JPH1023900A - Assay of dna synthase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To assay DNA synthase activity in high sensitivity by reaction of a DNA synthase gene-immobilized support bearing silicon oxide coating film with a labeled DNA complementary thereto followed by injecting linearly polarized light onto the support at a specified angle and by measuring the elliptically polarized light component of the reflected light. SOLUTION: A support 1 for DNA synthesis consisting of a silicon substrate bearing a silicon oxide coating film of a specified thickness and immobilized with nucleotide chain 2 as template of the DNA synthase is brought into contact with a reaction liquid containing a labeled deoxyribonucleotide 5 complementary to the above nucleotide chain, a primer 5 and a surfactant and a specimen to conduct a DNA synthesis, and the resultant support is brought into contact with a complex composed of an inorganic substance 9 and a second substance 8 specifically reactive with the label 6 of the deoxyribonucleotide. Thereafter, linearly polarized light is injected at a specified incident angle onto a reference support identical with the above support but immobilized with no nucleotide chain, and the resultant reflected light is, in turn, injected at the same incident angle as the above onto the support after the DNA synthesis, and the elliptically polarized light component of the resultant reflected light is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reverse transcriptase, D
The present invention relates to a method for measuring the activity of a DNA synthase such as an NA polymerase at a high sensitivity without using a radioactive label.

[0002]

2. Description of the Related Art A DNA polymerase enzyme is an enzyme that catalyzes a reaction for synthesizing a DNA chain using DNA as a template. Therefore, by measuring the activity of the DNA polymerase, a virus having DNA as a gene and having DNA polymerase can be detected. A method for measuring the activity of a DNA polymerase enzyme is as follows:
Taking advantage of this feature, hepatitis B virus (HB
V) and the like, and is used for the detection of virus in blood collected from patients infected with viruses. Further, when an unknown virus is isolated, it is also used to confirm whether the virus performs DNA-dependent DNA synthesis.

[0003] The measurement of the activity of such a DNA polymerase enzyme has hitherto been widely performed using a radioactive substance as a label. On the other hand, reverse transcriptase (hereinafter sometimes referred to as RT) is an RNA-dependent DNA.
Also called a synthase, it is an enzyme that catalyzes a reaction for synthesizing DNA using RNA as a template. Therefore, by measuring the activity of reverse transcriptase, a virus having RNA as a gene and having reverse transcriptase, ie, HIV,
Retroviruses such as HTLV can be detected. The method of measuring the activity of reverse transcriptase, which utilizes this characteristic, has been used in the detection of HIV isolated from lymphocytes collected from HIV-infected patients, drug sensitivity tests for the isolated HIV, and the like. ing. When an unknown virus is isolated, the virus
It is also used to confirm whether or not to perform dependent DNA synthesis.

[0004] Methods for measuring the activity of reverse transcriptase include, for example, 1) K. Suzuki, B. Craddock, N. Okamoto, T. Kano, and
R.Steigbigel, Poly Alinked colorimetric microtiter
plate assay for HIV reverse transcriptase, J. Vir
ol. Methods, 44 (1993), 189-98, 2) JP-A-4-148689, 3) Josef Eberle and Rudolf Seibl, A new method fo
r measuring reversetranscriptase activity by ELIS
A, J. Virol. Methods, 40 (1992), 347-356, 4) EP 393459 A3 and European patent application 90106847.8, 5) DE 41 09 599 A1, DD 298 603 A5 and J. Vi
rol. Methods, 31 (1991), 181-188; 6) WO 91/05066; and 7) Lee, Moon et a at the 6th AIDS International Conference.
l. report (the non-radioactive sensitive reverse t
ranscriptase assay, 6th International Conference o
n AIDS, San Francisco CA, Abstract 1028).

[0005] The publication 1) includes RNA as a template.
A method for immobilizing this poly A on a solid phase is disclosed. Figures shown in this publication
101 is a view schematically showing each step of this method.

In this method, first, a sample containing a virus is centrifuged twice with a microcentrifuge. That is,
Debris such as cells are precipitated in the first centrifugation, and the resulting supernatant is transferred to another centrifuge tube and centrifuged at a higher speed to precipitate the virus. Next, the obtained precipitate was subjected to oligo-dT (DNA) and biotin-labeled dUTP.
Together with a solution containing, for example, a polyA-immobilized container, for example, a well of a microtiter plate.

In the container, first, oligo-dT43 hybridizes to poly A42 immobilized in container 41, as shown in FIG. 5 (a). Next, if the sample contains reverse transcriptase, reverse transcriptase 44 binds to this complex and initiates DNA synthesis along the poly A42 from the end of oligo-dT43. At this time, biotin-labeled dUT
P45 is incorporated, and a DNA strand labeled with biotin 46 is generated. This reaction is carried out at 37 ° C. for about 15 minutes. After the reaction, the inside of the container is washed, and streptavidin 48 labeled with enzyme 47 is added. As a result, when the sample contains reverse transcriptase and the DNA strand 49 is formed, FIG.
As shown in (b), biotin 46 contained in DNA
And streptavidin 48 bind. Further, after washing the inside of the container to remove unbound streptavidin 48, the substrate 50 of the enzyme 47 used for labeling is added, and changes in color and absorbance are measured (FIG. 5 (c)).

