NL1003839C2 - Diagnostic test. - Google Patents

Description

DIAGNOSTIC TEST

The present invention relates to diagnostic methods, which are particularly intended to detect nucleic acid binding molecules, such as proteins. The invention further relates to kits for performing the method.

Diagnostic tests usually take advantage of the specific binding capacity of antibodies to demonstrate the presence of a particular molecule in a sample. The best known diagnostic tests are the ELISA and the EIA or RIA. To demonstrate the presence of antibodies to a known antigen, in an ELISA, the antigen is coupled to a solid support, such as a microtiter plate. By contacting the sample with the support, antibodies specific for the antigen will bind to it. After removal of unbound sample material and labeling of the bound antibody, the binding can be visualized and optionally quantified.

When it is necessary to demonstrate the presence of an antigen in the sample, a so-called ELISA sandwich is often used. Here, an antibody specific for the antigen to be detected is bound to a solid support. After contact with the sample and removal of unbound sample material, the antigen bound to the primary antibody is detected with a secondary antibody directed against another epitope on the molecule. However, the utility of this method requires the presence of at least two different, sufficiently immunogenic epitopes. However, some molecules to be detected are too small or insufficiently immunogenic to be detected in this way.

Such a molecule is, for example, the p7 protein of human immunodeficiency virus 1 (HIV-1). P7, also called NCP7 or nucleocapsid, is one of the four proteins that make up the nucleocapsid of virus 1003839 2. They arise from one 55 kDa protein (Pr55) encoded by the HIV gag gene. P7 consists of 55 or 72 amino acids and the mass is about 7 kDa. It binds, in principle at random, to a piece of nucleic acid 5 of at least 6 bases long in any order, for example, pieces of nucleic acid of at least 6 bases long spread over the entire HIV RNA, but with a higher specificity to the so-called T-structure. This is one of the hairpin structures at the beginning of the HIV-1 10 RNA, which are probably involved in the coupling between the two RNA molecules of a virus particle.

Because p7 is relatively small, it is also not very immunogenic. This makes detection of the protein by means of antibodies difficult. The normal diagnostic tests 15 are therefore not suitable to demonstrate the presence of p7 in a sample.

The aim of the present invention is to provide a diagnostic test for the detection of (small or little immunogenic), nucleic acid binding molecules in general and p7 in particular in samples, in particular of body fluids.

This is accomplished by the invention by a method comprising binding the molecule to be detected to a first ligand and detecting the bond between the molecule and the first ligand by binding a second ligand to the molecule, wherein at least one of both ligands is a nucleic acid formed by at least 3 bases. In this text, by "nucleic acid" is meant a piece of nucleic acid with a length of at least 3 bases in random order.

The basic method according to the invention has three general embodiments, each of which comprises several specific embodiments. For example, it is possible to use a binding nucleic acid and other means, in particular an antibody, for detection, but it is also possible to use a binding antibody and a nucleic acid for detection. Finally, 1003839 3, both binding and detection by nucleic acids can be accomplished.

The first embodiment comprises: a) contacting the body fluid with a carrier having at least one nucleic acid bound thereto as the first ligand in order to allow binding between the molecule to be detected and the nucleic acid, b) making visible the bond between the nucleic acid and the nucleic acid binding molecule by means of at least one antibody directed against the molecule as the second ligand, and c) visualizing and optionally quantifying the bond.

This method is illustrated in FIG. lwith p7 as protein to be detected.

In the second embodiment, the functions of the nucleic acid and antibody are exchanged. This method comprises: a) contacting the body fluid with a support having as a first ligand at least one antibody bound thereto directed against the molecule to be detected to allow binding between the molecules and the antibody, b) the visualizing the binding between the antibody and the nucleic acid binding molecule by means of at least one nucleic acid binding molecule as second ligand, and c) visualizing and optionally quantifying the binding.

Fig. 2 gives a schematic representation of this method.

The third embodiment (shown in Fig. 3) relates to a method using nucleic acids for both function (binding and detection), comprising: a) contacting the body fluid with a carrier having at least one as the first ligand 1003839 4 nucleic acid to allow binding between the molecule and the nucleic acid, b) visualizing the binding between the nucleic acid and the nucleic acid binding molecule 5 by means of at least one second nucleic acid binding nucleic acid, and c) visualizing and optionally quantify the binding.

