JPH08233822A - Detecting probe for antibody of anti-double-stranded dna in humor such as sle serum - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce measuring errors and enable correct measurements by labelling a linear dsDNA formed by cutting by means of a restriction enzyme, at its terminal. SOLUTION: A DNA tracer obtained by labelling a terminal of a linear dsDNA obtained by cutting of a cyclic dsDNA such as plasmid DNA or the like by means of a restriction enzyme is used. As a living body including the cyclic dsDNA such as plasmid DNA or the like, germs such as colon bacilli, Bacillus subtilis, etc., or yeast such as Saccharomyces-cereviciae or the like are utilizable. When the dsDNA is labelled only at its terminal, DNA molecules are never damaged by nicks, etc., and therefore the dsDNA can be labelled in the most stable state. As a labelling substance, deoxyribonucleotide phosphate labelled with radiactive substance, enzyme, fluorescent substance or chemical emission substance, or the like is used.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a probe for detecting anti-double-stranded DNA antibody in body fluid such as SLE serum.

[0002]

2. Description of the Related Art Quantitative measurement of anti-double-stranded DNA antibody in human body fluids, especially in serum or plasma, is carried out by systemic lupus erythematosus (SLE).
It is an extremely important test for the diagnosis of the theatoma). Anti-DN appearing in SLE patient serum etc.
A antibody includes 1) an antibody that reacts only with double-stranded DNA,
There are known 2) antibodies that react with double-stranded and single-stranded DNA, and 3) antibodies that react only with single-stranded DNA. Of these, the antibodies 1) and 2) are specifically found in the sera of SLE patients and are rarely found in other diseases, and are therefore important for diagnosis.

On the other hand, the antibody 3) appears in other diseases (rheumatoid arthritis, Sjogren's syndrome, etc.).
Although the utility of diagnosing E disease is low, the relationship with the antibody of 3) becomes a problem in detecting the antibodies of 1) and 2).

From the viewpoint of measurement, when double-stranded DNA is used as an antigen, the antibodies 1) and 2) react, and when single-stranded DNA is used as an antigen, the antibodies 2) and 3) react. Therefore, in order to diagnose SLE with high specificity, it is important to increase the purity of the double-stranded DNA of the antigen as much as possible in the anti-double-stranded DNA antibody measurement system using the double-stranded DNA as the antigen. Is.

As a method for measuring such an anti-double-stranded DNA antibody titer, erythrocyte pseudo-aggregation reaction method, radioimmunoassay method (RIA
Method) and oxygen immunoassay (EIA method).

Although the red blood cell agglutination reaction method is widely used because it is less expensive than the RIA method and the EIA method, it has a drawback that it lacks in sensitivity and quantitativeness. In contrast, RIA
The method and EIA method have good sensitivity and quantitativeness, and are highly clinically effective because they correlate well with the severity or recovery of SLE patients.

Among the RIA methods, the method using labeled DNA called the Farr method is currently widespread as the most sensitive and accurate method. In the Farr method, DN
After reacting the DNA tracer labeled with A with radioactivity and the anti-double-stranded DNA antibody in the patient sample, the final concentration was 50%.
Ammonium sulfate (ammonium sulfate) aqueous solution was added to achieve saturation, and after mixing, centrifugation was performed to precipitate antibodies such as immunoglobulin, and the amount of precipitation that was bound by binding to antibodies such as immunoglobulin was labeled on a DNA tracer. The amount of antibody is calculated from the radioactivity. This is because DNA that does not bind to the antibody does not precipitate in 50% saturated ammonium sulfate aqueous solution, and DN that binds to the antibody
The principle is that A precipitates.

[0008]

Problems to be Solved by the Invention The anti-double-stranded DNA antibody measurement system that has been used so far has the following problems. (A) Control of the number of antigen molecules in the measurement system (b) Contamination of single-stranded DNA into the anti-double-stranded DNA antibody measurement system (c) When evaluating the amount of antibody as an antibody titer using a standard substance Shows that the relationship between antibody titer and antigen binding rate does not show very good linearity,

With respect to these points, the prior art will be described. [Prior Art] (a) Controlling the Number of Antigen Molecules in the Assay System In conventional anti-double-stranded DNA antibody assay systems, human cultivated cells of calf thymus DNA (Japanese Patent Publication No. 58-56694) are used as the antigen. DNA (Pincus et al., The N
ew England Journal of Med
icine. (1969) 281 P701), Cri
Purified network DNA of the genus Thidia (JP-A-60-78349), and beef thymus DNA
What is cut by ultrasonic waves (Japanese Patent Laid-Open No. 57-42632) is used. All of these DNAs are composed of DNA molecules of nonuniform length (molecular weight). Since this heterogeneity (what length of DNA contains at what ratio) varies depending on the lots prepared, it is virtually impossible to control the molar concentration of such DNA.

In the antibody measurement system, the amount of antigen in the system is very important for the measured value. In particular, in the measurement system in which the amount of antibody is determined from the amount of bound antigen, such as the Farr method using a standard substance, the amount of bound antigen must be constantly controlled by the molar concentration. In a conventional antibody measuring system, for example, a rheumatoid factor (antibody against denatured IgG) measuring system, since the antigen has a constant molecular weight, if the antigen is controlled by weight concentration, it can be controlled by molar concentration at the same time. On the other hand, in the case of an antigen such as the above-mentioned heterogeneous DNA (DNA having a wide distribution of molecular weight), the weight concentration and the molar concentration do not show a constant relationship.