The publication 2) discloses a method for immobilizing oligo-dT on a solid phase such as a microtiter plate, contrary to the method disclosed in the publication 1). That is, together with poly-A and biotin-labeled dUTP, the above-mentioned 1) is placed in a container having oligo-dT immobilized thereon.
The sample (precipitated after centrifugation twice) prepared in the same manner as described above is added, and oligo-dT and polyA are hybridized. Thereafter, similarly to the method disclosed in the above publication 1), enzyme-labeled streptavidin is added, and further, a substrate of the enzyme is added, and color change and change in absorbance are measured.

The above publication 3) discloses a method using free poly-A and oligo-dT. In this method, first, a sample, polyA, oligo-dT, digoxigenin-labeled dUTP, and biotin-labeled dUTP, which are pretreated in the same manner as in 1) above, are mixed, hybridized between polyA and oligo-dT, and reverse transcriptase. To perform DNA synthesis. At this time, digoxigenin and biotin are incorporated into the synthesized DNA. Next, these products are placed in a container having streptavidin immobilized thereon, and the streptavidin is bound to biotin used for labeling.
Finally, after removing unbound substances, the amount of digoxigenin incorporated into the DNA and immobilized on the container surface is determined by EI.
It is measured by Method A.

The above publication 4) discloses a method using free poly A and oligo-dT. In this method, first, a sample pretreated in the same manner as in 1), polyA, oligo-dT, and biotin-labeled dUTP are mixed, and polyA
And oligo-dT, and DNA synthesis by reverse transcriptase. At this time, the synthesized DNA
Incorporates biotin. Next, these products are spotted on a nitrocellulose membrane, and the DNA is immobilized on the membrane. Finally, biotin incorporated in the DNA immobilized on the membrane is measured.

The above publication 5) discloses a method using free polyA and oligo-dT. In this method, first, a sample, polyA, oligo-dT, and a hapten-labeled nucleoside triphosphate (here, BrdUTP; 5-bromo-2'-deoxyuridine triphosphate) mixed with a sample pretreated in the same manner as in 1) above are mixed, Poly A and oligo
Hybridization with dT and DN by reverse transcriptase
A synthesis is performed. At this time, the synthesized DNA contains Brd
UTP is captured. Next, the synthesized DNA is separated on a microtiter plate on which an anti-bromo antibody is immobilized, and the separated DNA is added with an enzyme-labeled anti-bromo antibody and measured by the EIA method.

In the method disclosed in the above publication 6), reverse transcriptase itself is regarded as a protein, and the measurement is carried out by ELISA using an antibody against reverse transcriptase and a labeled antibody.

The publication 7) is similar to the method disclosed in the publication 4) above, except that the free poly-A and oligo-d
A method using T is disclosed. In this method, first, a sample pretreated in the same manner as in the above 1), polyA, oligo-d
T and digoxigenin-labeled dUTP are mixed, and hybridization between polyA and oligo-dT and DNA synthesis using reverse transcriptase are performed. At this time, digoxigenin is incorporated into the synthesized DNA. Next, these products are spotted on a nitrocellulose membrane, and the DNA is immobilized on the membrane. Finally, digoxigenin incorporated into the DNA immobilized on the membrane is measured.

[0014] In addition to the method for measuring the activity of reverse transcriptase, a method for measuring the antibody titer of an antibody against reverse transcriptase, ie, an anti-reverse transcriptase antibody is also known. When a virus having a reverse transcriptase, that is, a retrovirus such as HIV, enters a living body, an anti-reverse transcriptase antibody is produced by an immune reaction. Therefore, using this method, it is possible to determine whether there has been a retroviral infection in the past, regardless of the current presence or absence of the infection, and it is possible to understand the patient's medical history and disease progress .

[0015]

As described above, the measurement of transcriptase activity is usually performed as a means of detecting a virus. In general, the measurement of a reaction involving an enzyme is performed in a short time in consideration of the stability of the enzyme. In fact, even in the conventional measuring method described above, linearity that can be used for quantification of an enzyme reaction is obtained at most up to about 30 minutes, and furthermore, this linearity is stably obtained up to about 15 minutes. It is. For this reason, the enzymatic reaction is usually performed within 5 to 10 minutes, and the measurement is not performed for a longer time.

As described above, since the enzymatic reaction cannot take a long time, any of the conventional measurement methods has low sensitivity, and a sample having a very large amount of virus, such as the measurement of virus transcriptase activity in cultured cells, is used. Otherwise, measurements cannot be made. Therefore, in order to measure a trace amount of virus such as detection of a virus present in the blood of a patient, it is necessary to increase the concentration of the virus in the sample using some means.