Unlike the sandwich ELISA, the primary binding of the molecule to be detected to the support according to the first embodiment of the invention is accomplished by a nucleic acid. In that first embodiment, the secondary binding (detection) still takes place via an antibody. The advantage, however, is that the molecule to be detected need only have one epitope to be detected. The method according to the second embodiment also has the above-mentioned advantage that only one epitope is required. In the third embodiment, no epitopes are needed at all. The only condition is that the molecule to be detected binds to the nucleic acids used, but this condition naturally applies to all embodiments.

The nucleic acid can be RNA, single-stranded DNA or double-stranded DNA. The sequence of the nucleic acid preferably corresponds at least in part to that of naturally occurring nucleic acid, such as HIV-1 RNA, permitting modifications which do not negatively affect the binding capacity of the nucleic acid to the molecule to be detected.

Modifications can be sequence modifications or chemical modifications. Thus, in addition to naturally occurring nucleic acids or synthetic homologues thereof, sequence-modified versions can also be used. In this case, this means any molecule that does not occur in nature, but which has favorable properties for the test. As an example, a repeating sequence of the binding relevant 1003839 5 parts of a naturally occurring nucleic acid can be mentioned. For p7, for example, that could be a sequence of repeating f structures.

Chemical modification may be needed to protect the nucleic acid from degradation by sample nucleic acid-degrading enzymes, such as RNase or DNase. Presence of these enzymes in the sample could render the test useless. Examples of such degradation protective modifications are complexing the RNA with vanadium.

Protection against nucleic acid degrading enzymes can also be achieved by adding one or more RNase or DNase inhibitors to the sample or the carrier before contacting the sample with the carrier containing the nucleic acid. .

The detection nucleic acid according to the third embodiment can bind specifically to the molecule to be detected, but can also have very general (protein) binding properties.

The support can take various forms, such as a microtiter plate, a column, a membrane or granules. The latter can be, for example, magnetic beads, such as Dynabeads ™.

Binding of the antibody to the molecule to be detected in the first embodiment of the invention can be detected in various ways. The particularly preferred method is labeling the antibody with a radioactive or fluorescent label or with an enzyme, which can give rise to a color reaction, such as horseradish peroxidase. A method that works via an electrochemiluminescent label is possible if the antibodies or nucleic acids required for detection are labeled with an electrochemiluminescent label, the system for detection being the same as that of the NASBA RNA (Organon Technika, Oss ). This substance can be energetically excited by an intermediate substance, after electron transfer from and the electrochemiluminescent label and intermediate substance have taken place via an electrode in the analyzer. When the label falls back energetically to a normal level afterwards, a certain amount of energy is released, which is released in the form of a photon. These photons can be detected and measured in a so-called photon multiplier tube and quantified.

In the case of the second and third embodiments of the invention, the nucleic acid is labeled for detection. The same labels can be used here as for labeling an antibody for detection.

In addition to complete antibody, antibody fragments such as Fv, Fab, F (ab) 2, chimeric or bispecific antibodies can also be used.

It is quite easy for the skilled artisan to isolate or synthesize a suitable nucleic acid based on his knowledge of the molecule to be detected. Also production of the antibody for primary binding or detection is routine for a skilled artisan and is described, for example, in "Antibodies, a laboratory manual" (Harlow and Lane, Cold Spring Harbor Laboratory (1988). Binding of nucleic acids to various carriers can be carried out via a streptavidin-biotin bond Carriers can be coated with streptavidin and nucleic acid can be labeled with biotin via the incorporation of a biotin-labeled nucleoside triphosphate during the synthesis of the nucleic acid After the support is coated with streptavidin Nucleic acid labeled with biotin is added and the binding takes place. Binding of the antibody for primary binding to the support is standard technique and is described, for example, in Laboratory techniques in biochemistry and molecular biology. Practice and theory of enzyme immunoassays., P. Tijssen, Elsevier Amsterdam 1987. Labeling of the antibody for detection is also a self-contained nth technique (Tijssen, supra).

Nucleic acid labeling for detection can be done as follows. The nucleoside triphosphate GTP lends 1003839 7 for labeling with an enzyme or label coupled to an N-hydroxysuccini midine ester. The free amine group on this nucleoside is not used for hybridization of the nucleic acids to each other via hydrogen bonds and thus can be labeled. Another possibility is to label the nucleic acid at the 5 'terminal end with an amino linker so that the activated enzyme or label can react and bind with it.

In addition to the methods, the invention further provides diagnostic kits for performing it.

In general, a base kit includes a support with a bonded first ligand for binding the molecule to be detected and a second ligand for binding the molecule to detect the bond between the molecule and the first ligand, wherein at least one of the two ligands is a nucleic acid.

