JPH0276600A - Molecular probe with a mark, its production and use - Google Patents

Abstract

PURPOSE: To produce a labeled molecular probe comprising a molecular probe and a label enzyme by combining a molecular probe or a mixture of molecular probes with a transferase or a ligase.

CONSTITUTION: This labeled molecular probe is produced by combining a molecular probe or a mixture of molecular probes with a transferase (E.C.2) or a ligase (E.C.6). The primary obtained molecular probe is bonded to a transferase directly or through a spacer. Secondary, a labeled molecular probe is obtained by combining the molecular probe or a mixture of the molecular probes with an aminoglucoside-phosphoric acid transferase. The molecular probe is utilizable in a molecular biology, a medicine or a genetic biology, etc., for detecting an organic molecule by an enzyme reaction.

COPYRIGHT: (C)1990,JPO

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for detecting biomolecules by enzymatic reaction.
This invention relates to enzyme-labeled molecular probes, methods for their production, and methods for their use. The fields of application of the present invention are molecular biology, medicine, biology, genetic biology, plant pathology, genetic engineering, agriculture and veterinary medicine.

[Conventional technology]

There are known methods for detecting biomolecules using molecular probes bound to enzymes. In this case, up to now probes of immunological activity [e.g. Immunoenzymatic Technique
s-5, edited by Vramcas et al. (1983) Els
5science Publishers], hybrid reference probes [e.g. B, M
, Renz, ChrKurz: Nucl, Ac1
ds Res, (1984)■, 3435-344
In addition, various probes with special affinities for other biomolecules have also been attached to various enzymes. Binding enzymes mainly include alkaline phosphatase, galactosidase, glucoamylase,
Glucose oxidase, glu-3ronidase, raftate dehydrogenase, lactoperoxidase, peroxidase, ribonuclease and tyrosinase have been linked to various molecular probes by conventional cross-linking agents (S, Avrameas, T, Ternyck,
J 几, Guesdon; Magazine 5cns, J,
Immunol, (1978), p. 7, 27].

Conventionally, enzymes used in detection methods have been essentially limited to those mentioned above, that is, only a limited number of them have been put into practical use. One of the reasons for this limitation is the poor detection potential for various other substrates. Until now, substrates have been used whose enzymatic reactions yield colored reaction products. In detection methods using enzyme-conjugated probes for biomolecules such as those bound to various solid phases, the detection of the unreacted substrate has always been based on the presence of the biomolecule on it as well. It is carried out on the same solid state or in solution. The principle procedure of such a detection method is, for example, the Immunoenzymatic method mentioned above.
R, H on page 274 of Techniques.

Antigen determination (immunological detection) is explained by John Yolken as follows: 1) Antibody 1, which is the antigen to be measured, is immobilized on a microtiter plate.

2) Add the test substance and allow the antigen present to bind by the antibody.

3) Add enzyme-conjugated antibody 2 after washing several times. This reacts with the antigen bound to antibody 1 above.

4) Add substrate after several washes. Enzyme (AKI-antigen, Δ2) immobilized on the wall of the microtiter plate above.
- an enzyme) converts its substrate into a visible form. The color intensity that can be measured in the solution is proportional to the amount of antigen in the test substance.

In most cases, water-soluble reaction products are produced in such detection reactions. The water-insoluble products are separated out by precipitation on the carrier material. Conditioned by the properties of conventionally used enzymes, enzymes directly coupled to such probes cannot be used under extreme conditions (e.g. hybridization conditions, high temperatures or the presence of detergents). etc).

Until now, in detection reactions for biomolecules that are immobilized on a solid phase consisting of biomolecules, the detection has always been performed on the same solid phase on which the molecules of interest are present. (e.g. detection of antigen by plotting on nitrocellulose from polyacrylamide gels using antibodies conjugated with peroxidase)
.

During this detection, the antigen is first transferred to and immobilized thereon by polyacrylamide gel electrophoresis. The carrier material on which the antigen rests is then incubated with the enzyme-conjugated antibody, which then binds to the antigen. After several washes to wash away the enzyme-bound excess, a compatible substrate, such as diaminohentidine or 4-chloronaphthol, is added in the presence of hydrogen peroxide. Peroxidase converts this substrate into a water-insoluble, colored compound (
diaminohentidine: brown, 4-chloronaphthol: purple), which appears on the support at the site where the antigen-antibody-peroxidase complex is present, and thereby increases the concentration of the antigen in the separated protein mixture. Mark the location on the carrier material. The antigen is then colored and marked on the same carrier material on which it is marked. It is difficult to reuse the pronodes with dyes after such enzymatic detection reactions, since the bound dye molecules almost always undergo side reactions and are therefore Guide to background issues in stages (.

Detection methods using color reactions are always less sensitive than radioactive detection methods. Until now, detection of a specific nucleic acid sequence using a hybridization probe bound to an enzyme has been carried out using basically the same procedure.

Among these, the detection of nucleic acid sequences from extremely small amounts of biological material, for example, the detection of individual genes from minimal amounts of DNA i, essentially requires a more sensitive detection method.

Furthermore, it has hitherto been impossible to detect biomolecules using enzyme-conjugated probes on a support layer independent of the biomolecules.

Molecular probes include a large number of compounds and biomolecules that have special affinities for other biomolecules and are therefore suitable for their detection and affinity thalamatographic purification.

These probes include: o Immunologically active probes, e.g. antibodies, antisera, immunologically reactive proteins o Hybridization probes, e.g. nucleic acid fragments of different lengths, DNA molecules and RNA. Molecules, o Proteins that have special affinities for other biomolecules, such as avidin, lectin, streptavidin, etc. o Various organic compounds that have special affinities for various biomolecules, such as biotin, etc. In molecular biology, the detection of a specific biomolecule (target molecule) is carried out by the reaction of the target molecule with various molecular probes (e.g., by immunoreaction or hybridization), in which these probes are directly detected (e.g. a second detection system, which is labeled (with a radioisotope or a fluorescent dye) or which leads to the detection of the target molecule by means of another specific reaction (e.g. precipitation of a colored substance and subsequent photographic evaluation); combined. These detection methods are described in many published documents and belong to the well-known art. Coupling enzymes to probes is playing an increasingly important role in these detection methods [for example, the previously mentioned Tmmunoenzymatic Techniq
ues and R4, Burdon and P, H, van K.
Amsderdam edited by Nippenberg (Else
5 sciences Publishers)
Laboratory Techni published in 1985
questions in Biochemistry andM
Olecular Biology, Vol. 15, Chapter 11, "Preparation of Enzymes, Antibodies or Other Enzyme Macromolecule Conjugates", pp. 221-278].

A series of enzymes have been coupled to antibodies, for example (S, Avr
ameas, T., Ternyck, J.L.
Guesdon: alkaline phosphatase, galactosidase, glucoamylase, glucose oxidase, glucuronidase, raftate dehydrogenase, rough j. peroxidase, peroxidase, ribonuclease, tyrosinase)6 According to Avrameas et al., enzymes that should be used to bind to various molecular probes must meet certain general requirements such as: 1) high specific activity and permeation rate of the substrate (Durchsat
zrate) 2) Good stability at real and working temperatures 3) No or low loss of activity after conjugation 4) Readily available in high purity from readily available materials or easily accessible Preparability These requirements are only partially met by the enzymes used hitherto. Enzymes most often used in detection reactions are peroxidase and alkaline phosfactorase. The relatively low thermostability of these enzymes in this case makes them particularly difficult to use in hybridization reactions (e.g. 48 hours at 65°C).
or even make it completely impossible to use. Another drawback of these conventionally used enzymes is that they generally utilize color reactions for detection, in which case only relatively low detection sensitivities are achieved. 32p
It is believed that the transfer of the labeled phosphate to the substrate allows for a substantially more sensitive detection than in the various described methods. However, the transfer of 32p-labeled phosphates onto certain substrates using enzymes coupled to probes has not been previously reported.

When coupling enzymes to molecular probes, bifunctional molecules having the same functional group are generally used as coupling reagents, such as glutaraldehyde [e.g.
Bull, Soc, Chem, Biol
, (1968) 50. See page 1, line 169 et seq.] Benzoquinone [e.g. M, Reng, Ch,
Kurg: (1984) Nucl, 8c.
ids, Res.

8, 3485-3444], bisimide esters [e.g. W, C, Mentzer: J, Prot.
eLn Chem, (1982) It141-155
] or molecules with several different groups, such as azidoaryloxysuccinimide (e.g. T, H
, Ji and J, Ji: eightal, Biochem.

(1982) 121.286-289], azidoarylglyoxals [e.g. S, M, Politz, H. F., Noller, P, D.

McWirther: Biochemistry
(1981) -20, 372-378] or azidoarylthiophthalimides [e.g. RoB, M
oreland et al; Anal, Biochem, (
1982) plate 321/6] is used.

The biotin/avidin (streptavidin) system is gaining increasing importance, since it can be used to perform cascade double-intensity amplification up to a factor of 8 (e.g. as described in the US patent (See Publication No. 4463090).

This detection system most often consists of peroxidase or alkaline phosphacus coupled to streptavidin or biotin, so that again a color reaction is utilized for detection.

In today's most sensitive hybridization methods (e.g. for detecting individual genes in human DNA), detection methods using enzymes coupled to probes that do not use radioactive isotopes are clearly still directly labeled with radioactive elements. This method is inferior to detection methods using conventional nucleic acid probes. In this area, methods with inherently higher detection sensitivities are particularly required. With hitherto known probes, on the one hand, intensity amplification up to a factor of 8 can be achieved using a cascade with biotin/avidin;
The operation is carried out on a solid support. In the case of a detection method in which a colored substance is precipitated, intensity amplification as described above is not possible, and furthermore, the precipitated colored substance generally makes it impossible to reuse the biomolecule.

Detection using enzyme-conjugated probes has traditionally been coupled with color methods, in which the relatively high detection sensitivity of autoradiographic methods is not exploited. For radioactive detection using signal intensity amplification, enzymes that can withstand the conditions of all reaction steps are not available.

[Problem that the invention seeks to solve]

The object of the present invention is to provide a novel enzyme-labeled molecular probe, a method for its production and a sensitive detection method for biomolecules using such a new enzyme-labeled molecular probe. It is about creating.

The object of the present invention is to label various molecular probes with binding-enabled enzymes and to use the enzyme-labeled molecular probes and a special solid phase for binding and detection of the reacted substrate. The objective is to develop methods for detecting various biomolecules that provide detection sensitivity.

[Means for solving problems]

The method according to the invention for the detection of various biomolecules by means of labeled molecular probes is a method for detecting biomolecules present in solution or contained in a gel, or alternatively bound to a solid phase. identified by a molecular probe bound to an enzyme, reacted with a substrate in the presence of a cosubstrat consisting of the enzyme bound to or separated from the molecular probe, and the reaction It is characterized by detection on the solid phase ① or ②. The method according to the invention allows for the first time to carry out the identification and detection steps in the detection method for various biomolecules on different phases, for example on two flat supports, with different enzymes participating in the reaction. , and thereby a method can be realized in which the potential for intensity amplification by the enzymatic reaction on the second phase can be fully exploited.

A large number of variants of the method according to the invention are possible, which follow all the method steps based on its basics. According to the invention, therefore, the detection of various biomolecules is carried out in the following method steps: a) Identification step: In this step, the molecular probe labeled by the bound enzyme is present in solution and in the gel. or attached to a biomolecule that has a specific affinity for it by being bound to the solid layer.

b) Washing step: In this step the excess of the labeled molecular probe is washed away and/or separated off.

C) Enzyme reaction step: In this step the substrate reacts with the enzyme in the presence of certain co-substrates.

d) Separation step: In this step unreacted co-substrate and optionally substrate are washed away or separated off.

e) Evaluation stage: In this stage the reacted substrate is measured on a solid layer (1) or (2) or detected by a suitable detection method.