In the conventional measurement method, this point is compensated for by pre-processing the sample. That is, as described above, the sample is centrifuged twice and concentrated to increase the virus concentration.

However, such pretreatment requires dispensing of a sample before the first centrifugation, between the first and second centrifugation, and after the second centrifugation. And This is a very complicated and labor-intensive task, especially when the number of samples is large. In addition, since the number of samples that can be centrifuged at one time is limited, when the number of samples is large, the processing must be performed in a plurality of times, and the complexity is further increased.

As described above, the measurement of the activity of the DNA polymerase enzyme is conventionally performed using a radioactive substance as a label. However, handling of the radioactive substance requires a special facility and a skilled technician. Lack. In addition, the analysis method is complicated.

Further, as a method for measuring the antibody titer of an anti-reverse transcriptase antibody, a Western blot method has been conventionally used, but the measurement sensitivity of this method is not sufficient. In addition, this method simply measures the antibody titer of the whole anti-reverse transcriptase antibody contained in the sample, and the antibody of the antibody necessary to inhibit the activity of reverse transcriptase to prevent the establishment of virus infection. The value cannot be measured.

Therefore, the present invention provides a simple and high-sensitivity D
An object of the present invention is to provide a method for measuring the activity of a DNA synthase capable of measuring the activity of an NA synthase.

[0022]

Means for Solving the Problems In view of the above circumstances, the present inventor has made extensive studies and, as a result, has elongated the reaction time of DNA synthase by destroying a virus using a surfactant in a reaction solution. It was found that the sensitivity can be improved by increasing the amount of labeled nucleic acid taken up, and furthermore, utilizing the fact that the state of the reflecting surface can be measured by measuring the elliptically polarized light component contained in the linearly polarized reflected light As a result, the present invention has been completed. That is, the method for measuring DNA synthase activity according to the present invention comprises a silicon substrate on which a silicon oxide film having a predetermined thickness is formed and a nucleotide chain as a template for DNA synthase is immobilized.
Performing a DNA synthesis reaction by contacting a sample with a reaction solution containing a deoxyribonucleotide, a primer and a surfactant that is complementary to and labeled with the nucleotide chain, on a support for NA synthesis reaction; Contacting the DNA synthesis reaction support with a complex of a substance capable of specifically reacting with the label of the deoxyribonucleotide and an inorganic substance, comprising the same support as the DNA synthesis reaction support, and A step of causing linearly polarized light to enter the control support on which the nucleotide chain is not immobilized at a predetermined incident angle, and the first reflected light reflected by the control support is defined as an incident angle of the linearly polarized light. At the same angle of incidence, the DNA
And a step of measuring the elliptically polarized light component of the second reflected light reflected by the DNA synthesis reaction support after the synthesis reaction has been performed. I do.

In the method for measuring DNA synthase activity according to the present invention, the DNA synthesis reaction
The support for synthesis reaction may be subjected to measurement after washing with water and then removing water drops.

Hereinafter, the present invention will be described in detail. In the present invention, the DNA synthase means an enzyme that catalyzes a reaction for synthesizing DNA using DNA or RNA as a template, and specifically includes DNA polymerase or reverse transcriptase (RNA-dependent DNA polymerase). DNA polymerase uses DNA as a template, and reverse transcriptase uses RNA as a template. Therefore, different nucleotide chains are immobilized on the support depending on the enzyme whose activity is to be measured.

The type and molecular weight of the nucleotide chain to be immobilized are not particularly limited, but are usually poly A for reverse transcriptase because they are easily available and the reaction is simple. However, poly dA is used for DNA polymerase.

DNA on which this nucleotide chain is immobilized
The support for synthesis reaction is obtained by forming a silicon oxide film having a predetermined thickness on a silicon substrate. As such a silicon substrate, S whose surface is precisely polished is
An i single crystal substrate can be used. The Si single crystal substrate is excellent in that it is easily available and a uniform surface can be easily obtained.

The thickness of the silicon oxide film coated on the silicon substrate is preferably in the range of 1000 to 1900 angstroms (hereinafter abbreviated as A). This is because when the thickness of the silicon oxide film is within the above range, the peak of the absorbance spectrum is in the visible light region (wavelength range).
This is because it appears within 300 nm to 800 nm). Also,
The error range of the thickness of the silicon oxide film is preferably 10 A or less.

The formation of the silicon oxide film on the silicon substrate can be performed by a well-known method used in the manufacture of semiconductor devices such as thermal oxidation. Immobilization of the nucleotide chain on the support,
The method can be performed according to a usual method for immobilizing nucleotides on an inorganic substance, for example, a carbodiimide method or a glutaraldehyde method.

The reaction solution used in the measuring method according to the present invention contains deoxyribonucleotides, primers and surfactants that are complementary to and labeled on the nucleotide chain immobilized on the support. Any deoxyribonucleotide and primer may be any as long as they are complementary to the nucleotide chain immobilized on the DNA synthesis reaction support.For example, the nucleotide chain immobilized on the support may be poly A or In the case of poly dA, dUTP is used as a nucleotide and oligo dT is used as a primer. Also, the combinations shown in the following table can be used.