In the first general embodiment, the kit comprises a carrier having at least one nucleic acid bound thereto and means for detecting binding of the nucleic acid to the nucleic acid binding molecule to be detected. In a more specific embodiment, the diagnostic kit comprises a microtiter plate support for the nucleic acid, and means for detecting binding of the nucleic acid to the nucleic acid binding molecule to be detected. What these means can be has already been described above for the method. Preferably, the means of detection are (labeled) antibodies.

In another specific embodiment, the diagnostic kit comprises magnetic beads as a carrier for the nucleic acid and antibodies labeled for chemiluminescence as the means for detecting binding of the nucleic acid with the nucleic acid binding molecule to be detected.

In an even more specific embodiment, the diagnostic kit is intended to demonstrate the presence of HIV-1 in a body fluid, and includes Dynabeads ™ with a bound nucleic acid, consisting of a repeating sequence of the p7 binding psi. structure of HIV-1, and a chemiluminescence-labeled (monoclonal) antibody directed against p7 for the detection of p7 protein bound to the nucleic acid.

Kits according to the second general embodiment of the invention comprise a carrier having as first ligand at least one antibody bound thereto directed against the molecule to be detected and a nucleic acid binding the molecule to be detected for detecting binding of the antibody to the molecule nucleic acid binding molecule to be shown as second ligand.

Kits according to the third general embodiment include a support having the first ligand having at least one bound first nucleic acid capable of binding to the molecule to be detected, and as the second ligand a second molecule binding for detection to be detected for detection, for detection of binding of the first nucleic acid to the nucleic acid binding molecule to be detected.

In addition, all kits may contain agents for inhibiting potential nucleic acid-degrading enzymes present in the sample.

The methods and kits of the invention are particularly suitable for testing various body fluids, such as blood, serum, plasma, sperm, urine, saliva, tear fluid, liquor, or sweat.

The present invention can have a wide variety of applications. However, the examples will only discuss one specific embodiment for detecting p7 in serum. It will be clear to the skilled person that the invention is very general and does not lie in this specific embodiment. Using his average skill in the art, those skilled in the art will be able to apply the principle of the invention, which is to demonstrate a molecule using the molecule's ability to bind to nucleic acid. This can be done by primary binding to a nucleic acid and detection by 1003839 through secondary binding to an antibody, by primary binding to an antibody and detection by secondary binding to a nucleic acid or by both primary and secondary binding to a nucleic acid. The following 5 examples are therefore given for illustrative purposes only.

EXAMPLES EXAMPLE 1

Microtitre plate-based diagnostic test A 96-well microtiter plate was coated with complete HIV-1 RNA by incubating virus-purified RNA at a concentration of 105 molecules in 100 µl demineralized and autoclaved water at room temperature overnight. The following day, the wells were washed with an autoclaved wash buffer consisting of 25 Mg / ml yeast tRNA, 200 mM KCl and 40 mM MgCl 2 in dH 2 O.

Then 25 μΐ serum sample with 100 μΐ wash buffer and 8 serum samples with a known amount of HIV-1 p7 were placed in triplicate wells. After 45 minutes of incubation at room temperature, the sample was removed and the wells were washed with wash buffer. After this, 100 µl of horseradish peroxidase labeled anti-HIV-1 p7 antibody (manufactured as described in "Antibodies, a laboratory manual", supra) was placed in the wells at a concentration of 2 Mg / ml. After incubation at 37 ° C for 1 hour, the antibody solution was removed and the wells were washed with wash buffer.

The protein to be detected was then quantitated by placing 100 μΐ substrate solution (OPD) in the 30 wells. The color reaction, which then started, was stopped after 30 minutes by adding 50µl of a 0.5M sulfuric acid solution. The optical density of the contents of the wells was then measured in a spectrophotometer at 450 nm. The values of the 8 calibration samples were then plotted against the known concentration p7, whereby a calibration line was created and the p7 concentration in the serum samples could be determined by reading the value on the calibration line.