Again a large number of variants are possible for the detection of various biomolecules based on various techniques in each of these method steps. That is, for example, the biomolecule to be detected may be present in a solution or bound to a certain solid layer. In this case, the solid layer may be in the form of a granular or plate-like carrier as well as a gel in which the biomolecules are free to move but remain bound. These gels include agarose gels and polyacrylamide gels, which are often used in molecular biology, and are generally used for separation operations. A large number of variants are possible in the washing and separation stages, since in these stages a number of individual stages carried out in known manner are combined together. Thus, for example, washing in the solid phase can be carried out using various buffer solutions that are compatible with the system. However, it may also be necessary to separate a component or a number of components, which can be carried out using columns, separating gels or by chromatographic methods. On the other hand, it is also possible to separate only the complex between the biomolecule and the enzyme-bound probe and then detect it in the enzymatic reaction step. It is also possible to decompose the enzyme again from its probe in one of these method steps and then detect only this enzyme on a solid layer based on the reaction of this with substrate and co-substrate. be.

Various possibilities therefore also exist for carrying out the enzymatic reaction step. This step can likewise be carried out in solution, or it can also be carried out on a solid layer or in a gel. If this enzymatic reaction step is carried out, for example, in solution, the reaction product of the substrate must then be bound to some solid layer and detected there. This means that a support medium is used which selectively binds the reacted substrate. However, if this enzymatic reaction step is already carried out on a solid layer, i.e. the substrate and/or the complex consisting of the biomolecule and the molecular probe are still bound or have already been decomposed from this. When an enzyme, such as
It is then detected there, but unreacted co-substrates must be carefully separated. Even if the enzyme, which has been decomposed and separated from the complex consisting of biomolecules and molecular probes, is present in solution or confined in a gel, this reaction can occur in some solid layer. The reacted substrate can then be detected on this solid layer. Although separation of the unreacted substrate can be carried out in this case as well, this is unnecessary in most cases.

Some of the variants which are preferably carried out are described in more detail below: Variant I a) Separation of different biomolecules in a gel or other separation carrier material and containing the biomolecules A gel or other separation carrier material is contacted with a solution containing a molecular probe bound to an enzyme, whereby the biomolecule and the probe react with each other, thereby linking the biomolecule with the enzyme-bound molecular probe. A complex compound with is formed in the gel (identification step).

b) In a washing step, excess of the delabeled molecular probe is washed out of the gel or degraded.

C) In the enzymatic reaction step, the gel is contacted with a second gel or separation carrier material containing the substrate and co-substrate to react the substrate.

d) In a separation step, the second gel or separation carrier material is brought into contact with the solid phase (1) together with its added substrate and the reacted substrate is selectively bound thereto.

e) After several washing steps, the reacted substrate on the solid phase (1) is detected in a detection step by radioactivity measurements, by autoradiography or by other suitable detection methods.

In one special embodiment of this method steps a and b
After that, a gel containing a complex compound consisting of the biomolecule and a labeled molecular probe is brought into contact with a solid phase to which a substrate is bound in the presence of a co-substrate,
The enzymatically reacted substrate is then detected on the solid phase (2). This particular embodiment can also be used as an enzyme test for the detection of the activity of enzymes, thereby providing yet another possibility of application of the method according to the invention.

Extracting a) In the identification step, the molecular probes and biomolecules bound to an enzyme are in solution and are added to each other there.

b) Separating and removing the labeled excess molecular probe in a washing step.

C) In the enzymatic reaction step, a complex compound enzyme consisting of the biomolecule and a labeled molecular probe is used as a substrate;
The reaction is carried out in solution in the presence of a co-substrate.

d) Separation of the enzymatically reacted substrate from excess co-substrate in the separation step is carried out by means of a solid phase (2) to which the substrate is selectively bound. and e) in the evaluation phase the reacted substrate is measured on the solid phase (1) or by autoradiography.

Step b) above, i.e. the separation of the excess of labeled molecular probes, can be carried out by e.g. a column, especially an affinity ram.
Alternatively, it can be carried out with a particle suspension having an affinity effect.

The enzymatic reaction step can be carried out in this variant with an enzyme that is attached to the probe, which is also present attached to the biomolecule, or alternatively this enzyme can be carried out by some intervening step. It may be decomposed and separated from a complex compound consisting of the probe and the biomolecule.

In this case, this degradation can occur at the original binding site of the enzyme and the molecular probe (i.e., this is a reversible binding), but also on the molecular chain that was used to bind the enzyme and the molecular probe. (spacer molecule)
It is also possible to decompose the

Of course, such a decomposition is possible not only in the Kome variant, but also in various other variants of the method according to the invention.

In the identification step, the molecular probe labeled by the bound enzyme is added to the biomolecule bound to the solid phase I.

b) In a washing step, the excess of the labeled molecular probe is washed away and/or separated off.

C) In the enzymatic reaction step, the enzyme, which is a complex compound consisting of the biomolecule and the labeled molecular probe and bound to the solid phase (2), is reacted with the substrate and co-substrate present in the solution.

d) The unreacted co-substrate and optionally the substrate are also separated in the separation step, and the reacted substrate is combined with the solid phase (1). and e) in the evaluation step the reacted substrate is measured on the solid phase (1) or autoradiographically.

According to this variant, the biomolecule to which the molecular probe is attached and the reacted substrate are each present on a solid phase. The solid phases can also be identical, but in this case they are always adapted to each particular biomolecule and reacted substrate. The enzymatic reaction step can in this case again be reacted with the complete complex consisting of the biomolecule, the probe and the bound enzyme, or with the enzyme decomposed and separated as described in variant Ⅰ above. .

Another embodiment of this variant provides that the solid phase I, together with the biomolecules bound to it and the labeled molecular probes attached to it, is removed together with the gel after the process steps a) and b) or coated with another separating carrier material (iib
In this case, the substrate and cosubstrate are present, the enzymatic reaction takes place, and the reacted substrate is subsequently transferred from the gel onto the solid phase.

Variant: This variant represents one of the preferred embodiments according to the invention. According to this, each method step is carried out as follows.

a>m In another step, the bound enzyme-labeled molecular probe is added to some solid phase I and the bound biomolecule.

b) In a washing step the excess of labeled molecular probe is washed away and/or separated off.

C) In the enzymatic reaction step, a complex compound enzyme consisting of a biomolecule and a labeled molecular probe is bound to the solid phase I, and a substrate is bound to the solid phase II and a co-substrate present in the solution. react in the presence of

d) washing away or separating excess co-substrate in a separation step;

e) In the evaluation step, the reacted substrate is measured on the solid phase (2) or autoradiographically.

In this variant, it is possible to bring two solid supports, especially flat supports, into contact with each other and to start the enzyme reaction by adding a solution of a co-substrate, and this allows for the detection of various biomolecules. It is also a completely new technique to implement. Preference is given in this case to flat plate carriers, such as those described, for example, in East German Economic Patent Application No. C07G/296.754-2. However, it is likewise possible to cause differentially coated granules, such as latex particles made of synthetic resins with magnetic cores, to react with one another.

The enzyme may be coupled to (or decomposed from) the molecular probe in reaction step C). In this latter case, especially if ON8 is used as substrate and the enzyme is conjugated to, for example, a ligase or It is advantageous if it is another repair enzyme.

In the identification step, it is possible in all variants to initiate the branching of the reaction already here or to interpose another molecule between the biomolecule and the labeled molecular probe. It is. It may be pointed out that this is an advantage in the case of sensitive enzymes. In this case, it is possible to use the known avidin/biotin system and thereby take advantage of the possible intensity amplification already in the identification step. As a final step in this identification step, the labeled molecular probe in this case does not directly identify the biomolecule itself, but the partner molecule of the preceding reaction in the cascade. Therefore, in the identification step, a biomolecule is first identified by or together with one or more molecules with specific affinity in one or more identification systems and in one or more reaction steps. The possibility arises that a molecular probe used and then attached to an enzyme will react with at least one or more of these molecules.

Suitable solid phases are a series of substances which are known according to the state of the art and which are used as carrier substances for various biological molecules. The solid phase I, to which the biomolecules are bound, can be a support such that they are bound by covalent bonds, by ionic bonds or by electrostatic interactions. For example nitrocellulose and polyvinylidene difluorite by electrostatic interaction.

are bonded together, whereas polyamites (such as cinnabar membranes) or charged polyamides as well as diethylaminoalkyl cellulose are bonded by ionic forces. Covalently bonded carrier materials are, for example, cellulose derivatives, such as diazubenzyloxymethylcellulose,
cellulose activated by siamerc 1 collide (such as that according to East Germany Economic Patent No. 220,316), and the like. Additionally, it is possible to use glasses without or with activation of the polystyrene (for example by sulfonation or amination/diazotization, etc.) or chemically activated. For carrying out the method according to the invention, preferably a solid phase I is used, which is present in the form of a tabular structure. Solid phase (1) is a carrier that can bind substrates, depending on the enzyme that is bound to the molecular probe. Here again, depending on the substrate, it is possible to use carrier substances which are bound by covalent bonds, as already mentioned, by ionic forces or by electrostatic interactions. Therefore, the solid phase ■ again includes nitrocellulose, polyvinylidene difluoride, polyamide, charged polyamide, diethylaminoethylcellulose, cellulose activated with cyanuric chloride, activated polystyrene, and chemically activated glass. or other cellulose derivatives. Among them, celluloses that bond by ionic forces, such as phosphorous cellulose (a reaction product of cellulose and phosphoric acid, and thus a phosphoric acid group is bonded to the anhydroglucose unit of cellulose), carboxymethyl cellulose or cyanuric chloride, and amino groups. East German Economic Patent Application No. C07 modified with compounds having carboxylic acid groups, phosphoric acid groups or sulfonic acid groups
G/296.754-2 or East German Economic Patent No. 2
Examples include cellulose derivatives such as those described in No. 37.844.

In one particular embodiment, the solid phase may be in the form of an optically transparent medium, for example in the form of a film, to which the substrate can be bound. . Such substances are particularly suitable for everyday evaluation tasks, such as for example medical assays,
This is because a large number of samples can be mechanically evaluated simultaneously. The solid phase (3) is preferably used as a plate-like material for the detection, in which case the film described above represents the case of an optically transparent plate-like material. substrate and/or
Alternatively, the solid phase (2) capable of selectively binding the enzymatically reacted substrate can be a gel or other separation carrier material. Similarly, this solid phase (1) can be a carrier bound covalently, ionicly or by electrostatic interaction.

This includes known substances which are essentially identical to those already described above, since the reacted substrates can be of a very wide variety of properties, i.e. for example nitrocellulose, polyvinylidene difluoride. , polyamide, modified polyamide, diazobenzylogisimethyl cellulose, diethylaminoethyl cellulose, cellulose activated by cyanuric chloride, activated polystyrene, chemically activated glass, phosphorus or sulfonic acid groups An example of cellulose modified with a compound having the following. This solid phase (1) can also be used in the form of a technologically permeable medium. The same various possibilities and variants as in the case of solid phase (1) apply here as well. Detection using this solid phase (1) is preferably carried out as a flat material, if it is formed, for example, as a film, membrane or crimped fiber fleece.

The evaluation of the optically transparent solid phase, especially when it is configured in the form of a film, can be carried out by various optical methods, for example by densitometry in the case of colored products, by colorimetry, or by colorimetry. Done by photographic technology. Living cells can be placed on the solid phase (1) in a conventional manner.

In other words, these cells can be detected using the spot method (Tuepfe).
After separation in a gel or other separation carrier material by means of the so-called transfer or transfer method (plotting method). One of the preferred embodiments of the method according to the invention is? A biomolecule that may be present in a sample, contained in a gel, or bound to solid phase I is identified by an aminoglucoside-phosphotransferase-conjugated molecular probe; reacting an aminoglucoside-phosphate transferase bound to or decomposed therefrom with a substrate in the presence of a co-substrate;
The phosphorylated substrate is then transferred to the solid phase ■ or ■
and carrying out the method in several woody process steps as follows: a) Identification step: in this step the combined aminoglucoside-phosphate 1-transferase A labeled molecular probe is attached to a biomolecule that is present in solution, contained within a gel, or bound to a solid phase and has a specific affinity for it.

b) Washing step: In this step the excess of the labeled molecular probe is washed away and/or separated off.

C) Enzyme reaction step: In this step, phosphorylation of the substrate is carried out by reaction with the substrate in the presence of its aminoglucoside-phosphotransferase co-substrate.

d) Separation step: In this step the unreacted co-substrate and optionally the substrate are washed away or separated off.

e) In the evaluation step, the phosphorylated substrate is measured on a solid phase (1) or (2) or detected by special detection methods.