[0030] The substance labeled on the deoxyribonucleotide contained in the reaction solution is a substance capable of specifically reacting with the substance bound to the inorganic substance in the complex used in the next step. Any combination of these substances may be used as long as it specifically binds. For example, biotin-avidin, biotin-streptavidin, antigen-
Examples include an antibody reaction and an enzyme-substrate reaction.

In the present invention, the term "surfactant" refers to a DNA which destroys the outer coat of a virus or a bacterial cell, but which is present in the membrane.
Refers to a chemical substance having a property and a concentration that do not inhibit a synthesis reaction of a synthase. Examples of such surfactants include anionic surfactants such as glycodeoxycholic acid and lauryl sulfate, cationic surfactants such as cetylpyridinium chloride and dodecyltrimethylammonium bromide, CHAPS and CHAP
Examples include amphoteric surfactants such as SO and non-ionic surfactants such as NP-40, Triton X-100, Tween 20, and MEGA-8. It is excellent in that it hardly destroys the structure of the synthase. Further, the amount of the surfactant used in the present invention is 0.001% or more in the concentration in the reaction solution,
It is preferably 0.01% or more, but if the viscosity or foaming property increases, the treatment efficiency tends to decrease.
Preferably, it is used in a concentration range of 0.01 to 1%.
However, when a surfactant such as an anionic and / or cationic surfactant that may change the molecular structure of the enzyme itself is applied, it is preferable to use a surfactant within the above practical concentration range. It is preferable to use a low concentration, or use it at an extremely low concentration which is included in the above practical concentration range only when combined with a nonionic surfactant. These surfactants can be used alone, but if it is convenient to use the characteristics according to the type of surfactant, a plurality of types and concentrations can be appropriately used in combination.

[0032] In addition to the above-mentioned components, DN
Various additives which are added during the DNA synthesis reaction by the A synthase can be arbitrarily added. The inorganic substance forming a complex with a substance capable of specifically binding to the substance labeled with the deoxynucleotide is not particularly limited, but is preferably a colloidal metal particle, and particularly preferably a gold colloidal particle. preferable. When the inorganic substance is colloidal particles, the particle size is from 20 nm to 100
nm is the effective range, but 40 nm is required to improve the measurement accuracy.
It is preferably from 90 to 90 nm, particularly preferably from 50 to 80 nm. When the particle size is less than 20 nm, the effect of the present invention cannot be sufficiently exerted. When the particle size is more than 100 nm, the film thickness formed on the silicon substrate has a thickness that can be measured by an ellipsometer described later. Exceeds the maximum value.

The control support used in the measurement method according to the present invention is a support composed of a silicon substrate having a silicon oxide film having the same thickness as the support for DNA synthesis reaction, wherein the nucleotide chain is immobilized. Not what can be used. In addition, a support for a DNA synthesis reaction which has not been reacted with a sample, in which a silicon oxide film having the same thickness is formed and the nucleotide chain is fixed in exactly the same state, can also be used. However, it is difficult to immobilize nucleotide chains on the surface of the silicon oxide film in exactly the same state, and therefore the former is preferable as a control support.

In the measuring method according to the present invention, the first reflected light from the control support is incident on the DNA synthesis reaction support after the DNA synthesis reaction, and the resulting D
The elliptically polarized light component of the second reflected light from the NA synthesis reaction support is measured. Examples of the measurement method include measurement of an absorbance spectrum by a reflection type spectrophotometer, a CCD camera, and a photomultiplier. There is a method of measuring the amount of elliptically polarized light component by photoelectric conversion using a tube or a photodiode.

Hereinafter, the measuring procedure of the method for measuring DNA synthase activity according to the present invention will be described in detail. First,
The reaction solution is mixed with a sample such as human serum on a support for DNA synthesis reaction on which the nucleotide chain is immobilized, and left under conditions suitable for DNA synthesis to perform DNA synthesis. I do. The conditions at this time are not particularly limited as long as DNA synthesis is possible, but the reaction is usually performed at pH 5 to 8, at a temperature of 25 to 40 ° C., and for a reaction time of 15 minutes to 24 hours. Further, in order to increase the reactivity and perform the analysis efficiently, it is preferable that the pH is around 7.5, the temperature is around 37 ° C., and the reaction time is 90 minutes to 15 hours.

Thus, when a virus having a DNA synthase is present in the specimen, DNA incorporating the labeled nucleotide is synthesized. In particular,
First, the virus membrane is destroyed in a short time by the surfactant contained in the reaction solution, and a sufficient amount of the DNA synthase of the virus is released into the solution (for example, within 1 minute). On the other hand, a sufficient amount of the primer hybridizes to the immobilized nucleotide chain in a short time (for example, within several minutes), and the DNA synthetase released from the virus binds to the primer. Here, it is preferable that the amount of the primer used is sufficiently larger than that of the immobilized nucleotide, since the hybridization is increased accordingly. Next, the DNA synthesizing enzyme takes in the labeled nucleotide while moving along the nucleotide chain as a template to synthesize DNA. Here, as long as the synthesis process including these binding reactions proceeds, the order of mixing the respective reaction elements is arbitrary, but mixing at almost the same time has the highest measurement efficiency. In addition, by adjusting the mixing time of the primers, a plurality of samples can be efficiently specified.