1003839 10 EXAMPLE 2

Diagnostic test based on DynabeadsTM

12x75 mm round bottom tubes were filled with 25 μl of biotin-labeled RNA in dilution buffer (25 Mg / ml yeast tRNA, 200 mM KC1, 40 mM Mgcl2 and 5U / ml RNasin (Promega) in dH20), 25μ1 of a solution of an anti-HIV-1 p7 antibody (2 μg / ml) labeled with an electrochemiluminescent label, and a 25 μΐ serum sample. A calibration line was made by replacing the serum samples to be tested with 25 μΐ serum samples with a known amount of p7. After 30 minutes at room temperature with continuous shaking, 2.5 µg Dynabeads ™ (M-280, coated with streptavidin) in 25 µl dilution buffer was added. After 15 minutes of incubation at room temperature with continuous shaking, 200 µl of assay buffer (IGEN Ine., Gaithersburg, MD, USA) was added to this and the samples were analyzed in the ORIGEN analyzer (IGEN Inc., Gaithersburg, MD, USA).

20 EXAMPLE 3

Diagnostic test with sequence-modified nucleic acid

The test was performed in the same manner as in Example 2, but instead of complete RNA, a synthetic RNA was used consisting of the sequence of HIV-1n nucleotides 300 to 337, which sequence is the Ψ-hairpin, 10 times in succession. contains.

30 EXAMPLE 4

Diagnostic test with chemically modified nucleic acid

To inhibit nucleic acid degradation by RNase, the same RNA as in Example 3 was complexed with vanadyl sulfate according to the protocol described in Maniatis, Fritsc and Sambrook, Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, 1982.

1003839 11 EXAMPLE 5

Nucleic acid diagnostic test as a means of detection

Examples 1 to 4 concerned diagnostic tests in which the molecule to be detected was bound primarily by a nucleic acid and detected secondary by an antibody. In this example, the reverse is described, in which primary binding occurs by means of an antibody and detection by means of a labeled nucleic acid.

A 96-well microtiter plate was coated with anti-HIV-1 p7 antibody (manufactured as described in "Antibodies, a laboratory manual", supra) by demineralizing it with 100 µl of an antibody solution at a concentration of 2 Mg / ml and incubate autoclaved water overnight at room temperature. The following day, the wells were washed with an autoclaved wash buffer consisting of PBS / 0.5% Tween-20 ™

Then 25 µl serum sample with 100 µl 20 wash buffer and 8 serum samples with a known amount of HIV-1 p7 were placed in triplicate wells. After 60 minutes of incubation at room temperature, the sample was removed and the wells were washed with 25 µg / ml yeast tRNA, 200 mM KCl, 40 mM MgCl 2 and 5 U / ml RNasin (Promega) in 25 dH 2 O. After this, 100 μΐ of a solution of the horseradish peroxidase-labeled nucleic acid from Example 3 was introduced into the wells at a concentration of 105 nucleic acid molecules per 100 μΐ. After incubation at room temperature for 30 minutes, the nucleic acid solution was removed and the wells were washed with 25 µg / ml yeast tRNA, 200 mM KCl, 40 mM MgCl 2 and 5 U / ml RNasin (Promega) in dH 2 O.

The protein to be detected was then quantitated by introducing 100 μΐ substrate solution (OPD) into the 35 wells. The color reaction, which then started, was stopped after 30 minutes by adding 50 μΐ 0.5 M H2SO4. The optical density of the contents of the wells was then measured at 450 nm in a spectro 1003839 12 photometer. The values of the 8 calibration samples were then plotted against the known concentration p7, resulting in a calibration line and the p7 concentration in the serum samples could be determined by reading the value on the calibration line.

EXAMPLE 6

Diagnostic kit carbon black nucleic acids for both binding and detection. In a third alternative embodiment of the invention, both primary binding and secondary binding for detection are accomplished by means of nucleic acids. This example illustrates this embodiment.

A 96-well microtiter plate was coated with complete HIV-1 RNA by incubating virus-purified RNA at a concentration of 105 molecules in 100 µl demineralized and autoclaved water at room temperature overnight. The following day, the wells were washed with an autoclaved wash buffer consisting of 25 µg / ml yeast tRNA, 200 mM KCl and 40 mM MgCl2 in dHzO.

Then 25 μΐ serum sample with 100 μΐ wash buffer and 8 serum samples with a known amount of HIV-1 p7 were placed in triplicate wells. After incubation at room temperature for 45 minutes, the sample was removed and the wells were washed with wash buffer.

After this, 100 μΐ of a solution of the horseradish peroxidase-labeled nucleic acid from Example 3 was introduced into the wells at a concentration of 105 molecules per 100 μΐ. After incubation at room temperature for 45 minutes, the nucleic acid solution was removed and the wells were washed with wash buffer as described above.