Aminoglucoside-phosphate transferases are enzymes that cleave off phosphate residues from adenosine triphosphate (ATP) and transfer the phosphate residues onto substrates that can be phosphorylated. The substrate can be in solution, entrapped within a carrier, or bound to some solid phase. Acts as a co-substrate in the ATP reaction.

Instead of this ATPO, 32p-labeled ATP (“P-
When using γ-ATP), the aminoglucoside-phosphate transferase transfers the 32p-labeled phosphate onto the substrate. The enzyme attached to the molecular probe does not perform this reaction only once, but allows it to proceed until the following three conditions are met. In other words, ○substrate exists.O cosubstrate (i.e. ATP or 32P-γ-ATP)
However, the method according to the invention requires that each enzyme molecule transfers a number of phosphate residues to a number of substrate molecules, thereby providing an amplification factor of at least 10 or more. However, in most cases it is substantially higher than this. This method creates an enlarged replica of the molecular probe in the phase in which the substrate is present. Therefore, according to the method according to the present invention, detection is not carried out in the phase in which the molecular probe is present, but in the phase in which the substrate is present. If multiple phases containing substrates are desired in the presence of co-substrates for a given molecular probe (on both sides,
or back and forth), a number of magnified replicas can be created by a given biomolecule to be detected. The complex compound consisting of the probe and enzyme can be decomposed and separated from the original biomolecule again by conventional methods, and the detection can optionally be repeated using a molecular probe similarly labeled with aminoglucoside-phosphotransferase. can. Among the various transferases aminoglucoside-phosphate transferase surprisingly occupies a special position which can be expressed as follows: it has high thermostability; Maintained even after heating to 100°C,
Longer incubations at higher temperatures are therefore possible.

It has good surfactant stability: the enzyme still has enzymatic activity despite its secondary structure being changed by various surfactants.

○ Its specific affinity is very high.

o It is readily available and can be obtained relatively easily from the lysed product of microorganisms (Zellysat).

Therefore, 7-minoglucoside phosphate transferase is, for example, 3. Magazine Bu11. by llvremeas.
Soc.

Chim, Biol, (1968) 50. 1st page 1
This significantly satisfies the requirements for enzymes to be coupled to other biomolecules, such as those listed below. They are microbiologically e.g. B, Re1s
s.

R, Sprengel, H, Will and H, 5ch
magazine Gene ((1984) 30.
It can be manufactured as described on pages 211-218. A series of techniques for the detection of various biomolecules are possible based on the many possibilities, such as the ability to couple various molecular probes to aminoglucoside-phosphate transferases. First of all, various probes of immunoactivity, as molecular probes suitable for binding,
Compounds having affinity for nucleic acids, various proteins, or biomolecules can be used.

Immune activity probes include monoclonal and polyclonal antibodies, antisera, and proteins, especially protein 8,
Includes natural or synthetic peptides. Nucleic acids include single-stranded, double-stranded, or partially double-stranded DNA.
A or fragments thereof, oligodeoxyribonucleosides, RNA or fragments thereof or oligoribonucleotides can be used. Proteins that have specific affinity for certain biomolecules include, for example, avidin,
Streptavidin, pretamine, gelatin, fetuin, vepustin, lectins and numerous enzymes.

The last compounds that have affinity for certain biomolecules are biotin, n-aminophenylborate, and Clbacron.
It is Blue F30A. The protein with specific affinity for other biomolecules is preferably avidin or streptavidin in this case. The compound that has an affinity for a certain biomolecule is preferably biotin.

There are two major groups of enzymes: transferases (Group 2 according to E, C) and (Group 6 according to E, C1). Among transferases, acyltransferases (E
, C, 2, 3), enzymes that transfer 18-containing groups (E
, C, 2, 7) or polymerases (E, C.

Enzymes such as 2.7.7) are preferred. Among them, the following enzymes are considered particularly suitable for carrying out the method according to the invention: niprotiine kinase (E,C,2,7,L), aminoglucoside-phosphate transferase (E,C,2,7,
, 1), especially kanamine-phosphotransferase (E, C.

2.7, 1.95), T4-polynucleotide-kinase (E, C, 2, 7, 1, 78), nucleotidyl transferase (E, C, 2, 7, 7), DNA Δ polymerase (E ,C,2,7,7゜7), polynucleotide-adenyltransferase (E,C,2,7,
7,19), DNA-nucleotidyl oxotransferase (E, C, 2, 7, 7, 31>, rehertase (E, C, 2, 7, 7, 49), phosphate-forming antibody (repair enzyme) (E, C, 6, 5), DNA Δ-ligase (lE, c, 6.9.11).

All of these enzymes react with certain specific substrates. The various substrates listed below are therefore not effective for all enzymes, but only for those enzymes for which the substrate is specific. Although many substrates are reacted by many enzymes, however, each generally reacts by a different mechanism and in a different manner or with the formation of different reaction products. Substrates thus include chromphenicol, aminoglucoside antibiotics, inter alia kanamycin, neomycin, gentamicin and paromomycin, phosphorylatable proteins such as histones, DNA, RNA, deoxyribonucleotides or ribo-oligonucleotides, deoxyribopolynucleotides, etc. Nucleotide or rib polynucleotide, double stranded DNA to single stranded DNA + primer (ie starter).

These enzymes require cosubstrates in addition to their substrates.

This co-substrate is the building block used for substrate change. In the case of an acyltransferase, the cosubstrate can therefore be, for example, 14G- or 311-labeled acetate. In the case of phosphotransferases, the cosubstrate is generally adenosine triphosphate and/or triphosphate.
It is adenosine triphosphate (ATP) labeled with 2p. In addition, the four deoxynucleoside triphosphates (those of adenosine, guanosine, cytosine and thymidine, respectively) can also be transferred. Labeling of the deoxynucleoside triphosphate can be carried out in its alpha or gamma position and reacts with one or the other form, respectively, depending on the enzyme. Deoxynucleoside triphosphates other than by 32p can be converted to 35S, 3H or 1
Labeling with 25I is also possible, in which case each nucleoside may be labeled, or only one nucleoside may be labeled.

Furthermore, the co-substrate can be a biotinylated nucleotide, so that after the enzymatic reaction it is possible to react with an enzyme bound to, for example, avidin or streptavidin, thereby making it possible to react with an enzyme bound to, for example, avidin or streptavidin, thereby providing a means for evaluation with a colored substance. may be available.

Other co-substrates include T, which is already labeled.
NA or deoxyribopolynucleotides can optionally be used in a mixture with unlabeled molecules, which are mentioned in particular as co-substrates for ligases.

Preferred enzymes for carrying out the method according to the invention are aminoglucoside-phosphate transferases. These are inter alia kanamycin-phosphate transferase, or aminoglucoside-neomycin-phosphate transferase I, also designated APH6 or API13.
and ■. Substrates for aminoglucoside-phosphotransferases are aminoglucoside-antibiotics or aminocyclitol-antibiotics [for this, see L.

Rjnehardt: Magazine J, Tnfect, Dis
eases (1969).

345-50]. Examples of such agents include neomycin, kanamycin, gentamicin, and bramycin. Preferably, kanamycin or neomycin is used as the substrate. The co-substrate is adenosine triphosphate (ATP) with a suspended curtain of label or with no suspended label. 3Zp-γ-ATP is usually used for detection, but it may be mixed with unlabeled ATP.

A preferred embodiment of the method according to the invention for detecting various biomolecules using aminoglucoside-phosphotransferases consists of a number of method steps that can be carried out in different ways and thus A series of embodiments exist for enzymes. In this case, each embodiment depends on the type of biomolecule, its shape, and depending on the case itself, the mode of binding to a carrier, the mode of binding to a substrate, etc. The biomolecules to be detected can be in solution, in gels before or after separation operations, transferred onto solid supports by spotting methods, or onto flat supports etc. after separation methods from various gels. Exist = 48 = Can exist. The separation, transfer and immobilization of biomolecules on carriers are well known to those skilled in the art and will therefore not be described in detail here.

After the biomolecule has been brought into its form to be detected, the following method steps are carried out according to the invention: a) Identification step: in this step the molecular probe labeled by the bound aminoglucoside-phosphotransferase is attached to a biomolecule for which it has a specific affinity.

b) Washing step: In this step the excess of labeled molecular probe is washed away or separated off.

C) Enzymatic reaction step The aminoglucoside-phosphate transferase coupled to the bimolecular probe is reacted with the substrate with the participation of a co-substrate.

d) Separation step: In this step the unreacted co-substrate and optionally the substrate are washed away or separated off.

e) Evaluation stage: In this stage the phosphorylated substrate is measured or detected by special detection methods.

The implementation of each method step is combined with specific parameters that can be combined with one another and thus allows a large number of variants. Different conditions for each process step within each process variant are possible, so that the specific reaction conditions are not general, but can only be described in certain specific instances. In this regard, reference should be made to the embodiments given for the purpose of illustrating the invention.

Detection of phosphorylated substrates can also be carried out, for example, with a scintillation counter or by autoradiography. This autoradiography is particularly preferred in the case of flat carriers.

However, other detection methods are also possible, such as scanning or luminescence methods.

Similarly, the method according to the invention generally allows variations of its preferred embodiments in the individual steps of the method.

Some of these variants are described below.

Embodiment 1 Biomolecules are separated in a gel or other separation carrier material.

a) Identification Step 2 Contacting the separated material containing the biomolecules with a solution containing a molecular probe to which aminoglucoside-phosphotransferase is bound, so that the biomolecules and the probes become attached to each other. and thus allow a complex consisting of the biomolecule and the enzyme-conjugated probe to form in the gel.

b) Washing step: Excess labeled molecular probe is washed away from the gel.

C) Enzyme reaction step: A gel containing a complex consisting of a biomolecule and an enzyme-conjugated probe is contacted with a second gel containing a substrate and a co-substrate, thereby introducing such conditions. It phosphorylates the substrate.

d) Contacting the second gel containing the phosphorylated substrate with a solid phase (1) and selectively binding the phosphorylated substrate thereto.

e) After a washing step to remove excess substrate and/or co-substrate, the phosphorylated substrate on the solid phase (1) is detected by radioactivity measurements or by autoradiography.

In the second variant of Embodiment 1, step C), that is, the enzymatic reaction step, is carried out as follows: A gel containing a complex compound consisting of a biomolecule and a labeled molecular probe is heated as a substrate. This is carried out in such a way that the phosphorylated substrate is detected on solid phase (1) by contacting it in the presence of a co-substrate with solid phase (1) as bound thereto.

In this variant, the biomolecule is optionally present as a mixture of a number of biomolecules, and the labeled molecular probe is present in solution.

a) Identification step The molecular probe to which the niaminoglucoside-phosphate transferase is attached and the biomolecule to be detected are in solution and are added to each other in solution.

b) Washing step: Separating and removing excess labeled molecular probe.

C) Enzyme reaction step: the aminoglucoside-phosphate tranferase, which is bound to the molecular probe and whose complex is attached to the biomolecule, is reacted with the substrate and cosubstrate in solution.

d) Separation step: unreacted co-substrate and optionally substrate are separated off.

e) Evaluation step: measuring the activity of the phosphorylated substrate.

This variant represents another possibility when the biomolecule and the labeled molecular probe are present in solution but are to be detected on some solid phase.

a) Identification step: The biomolecule to be detected and the molecular probe bound to aminoglucoside-phosphotransferase are present in solution and react with each other there.

b) Washing step: Separating and removing excess labeled molecular probe.

C) Enzyme reaction step: the aminoglucoside-phosphate transferase, which is bound to the molecular probe and whose complex is attached to the biomolecule, is reacted with the substrate and co-substrate in solution.

d) Separation step Separation of the phosphorylated substrate from the excess co-substrate is carried out by means of a carrier (1) which selectively binds the two substrates.

e) Evaluation step: The activity of the phosphorylated substrate is determined or determined by autoradiography.

In carrying out this variant of the method according to the invention, the biomolecule is bound to some solid phase, such as a flat support or a dispersion of polymeric particles, and a labeled molecular probe is attached. It is then reacted with the substrate and co-substrate in solution.

a) Identification step: for biomolecules bound to a certain solid phase,
A molecular probe labeled by the bound aminoglucoside-phosphate transferase is attached.

b) Washing step: the excess of labeled molecular probe is washed away or separated off.