In this DNA synthesis reaction, the longer the reaction time is, the longer the synthesized DNA chain is. As a result, the amount of the inorganic substance incorporated into the DNA chain is increased, and the sensitivity of the measurement can be improved. According to the measurement method of the present invention, the upper limit of the reaction time is not particularly limited and can be as long as desired, but is practically preferably 24 hours or less.

After the completion of the DNA synthesis reaction, a complex of a substance capable of specifically reacting with a substance labeled with a nucleotide and an inorganic substance is brought into contact with the DNA synthesis reaction support.
At this time, when a DNA synthase is present in the sample, that is, when DNA is generated, the complex specifically binds to a substance that labels nucleotides incorporated in the generated DNA, and an inorganic substance is formed. Is fixed to the DNA.

The synthesized DNA is fixed on a nucleotide chain as a template. In other words, the inorganic substance forming the complex is incorporated into the DNA and fixed to the DNA synthesis reaction support via the nucleotide chain serving as a template.

Thereafter, the amount of the synthesized DNA is measured using polarized light. Specifically, the linearly polarized light is incident on the control support at a predetermined incident angle, and then the first reflected light emitted from the control support is subjected to DNA synthesis reaction at the same incident angle as the linearly polarized light. Then, the light is made incident on the completed support for DNA synthesis reaction, and the elliptically polarized light component contained in the second reflected light finally emitted from the support for DNA synthesis reaction is measured by an appropriate detector.

The linearly polarized light incident on the control support at a predetermined incident angle is polarized depending on the surface state of the control support, and becomes the first reflected light of elliptically polarized light. The first reflected light is
When the light is incident on the DNA synthesis reaction support at the same angle of incidence as that of the linearly polarized light, if the surface state of the control support and the surface of the DNA synthesis reaction support are the same, the first reflected light is again linearly polarized light. Return to However, when the surface state of the control support and the surface state of the DNA synthesis reaction support are different, the difference appears as an elliptically polarized light component in the second reflected light. Therefore, by measuring the elliptically polarized light component, the DNA containing the DNA chain bound to the immobilized nucleotide chain is obtained.
The state of the surface of the support for synthesis reaction can be accurately grasped as a difference from the surface of the support for control. Furthermore, D
By bonding an inorganic substance having a larger particle size as compared to NA molecules to deoxyribonucleotide constituting a DNA chain, the difference in surface state between a DNA synthesis reaction support and a control support becomes remarkable. Was found. As a result, the measured light amount of the elliptically polarized light component increases.

The above steps will be described in more detail with reference to the drawings, taking the measurement of the activity of DNA polymerase as an example.
1 and 2 are diagrams schematically showing steps of a specific example of the activity measurement of DNA polymerase according to the present invention.

In this example, first, as shown in FIG. 1 (a), oligo-dT 3 hybridizes to poly dA 2 immobilized on the support 1 for DNA synthesis reaction. next,
If DNA polymerase 4 is contained in the sample, D
NA Polymerase 4 binds to this complex and oligo-d
Initiate DNA synthesis along the poly dA2 from the end of T3. At this time, deoxyribonucleotide 5 labeled with biotin 6 is incorporated, and as shown in FIG.
DNA strand 7 to which biotin 6 is bound is generated. This reaction is carried out at 37 ° C. for about 15 minutes.

After the completion of the DNA synthesis reaction, the support for DNA synthesis reaction was washed with distilled water, and streptavidin 8
And a complex of gold colloid particles 9 is added. At this time, when biotin 6 is present in the generated DNA strand 7, as shown in FIG. 1 (c), biotin 6 and streptavidin
8 specifically binds to the DNA strand 7 produced by the colloidal gold particles 9 and is thus fixed to the DNA synthesis reaction support 1.

Thereafter, the DNA synthesis reaction support 1 is washed with distilled water, and the moisture adhering to the surface thereof is removed by spraying nitrogen gas. Thus, colloidal gold particles 9
The change in the polarization state that occurs when polarized light is reflected on the surface of the DNA synthesis reaction support 1 on which is fixed is measured as follows. For this measurement, as shown in FIG. 2, an ellipsometer using a control support as a control is used.

In the figure, reference numeral 21 denotes a light source which emits white light 22. First, the white light 22 passes through the polarizer 23 and is polarized into linearly polarized light 24 at −45 °. The linearly polarized light 24 is incident on the control support 25 at an incident angle θ. As the control support 25, for example, a silicon single crystal substrate having a 1100A-thick silicon oxide film formed on the surface is used.

