After stirring at room temperature for 5 minutes, immediately add a Sephadex G50 column (φ0, 5
cm x 25 cm), developed with ion-exchanged water,
Fractions with absorption at 260 nm were collected.

After recovery, the tris(benzoyltrifluoroacetonate) described in Example 1 was immediately freeze-dried.
Aminomethylated form of europium ([[+) chelate 6]
．． After the completion of the 0 reaction, 100 J of 3H sodium chloride-containing 0.1 M sodium carbonate buffer (pH 8,0) containing 14 was added and stirred at room temperature for 20 hours.
Bio-Rad P-100 column (φ1.0!40) equilibrated with f1M Tris-HCl buffer (pH 8,5)
cm) to remove unreacted tris(benzoyltrifluoroacetonate) europium(II).
I) The aminomethylated form of the chelate was removed to obtain a europium (III) chelate-labeled oligonucleotide probe solution.

Claims (2)

[Claims] (1) a biological reactant selected from an antigen or a nucleic acid;
a fluorescent rare earth metal chelate associated with the biological reactant, the fluorescent rare earth metal chelate being characterized by a long fluorescent decay lifetime compared to the longest fluorescent decay lifetime of competing unlabeled surrounding substances. Fluorescent biological reactants. (2) a biological reactant selected from an antigen or a nucleic acid;
a fluorescent rare earth metal chelate bound to said biological reactant, said fluorescent rare earth metal chelate having a long fluorescent decay lifetime compared to the longest fluorescent decay lifetime of a competing unlabeled surrounding material. A method for measuring a target substance by labeling a target substance with the fluorescent biological reactant and then detecting fluorescence from the fluorescent biological reactant-target substance conjugate. .