C) Enzyme reaction step: the complex of a biomolecule bound to a certain solid phase I and a labeled molecular probe is reacted with the substrate and co-substrate present in solution.

d) Separation step: unreacted co-substrate and optionally substrate are separated off and the phosphorylated substrate is optionally bound to the solid phase (1).

e) Evaluation step: The activity of the phosphorylated substrate is determined in solution or optionally on a solid phase (2) or determined radiographically.

This embodiment can be further modified by changing the enzymatic reaction steps, in which the solid phase r is combined with the biomolecules bound thereto and the labeled molecular probe bound thereto. is coated with a gel, in which the substrate and co-substrate are present to carry out the enzymatic reaction, and the phosphorylated substrate from the gel is subsequently deposited onto the solid phase ■. be transferred. Although the substrate and co-substrate exist in solution in the gel, they are also in a "sealed" state (entrapped state).

This variant represents a novel sandwich method in which not only the probe but also the substrate is bound to some solid phase and only the co-substrate is present in solution. This approach is particularly advantageous because, through the use of two solid phases, the binding of the molecular probe to the biomolecule, the binding of the substrate, and the enzymatic reaction can be carried out under very different reaction conditions; The solid phase remains unchanged with the complex compound bound to it consisting of biomolecules and labeled molecular probes and can therefore be used many times or the probe can be washed off and optionally This is because they can be replaced with others and thus detection using many probes can be performed.

a) Identification step: Biomolecules are bound to the solid phase (1), to which a molecular probe labeled with aminoglucoside-phosphate transferase is added.

b) Washing step: washing away and/or separating off excess labeled molecular probe.

C) Enzyme reaction step: Aminoglucoside-phosphate transferase, a complex compound consisting of a biomolecule bound to the solid phase (■) and a labeled molecular probe, is present in a solution with a substrate bound to the solid phase (■). The reaction is carried out in the presence of a co-substrate that is

d) Separation step: washing away or separating off excess co-substrate.

e) Evaluation step: The activity of the phosphorylated substrate on the solid phase (1) is measured or determined by autoradiography.

In all variants of the preferred embodiment of the method according to the invention, it is possible to carry out the reaction of the biomolecule of the identification step with the labeled molecular probe not directly, but via an intermediate step.

The biomolecule is, for example, first identified by a probe coupled to avidin and streptavidin or to biotin, which is then reacted with an aminoglucoside-phosphotransferase coupled to biotin, avidin or streptavidin. Can be done.

This principle states that in the identification step a biomolecule is first identified in one or more identification systems and in one or more reaction steps by one or more molecules with specific affinity, or by one or more can be generalized such that a molecular probe coupled to the aminoglucoside-phosphate transferase is reacted with at least one such molecule.

This gives yet another possibility, namely that the method can be repeated more than once.

Finally, the enzymatic reaction after the identification step and the washing step is used to decompose and separate the complex compound consisting of the biomolecule and the labeled molecular probe from the aminoglucoside-phosphotransferase and the molecular probe at the binding site again. It is likewise possible, for example, to carry out the coupling using different reactants which are themselves degradable or to carry out the coupling in such a way that the coupling is reversible for at least one of the two reactants. In this case, the enzyme can phosphorylate the substrate present in solution or after binding to another solid phase in the presence of a co-substrate, which is present in solution or bound to a solid. .

The carriers used for the binding of biomolecules, probes or substrates in preferred embodiments of the method according to the invention are again in different forms, e.g. powders, granules, gels, spheroids, fibers etc., and also dispersed. They can be constructed as liquids, emulsions, latexes, films, column packings, matrices, fleeces or planar carriers. The material can be a natural or synthetic polymer or a mixture thereof. The bonding takes place via covalent bonds if the solid phase is activated by a corresponding compound, such as cyanuric chloride or bromcyanide. Bonding can also be by ionic bonding or electrostatic interactions.

Support materials are therefore suitable as solid phase I, which make it possible to combine all three bonding principles. Examples of solid phases that are electrostatically, i.e., for example hydrophobic, dipole-dipole or bonded by van der Waals forces or interactions, are inter alia nitrocellulose or polyvinylidene difluoride. be. Examples of solid phases bound by ionic forces are, for example, modified and ionically charged polyamides, especially nylon membranes, cellulose derivatives substituted by ionic groups, such as diethylaminoethylcellulose, and Sulfonated polystyrene, etc.

Examples of covalently bonded solid phases are diazobenzyloxymethyl cellulose, cellulose activated with cyanuric chloride, cellulose activated with bromcyan, or activated polystyrene or glass. Furthermore,
Glasses can be used that are chemically activated and can be bonded by ionic and/or covalent bonds. Particular preference is given to flat supports consisting of the various materials described above for carrying out the method according to the invention.

The solid phase (1) is, inter alia, a carrier of negatively charged ionic bonds or a covalently bonded carrier. Anionically charged carriers include, among others, various cellulose derivatives, such as phosphorous cellulose, carboxymethyl cellulose, modified with cyanuric chloride, as described in East German Economic Patent No. 237 844, incorporated by reference. Cellulose, etc., which are subsequently reacted with at least one amino acid and a compound having one carboxylic acid group, phosphoric acid group, or sulfonic acid group in the molecule, are preferred. Suitable covalently bonded supports are the same as mentioned for solid phase I. Particularly preferred for the method according to the invention is a solid phase in the form of a plate.
It is.

The solid phase (2), which performs selective binding, is a negatively ionically charged carrier. Here, again, various materials coated with various cellulose derivatives or sulfonated polystyrenes or acrylates with carboxylic acid groups are passed. Phosphorous cellulose, carboxymethyl cellulose or activated with cyanuric chloride according to East German Economic Patent No. 237 844 and subsequently at least one amino group and at least one carboxylic, phosphoric or sulfonic acid group in the molecule. Preferred are cellulose derivatives, such as those made from cellulose by reaction with compounds containing the cellulose.

One preferred method for bonding the substrate to the solid phase is described in East German Economic Patent No. 254,012, which is hereby incorporated by reference. Attaching an enzyme to a molecular probe for its labeling can be accomplished by a variety of methods, such as those described for certain enzymes. Attachment of the probe to the enzyme can be carried out covalently, for example, by a series of bifunctional coupling reagents. Such coupling reagents include, for example, glutaraldehyde, bis-succinimidyl esters such as bis-sulfosuccinimidyl suberet I and disuccinimidyl tartrate, dimethyl suberimidate and dimethyl adipimidate. bisimidate,
Azides such as N-5-azido-2-nitrobenzoyloxysuccinimide or 4,4°-dithio-bis(phenyl azide). In this case, one binding molecule is 2
It can have two different groups or optionally several reducible or photochemically decomposable groups in the molecule. An example of such a coupling reagent is N-succinimidyl-(4-azidophenyl)
-1,3''-dithiopropionate or sulfosuccinimidyl-2-(m-azido-0-nido-I-cohenzamide)-ethyl-1,3''-dithiopropione-1.

Additionally, the molecular probe and the enzyme may be directly covalently linked to each other. Such bonding can be carried out, for example, in the presence of a condensing agent such as a carbodiimide.

Attachment of enzymes to molecular probes can likewise be achieved via ionic bonds. Ionic bonds may be formed directly between proteins and DNA, for example. However, in general, polyethyleneimines, polyelectrolytes or ampholytes with different molecular weights, such as
A variety of agents are used to enable binding.

Since enzymes range from hydrophobic to hydrophilic regions, it is also possible to couple them to molecular probes by electrostatic interactions. Electrostatic interactions should also be understood to include van der Waals forces, dipole-dipole interactions, and hydrogen bonds.

Using fusion proteins as labeled molecular probes = 64
= Used in special cases, it can be natural or genetically engineered. In this case the enzyme and the molecular probe are already linked to each other.

The binding site between the molecular probe and the enzyme can be degradable (eg, enzymatically).

The conjugation of the aminoglucoside-phosphotransferase used for the preferred embodiment of the method according to the invention to the molecular probe is novel and therefore the enzyme-labeled molecular probe so produced is also a novel product. . They form the second object of the method according to the invention. The production of molecular probes labeled with this aminoglucoside-phosphate transferase is likewise novel and constitutes yet another object of the method according to the invention. According to the invention, these molecular probes are attached to the aminoglucoside-phosphate transferase directly or via a spacer.

Surprisingly, it has been found that aminoglucoside-phosphate transferases have various properties that make them particularly suitable for detecting biomolecules with signal amplification. These special properties are as follows.

○ High thermostability: The enzymes maintain their activity even after warming to 100°C, meet the requirements for storage stability, and allow long incubations to differentiate between biomolecules and probes. Not only during the reaction between the enzyme and the substrate, but also during the reaction between the enzyme and the substrate.

O Detergent stability: These enzymes, unlike other enzymes, are still enzymatically active despite changes in their secondary structure by detergents.

o High specific affinity: A strongly radioactively labeled substrate can be created by transferring 32P-labeled phosphate to the substrate.

O Availability Niaminoglucoside-phosphate transferases can be obtained relatively easily from microbial lysate products (Zellysat).

The ability of aminogelcondo-phosphate 1-transferase to transfer radiolabeled phosphate without hindrance under various reaction conditions that are harsh for the enzyme and its normal hybridization conditions (i.e., up to 48 hours at 65°C) ) clearly indicates the special status of these enzymes and thus makes them particularly suitable for various detection methods.

Labeled molecular probes according to the invention create reaction products that are highly radioactive and therefore easily detectable. Detection is accomplished even at concentrations of the detection molecule that cannot be detected by conventionally known detection methods.

Suitable molecular probes for binding to aminoglucoside-phosphate transferases are as follows.

a) Immune activation probes, especially monoclonal,
or polyclonal antibodies, antiserum, proteins, especially protein A, peptides (not only natural ones but also those made synthetically or by genetic engineering) b) Nuclear M group or their derivatives or fragments thereof, among others Tree 1i DNA or fragments thereof, double-stranded or partially double-stranded DNVI or fragments thereof, oligodeoxyribonucleotides, tlN8 or fragments thereof or oligoribonucleotides or genetically engineered Hypolydization probesC) Proteins that have a specific affinity for other biomolecules, such as streptavidin, avidin, lectins, or specific various enzymes that can bind.d) Proteins that have a specific affinity for other biomolecules. Compounds with biotin, m-aminophenylborate or C1bacron, among others
Blue F3GA aminoglucoside-phosphate transferases are also called aminocyclitol-phosphate transferases. These include the relatively well-known neomycin-phosphate transferases I and ■ [kanamycin-phosphate transferases I and ■, lividomycin-phosphate transferases 〇 8- Regarding nomenclature, M, J, 1laas, J, E,
Dowding: Magazine Methods in En
Zymology (1975) 43.611-617
Page and Enzyme Nomenclature 19
84 i Nomenclature Committee
e, International Union
of Biochemistry in New York Ac.
Streptomycin-phosphate transferase (abbreviation: NPTI or NPTll), APR expressed by O-phosphorylation, such as API (6 (see above, L. Rinehardt)) Among the above-mentioned aminoglucoside-phosphate transferases, the enzyme-labeled molecular probes of soomycin-phosphate transferases I and 1 according to the present invention are preferred.

The enzyme can be isolated from genetically engineered host cells or from wild species; this method is described, for example, in B. Reiss, R. Sprengel, H.W.
ill+I1.5challer”: Magazine Gene
(1984) 30.21148, and T. Trie.
u-Cuot and P, Courvalin: J.
Antimior.

Chemother, (1986) 1B, 5up
p1. C, 93-102.

Coupling of this enzyme to a molecular probe can be accomplished using a number of methods and reagents. That is, in the first embodiment, a chemical reaction can be carried out by forming a covalent bond and, for example, by reaction with a difunctional reactant or by condensation in the presence of a condensing agent. . In a second embodiment, the ionic bonding between the molecular probe and the labeling enzyme is achieved by the presence of various agents that form ionic bonds with, for example, polyelectrolytes, ampholytes, or both molecules. formed by reacting with In another embodiment, the molecular probe and the labeling enzyme are bonded to each other by forming electrostatic interactions, for example by denaturing both molecules or by reacting them with a detergent. combine. In the fourth embodiment, a natural, synthetically produced, or genetically engineered fusion protein consisting of an enzyme and a protein as a molecular probe is used as a labeled molecular probe, or Create and use.