The incident linearly polarized light 24 is elliptically polarized and reflected as the first reflected light 26 depending on the surface state of the reference support 25. Thereafter, the first reflected light 26 is incident on the surface of the DNA synthesis reaction support 27 after the completion of the DNA synthesis reaction at the same incident angle θ as the incident angle θ of the linearly polarized light 24. At this time, the control support 25 and the DNA synthesis reaction support 27
The planes of incidence are arranged at right angles to each other.
For this reason, the polarization direction of the second reflected light 28 is rotated by 90 °, and the second reflected light 28 becomes + 45 ° linearly polarized light.

Thus, the first reflected light 26 is converted to the incident angle θ.
If the surface state of the control support 25 and the surface of the DNA synthesis reaction support 27 are the same, the first reflected light 26 returns to linearly polarized light again, and Is only linearly polarized light. However, the surface state of the DNA synthesis reaction support 27 differs from that of the control support 25 in that a DNA chain containing colloidal gold particles is formed on a nucleotide chain. Therefore, the first reflected light 26 is elliptically polarized.

The second reflected light 28 including the elliptically polarized light component is converted into an analyzer 29 having a polarization direction perpendicular to the linearly polarized light component (+ 45 ° polarized light) in the second reflected light. Introduce. Then, the + 45 ° linearly polarized light component of the second reflected light 28 does not pass through the analyzer 29, and only the elliptically polarized light component passes through the analyzer 29. In this way, the elliptically polarized light component is
Detect at 30. As the detector 30, for example, a reflection type spectrophotometer can be used to measure the absorbance spectrum of the DNA synthesis reaction support 27. In addition, a CCD camera, a photomultiplier, or the like may be used as the detector 30 to measure the amount of elliptically polarized light.

According to such a measuring method, D
Due to the presence of the NA synthase, the colloidal gold antibody 9 having an extremely large particle size as compared with the DNA chain is immobilized on the surface of the DNA synthesis reaction support 1 through the generation of DNA. Therefore, the D
The second reflected light 28 is higher than the surface of the support 1 for the NA synthesis reaction.
, The elliptically polarized light component increases. As a result, D
Measurement of NA synthase can be performed with higher sensitivity.

As described above, according to the measurement method of the present invention, the activity of DNA synthetase such as DNA polymerase and reverse transcriptase can be measured with high sensitivity without requiring pretreatment such as centrifugation. However, by using the measurement method of the present invention, the antibody titer of the anti-DNA synthase antibody can be easily measured.

As described above, anti-DNA synthase antibodies
It is produced by the body's immune system when a virus having a DNA synthase such as DNA polymerase or reverse transcriptase enters the living body, and inhibits the activity of the DNA synthase. Therefore, in the method for measuring DNA synthase activity according to the present invention, a DNA synthase whose activity is known in advance is added, and a sample containing an anti-DNA synthase antibody is further added to inhibit the activity of DNA synthase. The antibody titer of the anti-DNA synthase antibody can be known from the degree of inhibition. That is, the characteristic of the method for measuring the antibody titer of the anti-DNA synthase antibody is that a silicon oxide film having a predetermined thickness is formed and a nucleotide chain as a template of DNA synthase is immobilized on a silicon substrate. A DNA synthesis reaction is performed by contacting a sample with a reaction solution containing a deoxyribonucleotide, a primer, a DNA synthase and a surfactant, which are complementary to the nucleotide chain and are labeled, to a support for DNA synthesis reaction. Performing, a step of contacting the DNA synthesis reaction support with a complex of a substance capable of specifically reacting with the deoxyribonucleotide label and an inorganic substance, and the same support as the DNA synthesis reaction support. Making a linearly polarized light incident at a predetermined angle of incidence on a control support made of a body and not having the nucleotide chain immobilized thereon. Making the first reflected light reflected by the control support incident on the DNA synthesis reaction support after the DNA synthesis reaction is performed at the same incident angle as the incident angle of the linearly polarized light; Measuring the elliptically polarized light component of the second reflected light reflected by the support for synthesis reaction.

According to this method, when an anti-DNA synthase antibody is present in a sample, the activity of DNA synthase is inhibited, and the rate of DNA synthesis is reduced, or the synthesis ability is lost. For this reason, the amount of the inorganic substance fixed to the support for DNA synthesis reaction also decreases, and as a result, the elliptically polarized light component of the second reflected light also decreases. Since the intensity of the elliptically polarized light component indicates the degree of activity of the DNA synthase, the degree of decrease in the intensity directly indicates the antibody titer of the anti-DNA synthase antibody.

[0054]

【Example】

Example 1 (I) Pretreatment An 8-mm-square silicon wafer on which a 4000-A-thick silicon oxide layer was formed was covalently bonded to 2-aminoethyl-3-aminopropylmethyldiethoxysilane (KBM602, manufactured by Shin-Etsu Silicon Co., Ltd.). Modified by

Next, 40 mg /
1 ml of a 12.7 mM N-hydroxysulfosuccinimide (Pierce) solution of poly A (Boehringer Mannheim) in 50 ml.
ml, and further 50 ml of 10 mM 1-methyl-3- (3-
Dimethylaminopropyl) carbodiimide hydrochloride (Pier
ce) was dispensed and mixed. Then, the silicon wafer was left at room temperature overnight (12-18 hours), and poly A
Was immobilized to prepare a support for a DNA synthesis reaction.