The protein to be detected was then quantified by loading 100 µl of substrate solution (OPD) into the wells. The color reaction, which then started, was stopped after 30 minutes by adding 50 μΐ 0.5 M H2SO4. The optical density of the contents of the 1003839 13 wells was then measured in a spectrophotometer at 450 nm. The values of the 8 calibration samples were then plotted against the known concentration p7, resulting in a calibration line and the p7 concentration in the 5 serum samples could be determined by reading the value on the calibration line.

1003839

Claims (15)

1. Method for detecting nucleic acid binding molecules, in particular proteins, in a sample of, for example, a body fluid, comprising binding the molecule to be detected to a first ligand and detecting the bond between the molecule and the first ligand by binding a second ligand to the molecule, wherein at least one of the two ligands is a nucleic acid. 2. A method according to claim 1, comprising: a) contacting the body fluid with a carrier having at least one nucleic acid bound thereto as the first ligand in order to enable binding between the molecule to be detected and the nucleic acid, b) being visible making the bond between the nucleic acid and the nucleic acid binding molecule by means of at least one antibody directed against the molecule as the second ligand, and c) visualizing and optionally quantifying the binding. 3. A method according to claim 1, comprising: a) contacting the body fluid with a support having as first ligand at least one antibody bound thereto directed against the molecule to be detected in order to allow binding between the molecule and the antibody. b) visualizing the binding between the antibody and the nucleic acid binding molecule by means of at least one second nucleic acid binding molecule, and c) visualizing and optionally quantifying the binding. A method according to claim 1, comprising: a) contacting the body fluid with a carrier having at least one 1 0 0 383 9 nucleic acid as the first ligand to allow binding between the molecule and the nucleic acid, b) making visible of the bond between the nucleic acid and the nucleic acid binding molecule by means of at least one nucleic acid binding molecule as second ligand, and c) visualizing and optionally quantifying the binding. A method according to any one of claims 1-4, characterized in that the nucleic acid is formed by RNA and / or single-stranded DNA and / or double-stranded DNA, or modified versions thereof. 6. A method according to any one of claims 1 to 5, characterized in that the sequence of the nucleic acid 15 corresponds at least in part to that of naturally occurring HIV-1 RNA, wherein modifications which alter the binding capacity of the nucleic acid to the not negatively affect molecule, are allowed. A method according to claim 6, characterized in that the nucleic acid binding molecule to be detected is HIV-1 p7 and the nucleic acid contains at least one HIV-1 p7 binding sequence. Method according to any one of claims 1-7, 25, characterized in that the body fluid is blood, serum, plasma, sperm, urine, saliva, tear fluid, liquor or sweat. 9. Diagnostic kit for carrying out the method according to any one of claims 1-8, comprising a carrier with a bound first ligand for binding the molecule to be detected and a second ligand for binding the molecule for detection of the binding. between the molecule and the first ligand, at least one of which is a nucleic acid. Diagnostic kit according to claim 9 for carrying out the method according to any one of claims 1, 2 and 5-8, comprising a carrier having as first ligand 1003839 at least one nucleic acid bound thereto and means for detecting binding of the nucleic acid with the demonstrate nucleic acid binding molecule as second ligand. Diagnostic kit according to claim 10, characterized in that the nucleic acid support is a microtiter plate, and the means for detecting binding of the nucleic acid to the nucleic acid binding molecule to be detected are antibodies. Diagnostic kit according to claim 10, characterized in that the nucleic acid support is constituted by magnetic beads and the means for detecting binding of the nucleic acid with the nucleic acid binding molecule to be detected for chemiluminescence labeled antibodies. Diagnostic kit according to claim 12 for detecting the presence of HIV-1 in a body fluid, comprising Dynabeads ™ with the first ligand a nucleic acid bound thereto, consisting of a repeating sequence of the p7-binding psi structure of HIV-1, and a chemiluminescence-labeled monoclonal antibody directed against p7 as a second ligand for the detection of p7 protein bound to the nucleic acid. Diagnostic kit according to claim 9 for carrying out the method according to any one of claims 1, 3 and 5-8, comprising a carrier having as first ligand at least one antibody bound thereto directed against the molecule to be detected and a detectable molecule-binding, nucleic acid labeled for detection to demonstrate binding of the antibody to the nucleic acid binding molecule to be detected as second ligand. Diagnostic kit according to claim 9 for carrying out the method according to any one of claims 1 and 4-8, comprising a carrier having as first ligand at least one bound first nucleic acid which can bind to the molecule to be detected, and secondly 1003839 ligand a detectable molecule binding second nucleic acid for detection to demonstrate binding of the first nucleic acid to the nucleic acid binding molecule to be detected. 1003839