Among the numerous possibilities currently available for conjugation of enzymes and molecular probes, preferred methods and products so produced are described, provided that those skilled in the art will be aware of other methods listed herein. It is obvious to the person skilled in the art that besides bifunctional molecules or other methods, genetic engineering methods and constructs, among others, can be used as well and labeled molecular probes according to the invention can then be obtained. be. A first preferred embodiment of the method according to the invention for forming this labeled molecular probe by covalent bonding of the aminoglucoside-phosphotransferase and the molecular probe comprises As a bonding part, a spacer having the following structure is used, in which case one or more ring structures may be included in the molecular chain.

This binding essentially does not inhibit the probe's ability to discriminate for biological molecules or the enzyme's ability to operate.

Formation of covalent bonds to create labeled molecular probes according to the invention may include, for example, glutaraldehyde, p
-Henzoquinone, bis-succinimidyl ester, bisimide! - and/or azides. Bis-succinimidyl esters include, for example, bis-2-(succinimidoxylponyloxy)-ethylsulfone, bis-(sulfosuccinimidyl)-suberate, disuccinimidyl sulfate, disuccinimidyl tartare. 1., dithiobis-(succinimidyl propionate), 3.3'-dithiobis-(sulfosuccinimidyl)-propionate, ethylene glycol bis-(succinimidyl succinate), ethylene glycol bis-(sulfosuccinimidyl)-propionate cinimidyl succinate), bis-2-sulfosuccinimidoxylupoxyloxy)-ethyl sulfone, and disulfosuccinimidyl tartrate. Examples of bisimidates include dimethylazibimidate, dimethyl-3,3°-dithiobis-(
propionimidate), dimethyl-pimelinimidate,
Includes dimethyl suberimidate, etc. Examples of azides are N-5-azido-2-nitropenzoyloxysuccinimide, p-azidophenacyl bromide, p-azidophenylglyph 2-oxal, N-(4-azidophenylthio)-phthalimide, 4, 4゛-dithiobis-(phenyl azide),
4-(azidophenyl)-L4-dithio-butyric acid imide-
Ethyl ester, 4-fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoate, N-hydroxysuccinimidyl-4-azidosalicylic acid, 4-azidobenzoic acid imidomethyl ester, 2- Diazo-3,3,3-trifluoropropionyl chloride, p-nitrophenyl-2-diazo-3,3
, 3-trifluoropropionate, N-succinimidyl-6-(4'-azido-2''-nitrophenylamino)-hexanoate. This class also includes degradable linking reagents, which are described in the present invention. products which can be decomposed and separated again in the reaction step of the detection method by being used in the production of probes according to the invention, and whose enzymatic reactions can therefore be carried out with freely movable enzymes, resulting in mechanical benefits. is created.

These degradable coupling reagents include, for example, N-succinimidyl-(4-azidophenyl)-1,3'-dithioprobionate, sulfosuccinimidyl-2-(m-azido-0-2I-lovenzamide). )-ethyl-1,3'-dithiopropionate, sulfosuccinimidyl-(4-azidophenyl-dithio)-propionate-1, sulfosuccinimidyl-2-(p-azidozaritylamide) )-ethyl-1,3-dithiopropione-1.

Direct covalent linkage of enzymes and molecular probes is also possible. An example thereof is the condensation of both molecules in the presence of condensing agents such as carbodiimides.

Fusion proteins are a special case of covalent bonding of molecular probes and enzymes. Although they are most often created genetically, they can also be made synthetically,
At this time, the probe protein and enzyme bind to each other in the form of a peptide.

In this case, in the case of a genetically engineered fusion protein, the genetic information of the probe protein and the enzyme are adjacent, and a normal reading screen (Leseras t
er), which encodes a different amino acid sequence (e.g. for the protease cleavage site) between the probe-encoding DNA and the enzyme-encoding DNA. ) Not only can NA fragments be inserted, but also certain DNA fragments can be inserted.
Fragments may appear repeatedly (eg, the formation of a fusion protein consisting of multiple probe molecules and/or enzyme molecules).

The information present on these hectors is translated by the host organism, for example bacteria such as E. Co11, into a conventional amino acid sequence, and the fusion protein can be obtained by microbial engineering.

A second embodiment of the method according to the invention and the labeled molecular probe according to the invention is the binding of the probe and the labeled enzyme via an ionic group. In this case, various compounds can be used, such as polyethyleneimines, polyelectrolytes, or ampholytes, to which not only enzymes but also molecular probes are bound.

According to a third embodiment of the method according to the invention and the labeled molecular probe according to the invention, they are bound by electrostatic interactions. These electrostatic interactions include, for example, hydrophilic-hydrophobic interactions.
It also relates to a method for producing a molecular probe labeled using phosphotransferase. This method involves adding an aminoglucoside to a molecular probe or a mixture of several molecular probes.
This is achieved by binding phosphotransferase.

Probes include the various immunoreactive probes mentioned above, Nuclein-squeu
re), proteins and various compounds can be used. As aminoglucoside-phosphate transferase, neomycin-phosphate transferase ■ and ■
is preferred. The old method and other variants for carrying out this method are the same as before.

A first embodiment of the method according to the invention for the production of a labeled molecular probe comprises binding the molecular probe by forming a direct covalent bond consisting of an aminoglucoside-phosphotransferase; This is achieved by covalently bonding via a binding reagent.

As the binding reagent, those already mentioned can be mentioned. Reaction conditions depend on the type of molecular probe, aminoglucoside-phosphate transferase and its binding reagent.

However, the already mentioned standard recipes for forming the bond can also be used.

Preferred embodiments for various attachment aspects include spacer groups containing from 1 to 150 atoms in the chain and optionally structures in the chain that bind the labeled molecular probe to as many substrate molecules as possible. Care should be taken to use such things that give the opportunity to reach. Preferably glutaraldehyde, benzoquinone, bis-succinimidyl esters, as already described;
Coupling using bisimidates or azides is used.

A second embodiment of the method according to the invention for producing enzyme-labeled molecular probes is to carry out the binding by the formation of ionic bonds.

This binding can be carried out directly between the protein (enzyme) in this case and the DNA as molecular probe.

Preferably a variant is used in which this bonding is carried out by means of one compound in which both reactants are capable of forming an ionic bond. Such compounds are, for example, polyethylenimines with different molecular weights, polyelectrolytes or ampholytes, but also oligomeric or polymeric compounds with charges in the molecule.

A third embodiment of the method according to the invention for producing an enzyme-labeled molecular probe is to diversify the electrostatic interaction of the enzyme and the molecular probe for binding.

Such interactions may be hydrophilic-hydrophobic or dipole-dipole interactions.

A fourth embodiment of the method according to the invention for producing enzyme-labeled molecular probes comprises a fusion protein (Fus+) in which probe and enzyme are directly linked to each other.
on'viprotein) produced by genetic engineering or synthetic methods. T. for general procedures for the production of various hector and fusion proteins.

Maniaf, is, E, F, Fr1tsch,
J, Sambrook: Molecular C
loning''-Cold Spring Harbor
Laboratory (1982), pages 97 et seq. and pages 247 et seq. The details of the production and use of a novel fusion protein consisting of neomycin-phosphate transferase 1 and protein 8, which is also a new method, are described in the Examples below.

The detection method according to the invention of various biomolecules using enzyme-labeled molecular probes represents a new principle of biomolecule detection. The use of molecular probes with bound enzymes and detection of reacted substrates on a solid phase comprising a number of embodiments encompasses many embodiments. This method can be applied to a large number of biomolecules and therefore can be used universally. In its application, an amplification factor of at least 10 or more is achieved, although this is significantly exceeded in the design. That = 7
9- The achievable amplification factors (Vers. 5) depend on the materials used and the chosen method embodiment and can only be described strictly in special cases. With these different embodiments, it is possible that the biomolecules used in carrying out a particular variant can be detected by means of a number of probes, ie the plots, for example, can be used again. The use of the method according to the invention is not limited only to the few specific embodiments described here, but many other variations of this method are possible.

A preferred embodiment of the detection method using a molecular probe coupled to an aminoglucoside-phosphate transferase of a biomolecule according to the present invention allows various biomolecules to be detected by a molecular probe with higher sensitivity than previously known. It is.

Additionally, detection of various biomolecules is kept in a second, separate state. This indirect detection method has great significance in various detections in genetic biology, in the detection of various human genetic and biological diseases, and in the detection of virus degradation products. This is because the detection can be carried out with a large number of probes, the various biological molecules are not damaged by the mild incubation, and finally high amplification rates are obtained, which could not be achieved hitherto.

The labeled molecular probes made for a preferred embodiment of the method according to the invention are novel products with a variety of unusual properties and can be used in particular for the detection of biomolecules.

The enzymes preferably used, namely aminoglucoside-phosphate transferases, are distinguished by a high stability in all detection steps, i.e. the labeled molecular probe according to the invention can be used at temperatures of around 65°C in the hybridization process. It can be used in

By using this probe, extremely high amplification rates can be obtained in autoradiographic detection.

Until now, such amplification rates have not been achieved using enzyme-bound probes.

In other words, 10 gg of protein could be detected reliably.

In conventional detection of various proteins, +251-protein 8 is used at -7°C for amounts in the long range.
The autoradiography time was 1 to 3 days. In the method according to the invention, autoradiography times of 30 minutes to 1 hour at room temperature under the above conditions are typical for strong signals.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to various examples.

“Nagoya” 11-hour PBS: NaC18g, KHgPOt
0.2g 1Na2HPO, 12H202,9g and MCl0.2g are dissolved in water to make the whole 1p (pH=7.4) TBS: NaC10.15mol, Tris-
(Hydroxymethyl)-aminomethane (hereinafter abbreviated as Tris) 50 mmol (pH=7.4) GET Nidris/HCI 2.5 mmol (pH=8.
0) Glucose 5 mmol, ethylenediaminetetraacetic acid tetrasodium salt (EDT8) 1 mmol lysis buffer ditris/) lc1125 mmol (p
l(=6.8) Sodium dodecylsulfonate (SO5) 2%, glycerone 10%, β-mercaptoethanol 10% Elution buffer: Tris/HCl 20 mmol (pl(=
7.4) Dithiothreitol 10 mmol, SO50, 1% lysis buffer Ni Tris/HCl 20 mmol (ρ1 (=
8.8) Dissolve 48 g of urea in 100 mf of enzyme buffer Nitris/HCl 20 molar (pH=
7.2) NH4Cl 125 mmol, MgClz 20
Binding of Polyclonal Antibodies to Kanamycin-Phosphate Transferase, Dithiothreitol 10 mg of 1 mR anti-large serum albumin antiserum (homemade) containing 0.9% NaCl in 3 ml of ethanol.
of benzoquinone at room temperature for 1 hour. The excess benzoquinone is then separated by a Sephadex 625 column and tested in the benzoquinone/antibody immunoassay. The tested fraction is reacted with 1 mg of kanamycin-phosphate transferase in 1 molar bicarbonate buffer (pH=9) overnight at room temperature in the absence of light.

The reaction product is dialyzed against 0.9% NaCI solution overnight before use. After dialysis, divide into several equal parts and give 120
Alternatively, the antibody/benzoquinone/kanamycin-phosphotransferase product may be used immediately in immunological reactions.

H5A using an antibody conjugated with kanamycin-phosphotransferase to detect the reacted substrate on the solid phase
Immunological reactions of S. mixtures of various labeling proteins and ISA were separated by electrophoresis in polyacrylamide gels (FAA gels) and transferred onto nitrocellulose filters by electrotransfer (plots). Incubation for 4 hours at room temperature in TBS [0.9% saline buffered with Tris-(hydroxymethyl)-aminomethane] and 1% gelatin with the antibody conjugated to kanamycin-phosphotransferase according to Example 1 above ( Dilution rate' = 1:500)
. The plots containing the antibody conjugated to the enzyme were then washed four times for 15 minutes each with TBS and 20 mmol Tris/HCI (pH=7.2), 125 mmol NH, CI, 1 Contains mmol dithionysulfur, 20 mmol MgCl2, and 2 mmol adenosine triphosphate (octaTP), l mI! 1 μC per
Incubate for 30 minutes at 37° C. using a 1% agarose gel (made in reaction buffer) containing γ-32p-8TP and 20 ng kanamycin per I mR. This agarose gel was placed on a Keno Ibara cellulose plate carrier, and kanamycin was transferred to this phosphorus cellulose (cellulose amount: 0.5 kg/
3 hours/room temperature). Detection of 3Zp-labeled kanamycin is performed by autoradiography on phosphorous cellulose plate supports (2 hours, room temperature).