Further, a control support necessary for performing ellipsometric analysis was prepared. The control support was D
It is made of the same silicon wafer as the support for NA synthesis reaction, and the thickness of the silicon oxide layer is also 4000A.

The thus-prepared DNA synthesis reaction support and control support were combined, ellipsometric analysis was performed using an ellipsometer (manufactured by Photo Device) having the configuration shown in FIG. 2, and the obtained output was recorded. did. (II) Reverse transcriptase reaction Commercially available reverse transcriptase (Catalog No. 1465 333, Boehring
er Mannheim) was diluted with PBS to a concentration of 0,0.
Five enzyme solutions of 04, 0.1, 0.2 and 1.0 mU were prepared.

Next, the DN prepared in the above (I) was used.
A On a support for synthesis reaction, 10 ml of an enzyme solution and a reaction solution (4.16 mM biotin-dUTP (Boehringer Mannheim)
8.33 mM TTP (Boehringer Mannheim), 167.5 mM KCl (Sigma), 12.5 mM
MgCl ₂ (Sigma), 50 mM Tris, pH 7.8
(Sigma), 5.625 mM dithiothreitol (Sigm
a), 0.437 mM EDTA (Sigma), 0.2
87% Triton X100 (Sigma) and 0.0625 OD / m
40 ml of 1 oligo dT12-18 (Pharmacia LKB) was mixed in a test tube and reacted at 37 ° C. for 30 minutes. After the reaction,
The DNA synthesis reaction support was washed with a washing solution (10 mM Tris-HC).
1, pH 7.5, 0.15 M NaCl, 1 mM EDTA and 0.01% Tween 20).

Next, the diluted solution (10 mM Tris, pH 7.5, 0.15 M NaC) was added to the washed DNA synthesis reaction support.
l, 1 mM EDTA and 1% BSA) diluted 1: 5000 with streptavidin-gold complex [Streptavidin-g
old conjugate] (EY Laboratories Inc.) 100 ml was dispensed and allowed to stand at 37 ° C for 10 minutes. After the reaction, the DNA synthesis reaction support was washed with a washing solution (10 mM Tris-HCl, pH 7.
5, 0.15 M NaCl, 1 mM EDTA and 0.01% Tw
(en20) for 6 times. (III) Ellipsometry Analysis The DNA synthesis reaction support after the reverse transcriptase reaction was recombined with the control support used in the ellipsometry analysis performed before the reaction, and the measurement was performed with an ellipsometer. The difference between the output obtained and the output obtained before the reaction was taken as the measured output. FIG. 3 shows the results of the measurement using the enzyme solutions of each concentration.

As is clear from the figure, the enzyme concentration was 0.2 m
The change in the measurement output is particularly remarkable in the low concentration region below U,
It can be seen that measurement is possible even at a concentration of 01 mU or less. As described above, the measurement of the transcriptase activity is usually performed as a means of detecting a virus. Therefore, comparison of the sensitivity between a commonly used virus detection method and the measurement method according to the present invention was attempted in the literature.

[0061] As a virus detection method currently used, "HIV antigen / E" sold by Dynabot Co., Ltd. is used.
IAII Abbott "kit and the method of Robin L. Dewer et al. (Robin L. Dewer et al., J. Infectious Disease
s, 170: 1179-9 (1994)).

The "HIV antigen / EIA II Abbott" kit is a kit in which beads to which antibodies against DNA in HIV are immobilized are added to the sample and the measurement is carried out by the EIA method. 20p for
g / ml or less.

On the other hand, in the method of Robin et al.
The RNA inside is captured on the surface of the microplate by a target probe, and a bDNA (branched DNA) amplifier is hybridized with the target probe and bound. Next, a base labeled with an alkaline phosphate is hybridized to the bDNA amplifier and reacted with a chemiluminescent substrate such as dioxetane, and the absorbance is measured. The sensitivity obtained by this method is said to be 5000-10000 HIV-I RNA Eq / ml.

When the sensitivity of the above-mentioned conventional virus detection method is converted into the activity of a DNA synthetase to be measured in the present invention, each becomes 0.4 mU. Therefore, by using the method of the present invention, it is possible to detect a virus with extremely high sensitivity that cannot be obtained conventionally. Example 2 Comparison of Sensitivity Depending on Presence or Absence of Gold Colloidal Particles (I) Pretreatment A 6 mm square silicon wafer on which a 4000 A thick silicon oxide layer was formed was reacted with aminopropyltrimethoxysilane to prepare a modified silicon wafer.