Immunological Detection of H3^ with Kanamycin-Phosphate Transferase Labeled Antibodies on Solid Phase ③ Large serum is separated electrophoretically in a 1.5% agarose gel. The gel is incubated with antibodies conjugated to kanamycin-phosphotransferase for 1 hour at room temperature in TBS containing 2% bovine serum albumin (ISA) and then washed for 1 hour with TBE. The gel was first prepared as in Example 2 above in 20 mmol Tris/llCl (pH=7.2), 125 mmol Nl+4. CI, 1 mmol dithioerythrol, 20 mmol MgCh and 2 mmol ATP, incubated for 30 min in reaction buffer containing 1 μCi of PT-ATP, followed by cyanuric chloride and then sulfanilic acid. East German Economic Patent Application No. C0 consisting of reacted cellulose and having kanamycin ionically bound to this product.
7G/296.754-2 and reacted for 30 minutes at 37°C. This substrate and the carrier containing the phosphorylated substrate were washed (30 min in water).
After 1 minute), it is autoradiographed for 1 hour at room temperature and the 'P-labeled kanamycin on the support 1 is detected, thereby demonstrating the presence of ISA in the separating gel.

Coupling of neomycin-pv+-transferase to protein using glutaraldehyde in H. coli (NPT U)
A 10 ml cell pellet (Zell) of a cell culture (pRK89) of
pellet) into 1 mR of lysis buffer,
Ultrasonic irradiation for 1 minute, boil for 3 minutes, and ultrasonic irradiation again. The remaining cells are removed by centrifugation for 5 minutes at 4°C. This lysis product was dissolved in polyacrylamide/SO
Place it on the gel prepared in step 3 and separate it by electrophoresis.

The gel is rinsed with ice-cold water and placed in a 0.25M Kc+ solution until it shows good discrimination against the dark substrate.

Cut the NPT II band with a scalpel and cut the gel into small dice. For elution of proteins from the gel, it is covered with elution buffer and shaken for an initial 3 hours at room temperature. Elute once again overnight after changing the buffer.

The solution W so obtained is centrifuged at -rooOrpm, and 4 volumes of cold acetone are added to the supernatant and left to stand at -20°C for 2 to 3 hours. Next, centrifuge at 4000 rpm in an o'cp chamber.

The precipitate was dissolved in 20 mmol Tris/HCI (pl+=7.
Wash in the cold with a solution consisting of -4 parts of step 4) and 4 parts of acetone, and centrifuge once more. The cell pellet is dissolved in 200 μp lysis buffer and then dialyzed overnight against 20 mmol pH 7 sodium phosphate buffer (500 μp
mff, 2 changes, 4°C). The final volume is approximately 200μ! and contains approximately 200 μg of NPT ff with little residual impurity of E. coli protein.

b) Coupling with glutaraldehyde 400 μg protein A (Pharmacia A
B) and the NPT purified in a) above [400,
17g of PB in a dialysis tube with a volume of 1.5ml.
Dialyze against S (4°C, 800 min, 2 changes, 16
time). After the dialysate was transferred into a small glass tube, 6 μρ of glutaraldehyde (25% concentration solution:
5erva) and allowed to react at room temperature for 2 hours with gentle shaking. This product was then filled into the dialysis tube again and first added to PBS (4 hours at 4°C) and then to TBS (4°C 1800 m
1, 2 changes) for 16 hours. After the dialysis, the reaction product is transferred into glass tubes and stored at 4°C. Control-electrophoresis (10% SDS/
Separate and analyze 20 μp each sample taken before and after binding by polyacrylamide gel (solid phase -NPT- according to East German Economic Patent No. 254,029).
II test). It is shown that NPTU and protein 8 are crosslinked into a high molecular weight product. Its enzymatic activity is almost completely maintained after the above conjugation.

If the linkage is incomplete, it may be crosslinked again with glutaraldehyde, or the unreacted prodein octavo may be separated off over Sephadex G-50 using a small column.

The products thus produced are used for the detection of antibody-labeled biomolecules.

■ Results of neolysine-phosphate transferase ■ using glutaraldehyde on anti-potato virus-immunoglobulin NPT prepared and purified according to a) above.
Dialyze 4008 g of II with 1 mg anti-potato virus-immunoglobulin in dialysis tubing against PBS in 1 tube volume (16 h, 4°C
1C13O0, replaced twice). After the dialyzed sample is transferred into a small glass tube, 5 μβ glutaraldehyde (25% concentration solution: 5erva) is added and reacted for 2 hours at room temperature with gentle shaking.

The binding reaction mixture was then placed back into the dialysis tubing and first exposed to PBS for 4 hours (-overnight) and then to T.
Dialyze against BS (4°C, 800-12 changes, 16 hours). The reaction product is then transferred to small glass tubes and stored at 4°C. After aging (1 day), the conjugated product, an antibody conjugated via glucuraldehyde labeled with NPT 11, was used for the immunological detection of potato virus from plant fluids in the picogram range. can be used.

``Flame'' protein/l/NPT I + healing fusion protein Tsukuku composition, expression and recovery of the fusion protein fusion protein hector pRIT 2 (B, Ni1so
n, L.

Abrahamsen, N., Uhlen: E.M.B.
OJournal 4. (4)+(1985) 1
E, NPT H-gene from Co11 (Tn
5) (S, Brenner: Nature 32
9 (1987), p. 21] in an open reading screen by classical rerouting methods (BamHI, EcoRr-degradation, T4-ligase reaction). E, Colt (NPI) transformation and corresponding ampicillin resistance selection by conventional methods (T, Maniatis, E, F.

Frltsch, J., Sambrock; Mo
1ecular Cloning-Cold Spri
ng Harber-Laboratory (1982)).

7” otin A/NPT II fusion protein-clones were cultured in liquid medium NBI (
After raising the temperature to 42°C, the fusion protein is exprimieren (λ-PR promoter of the phage). After 2 hours, the cells are are centrifuged, washed once with GET buffer, suspended once more in GET, and centrifuged in portions of 1010 cells in Esobendorf tubes. ℃ and used to obtain the fusion protein.
Cells were added to 1 ml of 20 mmol Tris/)ICI buffer (pH 6,8) in an Ethopendorf tube.
Resuspend at 0.degree. C. and briefly externally sonicate 5 times. After cooling with ice, ultrasonic irradiation is performed again three times. The remaining cells are removed by centrifugation for 5 minutes at 4°C. Carefully pipette the supernatant into a second Esopendorf tube. The lysis product solution (Zell
ysat) is used as is in the detection reaction or stored at -20°C.

Protein A/NPT11- produced by genetic engineering
Crosslinking of the fusion protein with glutaraldehyde A lytic protein is prepared as described in Example 6. The lysate is pipetted into a Pendorff tube for control electrophoresis.

This lysate was filled into a dialysis tube and dialyzed against PBS (4°C, 116 hours, 800 ml, 2 changes).The dialyzed lysate was transferred into a small glass tube, and then 3 μp of Gluculaldehyde (25% strength solution: 5erva) is added and incubated for 2 hours at room temperature with gentle shaking. The reaction mixture was then returned to the dialysis tube and initially P
(4°C against BS) and then dialyzed against TBS overnight (4°C, 800-12 changes). The dialyzed binding reaction mixture was removed from the dialysis tubing and incubated at 4°C in a small glass tube. Store. Control - Remove 50 μl for electrophoresis.

The samples taken above are separated by control electrophoresis (10% SDS/polyacrylamide gel) and analyzed by solid phase-NPT If test using 32p-γ-ATP and Kanama Frasin. It is shown that during the second binding reaction, all of the protein A-NPT II fusion protein (molecular weight approximately 60 kD) was cross-linked to the high molecular weight binding product. The enzymatic activity of the conjugated product is fully maintained. The resulting product can be immediately used for the detection of antibody-labeled antigens.

±” Genetically engineered protein A/NPTII-
[i (Synthesis protein combined with another NPT II molecule with glutaraldehyde. A lysis product solution was prepared in the same manner as in Example 6 and added to 100%
Add NPT II made according to Example 4 above and electrophoretically purified in pg. This mixture is dialyzed for 16 hours against PBS (dialysis tube volume 1.5-800
ml, 2 changes, 4°C). Add 3 μl of glutaraldehyde (25 μl) to this dialyzed mixture in a small glass tube.
% concentration solution: 5erva) and incubate for 2 hours at room temperature with shaking.

Binding integrity is determined by electrophoresis and solid phase/NPT tests.

After binding, dialysis is carried out again, first against I'BS and then against TBS (4°C, 800°C, respectively).
ml, changed twice). The dialyzed conjugation product, stored at 4° C., is used for detection of various antigens labeled with antibodies.

Conjugation of Streptavidin and NPT U with Glutaraldehyde 100 μg of streptavidin and 400 pg of NPT U made according to Example 4 were mixed with P as in Example 8 above.
Dialyzed against BS (tube volume 1−), then 5μ
l of glutaraldehyde (25% concentration solution; 5erv
Add a) and mix at room temperature with gentle shaking.
Allow time to react. This reaction mixture was first reconstituted with P
Dialyze against BS and then TBS (4 ml each)
°C, 800-14 hours, 2 changes). The dialyzed product is stored at 4°C. The integrity of the binding is checked by control-electrophoresis and solid phase-NPT II test.

The bound product is used for the detection of biotin-labeled biomolecules (eg, biotinylated DNA).

Coupling of Danretsu NPT n and Protein 8 by toluylene diisocyanate prepolymer a) Preparation of the coupling reagent 17.4 g of freshly distilled toluylene diisocyanate 2,4 are warmed to 50° C. under nitrogen atmosphere.

Without stirring, at 50-55° C., 30 g of polyethylene glycol 600 (purified on ion exchange resin and dehydrated at 80° C., 1 Torr for 3 hours) and 0.05 g of cyanuric chloride are slowly added dropwise (about 30° C.). minutes). After this addition is complete, an additional 4 hours 60
Stir at ℃. Then degas at 50°C and 10.1 Torr (isocyanate equivalent -453)
.

This prepolymer 100B is dissolved in 500 mg of acetone. 0,3 of this solution after complete dissolution
Spray d into 7oomrp water while stirring rapidly. The thick emulsion thus obtained is used immediately.

b) Binding 800 μβ of the lysate solution containing 100 μg of NPT n prepared according to Example 4 above was added for 15 minutes in an Esopendorf tube.
Mix with 150 μg protein Δ in 0 μβ water.

Add 200 μl of the binding reagent prepared above to this solution and
Shake gently for an hour. Mix well using Virtex every 15 minutes. After 1 hour reaction time, 10
Add 0μ of freshly prepared binding reagent and incubate for an additional hour. Next, to stop the reaction, a 150 μβ lysine solution (200 μg) is added and shaken at room temperature for 30 minutes. The integrity of the binding was confirmed by control-electrophoresis and solid phase/NPT II tests. The high molecular weight conjugated product has full enzymatic activity.

This device is used to detect various biomolecules labeled with antibodies.

Biological cross-linking of fusion proteins of starved Protein A/NPT IT Protein A/N['T t
Add 5 μe of rabbit anti-mouse antiserum to 5 μp of the lysate containing the I-fusion protein and incubate overnight at 4°C. Produced protein A/
The complex formed consisting of NPT II-fusion protein and rabbit-anti-mouse antibody is used as antibody 2 after detection of the antigen with antibody 1 (mouse). This results in enzymatic detection (NPT 1).