Next, about 3000 mer polymer of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meso-p-toluenesulfonate and adenine (hereinafter simply referred to as poly A) was mixed with a phosphate buffer, The modified silicon wafer prepared above was immersed in this mixed solution.
In this state, after standing at room temperature for 1 hour, the silicon wafer was taken out of the phosphate buffer and washed to prepare a support for DNA synthesis reaction.

A control support was prepared in exactly the same manner, combined with the DNA synthesis support described above, and subjected to ellipsometry with an ellipsometer having the structure shown in FIG. 2 to obtain an output. Was recorded. (II) Reverse transcriptase reaction Commercially available reverse transcriptase (Boehringer Mannheim) was used in PB
Dilute with S, concentration 0, 0.04, 0.1, 0.2, 0.4 respectively
And six types of enzyme solutions of 1.0 mU / ml were prepared.

Next, the reaction solution (50 mM Tris, pH 7.8)
(Sigma), 167.5 mM KCl (Sigma),
12.5 mM MgCl ₂ (Sigma), 5.625 mM D
TT (Sigma), 0.437 mM EDTA (Sigma), 0.287% Triton X100 (Sigma), 8.33 μm
M dTTP (Boehringer Mannheim), 4.16 μM
Biotin-dUTP (Boehringer Mannheim) and 3.125 μg oligo-dT _12-18 (Sigma)) 1
The support for DNA synthesis reaction prepared in the above (I) was immersed in 66.7 μl, and the enzyme solution 33.3 prepared above was further immersed.
μl was mixed and reacted at 37 ° C. for 30 minutes. After the reaction, D
The support for NA synthesis reaction was removed from the reaction solution and washed. (III) Ellipsometric analysis The support for DNA synthesis reaction after the reverse transcriptase reaction was recombined with the control support prepared in the above (I), and measurement was carried out using an ellipsometer. The difference from the previously obtained output was taken as the measured output. The results of the measurement using the enzyme solutions of each concentration are shown by black squares in FIG.

After this measurement, the DN used for the above measurement was placed in a buffer containing avidin-modified gold colloid particles.
The support for synthesis reaction A was further immersed and reacted at 37 ° C. for 30 minutes, and then taken out of the buffer and washed.

The colloidal gold-modified DNA synthesis support thus obtained was combined with the control support similarly prepared in the above (I) and subjected to measurement with an ellipsometer. The difference between the output obtained before the reaction and the output obtained before the reaction was determined. The results of the measurement using the enzyme solutions of each concentration are also shown in FIG. 4 (solid triangles). As is apparent from FIG. 4, a clear increase in sensitivity is observed when the particles are modified with colloidal gold particles, as compared with the case where no colloidal gold particles are used.

[0070]

The method for measuring DNA synthase activity of the present invention is simple and very sensitive, and can detect DNA synthase activity even if it is too weak to be detected by conventional methods. Is possible.

[Brief description of the drawings]

FIG. 1 is a diagram schematically showing a process until a colloidal gold particle is fixed to a DNA synthase reaction support in a specific example of the method for measuring DNA synthase activity of the present invention.

FIG. 2 is a diagram schematically showing an optical system of an apparatus for measuring the state of the surface of a support for DNA synthesis in a specific example of the method for measuring DNA synthase activity of the present invention.

FIG. 3 is a graph showing the correlation between the concentration of DNA synthase and the output of an elliptically polarized light component, obtained by an example of the method for measuring DNA synthase activity of the present invention.

FIG. 4 shows the results of the DNA synthetase activity measurement method of the present invention using colloidal gold particles as the inorganic substance (black triangles) and the results of the measurement performed in accordance with the present invention except that no inorganic substance is used. FIG.

FIG. 5 is a diagram schematically showing a specific example of a conventional method for detecting a DNA synthase.

Claims (3)

[Claims] 1. A DNA synthesis reaction support comprising a silicon substrate on which a silicon oxide film having a predetermined thickness is formed and on which a nucleotide chain as a template for a DNA synthase is immobilized, is coated on the nucleotide chain. Contacting a sample with a reaction solution containing complementary and labeled deoxyribonucleotides, primers and surfactant
Performing a NA synthesis reaction; contacting the DNA synthesis reaction support with a complex of a substance capable of reacting specifically with the deoxyribonucleotide label with an inorganic substance; and the DNA synthesis reaction support. Consisting of the same support as
And a step of causing linearly polarized light to enter the control support on which the nucleotide chain is not immobilized at a predetermined incident angle; and the first reflected light reflected by the control support is incident on the linearly polarized light at an incident angle. Irradiating the DNA synthesis reaction support after the DNA synthesis reaction is performed at the same incident angle as above, and measuring the elliptically polarized light component of the second reflected light reflected by the DNA synthesis reaction support. And a method for measuring DNA synthase activity. 2. The method according to claim 1, wherein the inorganic substance is a colloidal metal particle. 3. The particle size of the colloidal metal particles is 20 nm or more.
The method for measuring DNA synthase activity according to claim 2, which is 100 nm.