Binding of phaseolus lectin to neomycin-phosphate transferase 2 by glutaraldehyde Phaseolus-vulgais-lectin 200
Dialyze against PBS together with p gO purified NPT n obtained according to example 4 (dialysis tube volume L
mR 54°C, 800 mR 32 exchanges, - night). The contents of the dialysis tube are filled into a small glass tube, to which 5 μl of gluturaldehyde (25% strength solution: 5erva) are added and allowed to react for 1 hour at room temperature with gentle shaking. The reaction mixture is then dialyzed again against PBS and subsequently against TBS (2 changes of 800 mβ for 4 hours at 4°C, respectively).
. The dialyzed reaction product is stored at 4°C. Binding integrity and enzyme activity are checked by control electrophoresis and solid phase/NPTII tests. The conjugated products are used to detect the corresponding carbohydrates on glycoproteins.

± Nilotin A/N under shellfish protein-free blocking conditions
Use of PT IT fusion proteins) Serial dilutions of proteins containing human serum albumin (IsA) in the nanogram-picogram range were electrophoretically separated in SO5/FAA gels and diluted by electrotransfer. I. transferred onto the cellulose membrane. The nitrocellulose membrane (plot) was then air-dried for 30 minutes -1,OO- and then blocked in TBS containing 1% Tween 20 by volume for 1 hour at 37°C.

Blocking was then performed once more within 30 minutes in TBS containing 0.2% by volume of Tween 20 (Block).

Incubation with anti-HSA-immunoglobulin (rabbit) albumin for 1 hour at room temperature.
:200 dilution. 1% at room temperature with TBS containing 0.1% by volume of Tween 20.
Washed for hours (changed 6 times). After blocking again (30 minutes at room temperature) with TBS and 0.2% by volume of Tween 20 (block ■), a fusion protein consisting of protein A and NPT TI prepared as in Example 6 above was contained. Add 50 μC of the lysis product solution and
ml! and shake gently at room temperature.
Incubation was carried out for an hour. Next, the nitrocellulose membrane (Hybond C; Amersham Inte
rnationaX) for 1 hour with TBS (6
times exchange).

A carrier prepared from cellulose activated with cyanuric chloride and sulfanilic acid and impregnated with kanamycin according to East German Economic Patent No. 254,012 was placed on a glass plate, and a concentration of 2 M1o·cpm/ml (specific radioactivity) was placed on a glass plate. 150 TBq/mmol) at 32P-r-AT
Impregnate with enzyme buffer containing P. The nitrocellulose plot made above is placed on the wetted, kanamycin-containing carrier with the side that was the gel side during electrophoresis. The nitrocellulose membrane is then once again impregnated from above with the enzyme buffer containing 32p-γ-ATB and then covered with a glass plate of the same size. The system is closed on all sides with PE membranes and first incubated for 1 hour at 37° C., during which time the glass plate is lightly pressed down with a weight. After this incubation, the kanamycin-containing carrier is removed from the system and washed with double distillation for 1 hour. The nitrocellulose membrane was immediately placed on the carrier containing kanamycin again and 32P-r-
Moisten with enzyme buffer containing ATP and incubate at 37°
Incubate overnight at C. Then wash with double distilled water. Evaluation is performed by autoradiography using a carrier impregnated with kanamycin and used in close contact with the carrier. The nitrocellulose membrane was coated with double distilled water (32P-r-ATP).
(contains no chlorine) and then subjected to autoradiography along with the control.

The autoradiogram of this closely applied support shows the position of the 115A-band as a blackened hand on the X-ray film to the picogram range (exposure time: 30 minutes at room temperature, XvA film H311, VE
B0RWO Wolfen).

Since protein-free blocking conditions were used, the proteins on the nitrocellulose membrane were
(Coloring = 2 minutes, decoloring with water, 5 minutes). For evaluation, autoradiography of the closely applied support is mounted on the colored nitrocellulose plot with each band in the correct position.

Immunological detection of 1 (SA) by protein plotting on plain nitrocellulose under isolated protein-blocking conditions.Carry out as in Example 13 above, except that as Block I, 0.
TBS containing 5% by weight gelatin (1 hour) and 0.5% by weight of gelatin.
TBS containing 25% by weight of gelatin (30 minutes) and TBS containing 0.25% by weight of gelatin (30 minutes) as block (2) are used.

Evaluation is carried out as in Example 13 by autoradiography of the intimate support.

Immunological detection of α-fetoprotein in a spot test on flame length nitrocellulose.
ersham Int, Inc.) and allowed to dry for 30 minutes at room temperature. Blocking is performed in block I with TBS and 1% Tween 20 for 1 hour at 37°C and then in TBS with 0.2% Tween 20 for 30 minutes at room temperature. First of all, 115A (dilution level -1: 1000)
Incubate with monoclonal antibody (mouse) against 0.2% Tween.
20 in TBS and incubation for 1 hour with the second antibody (kanamycin-anti-mouse) (dilution -1:500). After washing with TBS containing 0.1% Tween 20 at room temperature for 1 hour, blocking is performed again with TBS containing 0.2% Tween 20.

Proteins A and N were then prepared according to Example 6 above, and
Add 35 μl of E. coli lysis product containing PT II fusion protein and incubate for 1 hour at room temperature. After washing with TBS (1 hour at room temperature),
The same detection as described in Example 13 is carried out by contacting the nitrocellulose membrane with a kanamycin-containing carrier (made according to East German Economic Patent No. 254,012). Detection limits lie in the picogram range.

Detection of potato virus by Suboso 1 in plant body fluids on nitrocellulose using antibodies conjugated to NPT II (detection on a carrier according to East German Economic Co., Ltd. No. 2511, 029) made according to example 4a above, Purified NPT II 4
00 μg was added to PBS in a dialysis tube with 1 mg of anti-potato virus-immunoglobulin for 16 hours.
Dialyze in a tube volume of 1 mA (°C1Boo mR, 2 changes). After transferring the dialyzed sample into a small glass tube, 5 μl of glucuraldehyde (
A 25% concentration solution (5erva) is added and allowed to react at room temperature for 2 hours with gentle shaking.

The binding mixture is then placed back into the dialysis tubing and dialyzed first against PBS at 4° C. (-night) and then against TBS at 4° C., 800 m2, 2 changes, 16 hours).

The reaction product is divided into small glass tubes.

b) Carrying out the detection, b A series of dilutions of the pressed hemolymph of bell pepper leaves are spotted onto a strip of nitrocellulose membrane and allowed to dry for 20 minutes. The so treated membrane was treated with 1% Tween 2
0 in TBS for 1 hour at 37° C., followed by 30 minutes at room temperature in TBS containing 0.2% Tween 20. The conjugation product made above is then added at a dilution of 1:200 and incubated for 1 hour. After washing with TBS, radioactive detection is carried out on the kanamycin-containing carrier layer by the contact method as in Example 13. Detection limits are in the picogram range.

Detection of potato virus from plant body fluids using the anal protein A-NPT II fusion protein by the spot method on nitrocellulose (adhesive carrier P81) A series of dilutions was carried out with the pressed body fluid of potato leaves as in Example 16. make. However, after blocking, anti-10T-potato virus-immunoglobulin (rabbit) (dilution rate 1)
: 1000) for 1 hour.

After washing with TBS containing 0.1% Tween 20, blocking is performed again with TBS containing 0.2% Tween 20, and then 50 μβ of the lysis product according to Example 6 is added and incubated for 1 hour. After washing with TBS, a contact method similar to Example 13b) is carried out for detection. However, phosphorous cellulose (P81: Whatman, UK) is used as the kanamycin-containing carrier.

A specific activity of 10 Mio cpm/me of "P-r-ATP" is used in the enzyme buffer.

Evaluation is performed by autoradiography after washing the phosphorus cellulose support with water for 1 hour. Sensitivity is again in the picogram range.

Immunological detection of HSA by spotting method on nylon membrane using Protein A/NPT II fusion protein (adhesive carrier: phosphorous cellulose containing kanamycin) tlybo
and dry for 20 minutes. blocking r
2 hours in TBS containing 3% live serum albumin (HSA) and then 30 minutes in 1%. Blocking TIN; 1: Perform for 30 minutes in TBS containing 1% R3A. All operations are performed as described in Example 13. The evaluation is carried out by autoradiography on a phosphorous cellulose (P81) carrier.

Immunological Detection of HSA by Protein Plotting on a Plain Nitrocellulose Membrane under Non-Protein Blocking Conditions The same method as described in Example 13 is carried out. However, phosphorus cellulose (P81) is used as a carrier containing kanamycin.
: Whatman) is used. Immunology of 115A by protein plotting on nitrocellulose membrane under protein-free blocking conditions (15 min exposure time, room temperature). Detection (contact method with phosphorous cellulose containing neomycin) The same method as described in Example 13 is repeated. However, phosphorous cellulose containing neomycin is used as a carrier containing antibiotics (400 mg in 60 mf water).
Incubate the phosphorus cellulose with neomycin for 30 minutes, then wash with water for 30 minutes)
. The evaluation is carried out by autoradiography of the phosphocellulose using X-ray film (exposure time 15 minutes, room temperature).

Immunological detection of HSA by protein plotting on nitrocellulose under protein-free blocking conditions (
The method described in Example 13 (containing kanamycin in the enzyme reaction buffer) is carried out analogously. However, phosphorus cellulose (P 81; Whatman) is used as an adhesive carrier.
used without prior antibiotic impregnation treatment.

Add kanamycin to the enzyme buffer at a high concentration of /In+g/me.

The evaluation is carried out by means of phosphor-cellulose radiography.

Reuse of Protein Plots Protein plots on nitrocellulose (Ilybond C) were prepared with 0.1 molar β in TBS at 70°C after carrying out the same method as described in Example 13 (cycle ■). - Wash twice for 15 minutes each with elution buffer containing mercaptoethanol and 3% SDS, and thereby elute the first antibody including the enzyme-labeled probe. This wash was followed by another bronking with TBS containing 0.1% Tween 20 and Example 1
Repeat the same method described in 3.

The intimate supports from the first and second cycles are autoradiographed together. No loss of intensity is observed after using the plot again.

Immunological detection of HSA by protein plotting on nitrocellulose (adhesive carrier: carboxymethylcellulose containing kanamycin) under unrestricted protein-free blocking conditions The same method as described in Example 13 is carried out. Carboxymethylcellulose CCM-Membran impregnated with kanamycin as a cohesive carrier; Renner G
mbll) is used. The evaluation is this CM-Membran
performed by autoradiography.

Immunological detection of ISA by protein prosotyping on nitrocellulose under Arashigen protein-free blocking conditions (using a conjugated product consisting of protein 8 and NPT II with toluylene diisocyanate-2,4 prepolymer) (A') Preparation of enzyme-labeled probe a) E, c
Electrophoretic purification of NPT II from E. coli lysis product at 10 mA, cell pellets of E. coli (overextracted with NPT II) cell culture (pRK 89) were taken up in 1 mR lysis buffer. Apply ultrasound. The rest of the cells are centrifuged at 4°C. The lysate solution is placed on a preparative FAA/SDS gel and separated by electrophoresis. After washing the gel with water, it is placed in a cold solution of 0.25 molar KCl and the visualized NPT'IT band is cut out and subdivided with a scalpel. The protein is eluted with elution buffer for 3 hours at room temperature. The solution obtained by this elution is centrifuged, and 4 times the volume of cold acetone is added to the supernatant, and the mixture is allowed to stand at -20°C for 3 hours. then 0
Centrifuge at °C. After washing, the pellet is
Dissolve in 00 μβ lysis buffer and dialyze overnight against 20 mmol sodium phosphate buffer (4° C.). Approximately 2
Approximately 200 μg NPT II in a final volume of 00 μl
is found.

b) 17.4 g of freshly distilled toluylene diisocyanate-2,4 and 30 g of polyethylene 60 for bond bonding
A prepolymer consisting of 0 is prepared at 50-60°C. 100 mg of this prepolymer was dissolved in 500 mg of acetone, and after complete dissolution 300 mg of this solution was dissolved in 500 mg of acetone.
Emulsify mg in 700 mg water.

The 800μβ lysate solution obtained in a) above
Add 150 μg of protein 8 in 50 μl of water in an Enopendorf tube. 200 μl of the emulsion prepared as described above is added to this solution and reacted for 1 hour with shaking. After 1 hour, add 100 μβ of the freshly made emulsion again and allow to react for another hour. After that, 150μ! The reaction is stopped with a solution of 200 μg of lysine in water at room temperature for 30 minutes.

C) Implement the same method as described in Detection Example 13. However, instead of the genetically engineered Protein A/NPT It fusion protein, the conjugated product 50- produced as described above is used as an enzyme-labeled probe.

Detection is again performed by autoradiography.

Immunological detection of ll5A by spotting on CCA paper (adherent carrier according to East German Economic Patent No. 2s+, o''4) of the dilution series to IIs on CCA paper (EP Old No. 34).
, made according to No. 025), and 3
Expose to air for 0 minutes to dry. Next, add 10
% sulfanilic acid and 10% triethanolamine (ptl=7.5) for 1 hour at room temperature with gentle shaking.

Block ① is carried out in TBS containing 0.5% gelatin with an autoradiograph on a cohesive carrier according to East German Economic Patent No. 254,012 and CCA paper as described above. Kanamycin labeled with P-γ-ATP can be detected on both carriers.

Immunological detection of ISA by spotting on spot-cut negative treated paper (adhesive carrier: phosphor cellulose) A negative carrier is made from CCA paper and sulfanilic acid according to East German Economic Patent No. 237,841.

On this support we spotted a series of dilutions of 1 (block I and ■) and added a volume of 10% sulfanilic acid and 10% triethanolamine as blocking agents (blocks I and ■). 7.5) is used. Phosphorous cellulose containing kanamycin (P 81 ; Whatman) is used as a carrier impregnated with kanamycin.

Detection is then carried out as in Example 13. Detection of +ISA is carried out by orotraradiography on both supports.

Immunological detection of SA by spotting on a hydrophobic carrier (adhesive carrier: according to East German Economic Patent No. 254,012) A hydrophobic carrier was placed on CCA paper (E
D 0134,025) in equimolar amounts of di-n-
It is made by reacting with octylamine and sulfanilic acid as the sodium salt. IS on this carrier
Include the dilution series of series A. After 30 minutes at room temperature the spots are dry on the carrier and the spots are allowed to dry for an additional 15 minutes. Blocking was performed using TBS gelatin in blocks ■ and H as in Example J4.
Do the same as described in . This method is carried out in the same manner as described in Section 3 in other respects.

Detection is carried out by autoradiography on both carriers.

(by protein plotting on cage nitrocellulose)
Immunological detection of ISA (adhesive carrier: phosphor cellulose) and amplification of the detection reaction.
C; Blotting and subsequent blocking as described in Example 13 above (Amersham).

After incubation with the first antibody, it was first washed with TBS containing 0.1% Tween 20 for 1 hour and then blocked again (with 0.2% Tween 20).
TBS containing 20) and 10 μg of protein 8 in 10ml) and shake gently. This is followed by another wash with TBS containing 0.1% Tween 20 and blocking again.

The above pre-made Its8-anti-human serum albumin-protein A-NPT IT fusion protein [μl of the lysis product according to Example 6, Its85 to ttB, and anti-11s
A-Made from 50 μρ of immunoglobulin (rabbit) and 4
100 .mu..beta. of the proprietary compound (incubated overnight at 0.degree. C.) was then added and incubated for 1 hour. It is then washed with TBS and subjected to the contact method according to Example 15 using a kanamycin-containing carrier according to East German Economic Patent No. 254,012. The evaluation is carried out by autoradiography of this cohesive carrier (30 minutes at room temperature).

Immunological detection of α-fetoprotein by spotting on targeted nitrocellulose (adhesive carrier: according to East German Economic Patent No. 254.012) and augmentation with antibody-crosslinked protein A-NPTU fusion proteins. The first step is carried out similarly to that described in Example 15. After incubation with the first antibody (monoclonal), 0.
Wash with TBS containing 1% Tween 20 and 0.
．． Block with TBS containing 2% Tween 20. Next, biologically cross-linked protein A-NPT I
Add 50 μβ of fusion protein I (see Example 11) and incubate for 1 hour.

After washing with TBS (1 hour), the procedure is further carried out as in Example 15. Evaluation is carried out by autoradiography of a close-contact carrier at room temperature for 20 minutes.

Immunological detection of ISA (enzyme reaction in solution) A series of dilutions of ISA was detected using nitrocellulose (Hybond).
C; Amersham Int.) and initially treated as in Example 13. After incubation with protein A-NPTH fusion protein, the filter papers are washed in TBS for 1 hour and then transferred individually into plastic containers. This was supplemented with 0.5++ffi of enzyme buffer, I mg of kanamycin and 32P-r-ATP (
10Mio cpm7mR1 specific radioactivity 5000Ci/m
Add M) and react at 37°C for 1 hour. The nitrocellulose membrane was taken out and phosphorous cellulose (P8
Add 1.1 cm2 round filter paper and shake for 30 minutes. The phosphorus cellulose filter papers are removed, washed with water for 1 hour, dried and measured in a scintillation counter.

The evaluation is carried out directly by measuring the radioactivity (nitrocellulose filter paper without ISA is also sent as a control).

Claims (30)

[Claims] (1) In a labeled molecular probe consisting of a molecular probe and a labeled enzyme, these are bound to transferase (EC2) or ligase (EC6) directly or via a spacer. A molecular probe with the above-mentioned label, characterized in that it consists of a molecular probe. (2) The labeled molecular probe according to claim 1, which comprises a molecular probe bound to aminoglucoside-phosphotransferase directly or via a spacer. (3) Claim 1 or 2, wherein the aminoglucoside-phosphotransferase is neomycin-phosphotransferase I or II.
Molecular probes labeled as described. (4) The labeled molecular probe according to claim 1 or 2, wherein the molecular probe is bound to the enzyme through a covalent bond or through a certain spacer. (5) The labeled molecular probe according to claim 1 or 2, wherein the binding is reversible. (6) The labeled molecular probe according to claim 1 or 2, wherein the labeled molecular probe is a fusion protein of a protein and an enzyme. (7) Add transferase (E.C.2) or ligase (E.C.2) to the molecular probe or mixture of molecular probes.
．． 6) A method for producing a labeled molecular probe, which comprises binding. (8) The method for producing a labeled molecular probe according to claim 7, which comprises binding an aminoglucoside-phosphotransferase to the molecular probe or a mixture of molecular probes. (9) Claim 8, wherein neomycin-phosphotransferase I or II is used as the aminoglucoside-phosphotransferase.
Method described. (10) The method according to claim 8, wherein the binding of the aminoglucoside-phosphotransferase to the molecular probe is carried out by the formation of a covalent bond or by the formation of several covalent bonds via a spacer. (11) The method according to any one of claims 6 to 8, wherein the fusion protein consisting of a protein and an aminoglucoside-phosphotransferase is produced by genetic engineering. (12) adding a molecular probe coupled to the enzyme to identify biomolecules present in solution, contained within a gel, or bound to solid phase I;
characterized in that the enzyme bound to the molecular probe or separated therefrom is reacted with a substrate in the presence of a co-substrate and the reacted substrate is detected on solid phase II or III. , a method for detecting biomolecules using labeled molecular probes. (13) Detection of biomolecules in the following method steps, namely a)
an identification step in which a molecular probe labeled by a bound enzyme is attached to a biomolecule present in solution, contained in a gel or bound to solid phase I by virtue of its specific affinity; b) a washing step in which excess labeled molecular probe is washed away and/or separated; c) an enzymatic reaction step in which the substrate is reacted with the enzyme in the presence of some co-substrate; d) unreacted co-substrate. 13. The separation step of washing or separating off the substrate and optionally the substrate; e) the evaluation step of measuring the reacted substrate on the solid phase II or III or detecting it by a suitable detection method. the method of. (14) In identification step a), the biomolecule is separated in a gel or other separation-support material and the separation-containing biomolecule is separated from the carrier material in a solution containing the enzyme-conjugated molecular probe. contact, during which the biomolecule and the labeled molecular probe react to form a complex of the biomolecule and the enzyme-conjugated probe in the gel or separation carrier material, and in the washing step b) Washing away the excess of labeled molecular probe from the gel or separation-carrier material and transferring the gel or separation-carrier material to a second gel or separation-carrier material containing said substrate and co-substrate in enzymatic reaction step c). in separation step d) contacting a second gel or separation-support material containing said reacted substrate with solid phase III;
the reacted substrate is bound thereto and, after a washing step, in an evaluation step e) the reacted substrate on said solid phase III is detected by measuring radioactivity or by autoradiography or by other means. 14. The method of claim 13. (15) After steps a) and b), the gel or separation-carrier material containing the complex of the biomolecule and the enzyme-labeled molecular probe is transferred to the solid phase to which the substrate is bound.
Claim 1 wherein the enzymatically reacted substrate is detected on the solid phase II.
The method described in 4. (16) In the identification step a), the enzyme-bound molecular probe and the biomolecule are present in solution, where they add to each other, and in the washing step b) the excess of the labeled molecular probe is removed. In the enzymatic reaction step c), the complexed enzyme of the biomolecule and the labeled molecular probe is reacted with the substrate in the presence of a co-substrate in solution, and in the separation step d), the enzyme is reacted with the substrate in the presence of a co-substrate. Separation of the enzymatically reacted substrate from excess co-substrate is carried out by means of a solid phase III which selectively binds the substrate, and in evaluation step e) the reacted substrate and solid phase I are separated.
14. The method of claim 13, wherein the method is measured on II or autoradiographically. (17) Adding the molecular probe labeled with the bound enzyme in the identification step a) to the biomolecule bound to the solid phase I, and removing the excess labeled molecular probe in the washing step b). washing away and/or separating and removing enzymatic reaction step c)
In the separation step d, the complexed enzyme consisting of the biomolecule and the labeled molecular probe bound to the solid phase I is reacted with the substrate and co-substrate present in the solution.
), the unreacted co-substrate and optionally the substrate are separated off and the reacted substrate is bound to the solid phase III, and the reacted substrate is measured on the solid phase III in evaluation step e). or measured autoradiographically, the method according to claim 13. (18) After steps a) and b), in the enzymatic reaction step c), the solid phase I is combined with the substrate and co-substrate together with the biomolecules bound to it and the labeled molecular probe attached thereto. and then in a separation step transfer the reacted substrate from the gel or separation-carrier material onto the solid phase III, and in an evaluation step e. 18. The method of claim 17, further comprising detecting in ). (19) In the identification step a), the molecular probe labeled with the bound enzyme is added to the biomolecule bound to the solid phase I, and in the washing step b), the excess of the labeled molecular probe is removed. In the enzymatic reaction step c), the complex compound enzyme consisting of the biomolecules bound to the solid phase I and the labeled molecular probe is bound to the solid phase II. in the presence of a co-substrate present in solution, in a separation step d) the excess co-substrate is washed away or separated off, and in an evaluation step e) the reacted substrate is transferred to a solid phase II. 14. The method according to claim 13, wherein the method is measured on or autoradiographically. (20) in identification step a) identifying the biomolecule in one or more identification systems and in one or more molecules with special affinity, or reacting with one or more molecules;
The method according to any one of claims 13 to 19, wherein the molecular probe bound to the enzyme is reacted with at least one of these molecules. (21) The method according to claim 12, wherein a fusion protein of an enzyme and a molecular probe is used as the labeled molecular probe. (22) Claim 1, wherein the enzyme-bound molecular probe is a molecular probe that is reversibly bound to the enzyme.
The method described in 2. (23) The method according to any one of claims 12 to 20, wherein transferase (EC2) is used as the enzyme. (24) Enzymes that transfer phosphorus-containing groups (E.C.
．． 24. The method according to claim 23, wherein 2,7) is used. (25) The method according to claim 24, wherein aminoglucoside-phosphotransferase (E.C. 2, 7, 1) is used as the phosphorus-containing group transferase. (26) The method according to claim 24, wherein nucleotidyl transferase (EC 2, 7, 7) is used as the phosphorus-containing group transfer enzyme. (27) The method according to any one of claims 12 to 20, wherein ligase (EC6) is used as the enzyme. (28) The method according to claim 27, wherein a phosphorus ester-forming enzyme (repair enzyme) (EC6, 5) is used as the ligase. (29) The method according to claim 28, wherein DNA-ligase (EC 6, 5, 11) is used as the phosphorus ester-forming enzyme. (30) The method according to any one of claims 12 to 20, wherein the molecular probe according to claims 1 to 5, which is bound to an aminoglucoside-phosphotransferase, is used as the enzyme-bound molecular probe.