The inventor of the present invention deteriorates the accuracy of measurement in the conventional method for measuring anti-double-stranded DNA antibody, where the amount of antigen in the measurement system is not managed in a constant manner as the number of moles, that is, the number. I have noticed that there is a major cause for this.

(B) Contamination of single-stranded DNA As described above, the antibody against single-stranded DNA is SLE.
Since it appears in diseases other than the above, in the measurement system for anti-double-stranded DNA antibodies, double-stranded DNA with minimal contamination with single-stranded DNA must be used as an antigen. For this purpose, SI nuclease treatment in JP-A-57-42632 and S in JP-A-58-56694 are used.
After treatment with I-nuclease, purification with hydroxyapatite is performed. SI nuclease is known as an enzyme that specifically decomposes single-stranded DNA, but in reality, single-stranded DNA cleavage (Nic
Put k). (Shishido et al., Protein Nucleic Acid Enzyme, 3
0, p981 (1985)). As you can see, Nic
Double-stranded DNA with k is decomposed during storage to single-stranded D
It may cause NA, and such instability of the antigen has already been pointed out. (Tojo et al., Japanese Clinical 1
985 Autumn special edition (bottom), p197). Furthermore, it has been pointed out that even linear DNA obtained by cutting a circular double-stranded DNA is denatured at the cut end to give single-stranded DNA. By the way, it is known that double-stranded DNA treated with SI nuclease contains some single-stranded DNA moieties at the ends (Shishido et al., Supra).
The purity and stability of A are not always perfect.

Purification with hydroxyapatite is suitable for separating a complete double-stranded DNA molecule and a complete single-stranded DNA molecule, but some single-stranded DNA may be present at the terminal or central part of the double-stranded DNA. It is difficult to separate a molecule containing a portion from a complete double-stranded DNA.

A further serious problem is that it is extremely difficult to evaluate the contamination amount of single-stranded DNA when a DNA molecule having a heterogeneous molecular weight is used as an antigen. Heterogeneous double-stranded DNA and single-stranded DNA derived from it or double-stranded DNA partially having a single-stranded portion can be analyzed by ordinary DNA such as electrophoresis, gel filtration, ultracentrifugation, and hydroxyapatite column. It cannot be separated and analyzed by the method used for. Therefore, in the conventional method, it was only possible to consider that the single-stranded DNA does not exist after the treatment for removing the single-stranded DNA. For this reason, it has always been pointed out that single-stranded DNA may be generated during contamination of single-stranded DNA with an antigen or during storage (Tojo et al., Supra), and no clear objection has been made to deny it.

(C) Linearity between the antibody titer and the amount of bound antigen in the case of measuring the amount of antibody using a standard substance In the case of measuring the amount of antibody by the Farr method using a standard substance,
It has been a problem in the conventional method that the antibody titer and the amount of bound antigen do not always show linearity. As described above, the Farr method is a method of calculating the amount of anti-double-stranded DNA antibody from the amount of antigen bound to the antibody. Therefore, in order to accurately determine the antibody titer, it is most ideal that the antigen and the antibody bind at a ratio of 1: 1; that is, it is ideal that only one antibody molecule binds to one antigen molecule. The number of antigen molecules is equal to the number of bound antibody molecules. In the measurement system for anti-double-stranded DNA antibody, if the antibody is sufficiently less than the antigen DNA, the antigen and the antibody are 1
There is a high possibility that they will bind to each other, but when the antibody concentration becomes high, multiple antibodies will bind to one antigen. In such a state, even if the amount of anti-double-stranded DNA antibody increases,
The amount of bound antigen does not increase so much, and the linearity between the antibody titer and the amount of bound antigen deteriorates. This is a so-called “standard curve goes to bed”, which is a major cause of deterioration of measurement accuracy.

The present inventor has studied this point and found that the length of the DNA used as an antigen is, for example, 2
I noticed that it is due to the fact that it is too long, such as 0 kbp or more, so that the probability of binding of multiple antibodies to one antigen increases.

Further, a DNA-binding protein in serum proteins and a single-stranded DNA antibody mixed in SLE patient serum, etc. are one of the causes of worsening the linearity between the amount of antigen binding and the antibody titer. When an anti-DNA antibody measuring system is used as a diagnostic agent, it is usual to use human body fluids, especially serum and plasma as samples, and it is important to take into consideration the influence of DNA binding proteins in blood. This has been pointed out in the review article by Tojo et al. (Clinical Immunity 12, p77).
6, (1980)). On the other hand, Ballard et al. Used a system using a mouse monoclonal anti-DNA antibody for four types of D
After showing that NA-binding proteins interfered with the results of the Farr method, pure antibodies and DN without these proteins were removed.
Although the experiment in the system of A is described, (Ballard e
t al, J .; Biolog. Chem. (19
84) vol. 259. p3492-3498), thus removing the DNA-binding protein, pure double-stranded DN
The method that requires preparation of only the A antibody cannot be performed in the clinical laboratory. Such non-specific DN
The inventor of the present application has also found that the longer the DNA length, the greater the influence of the A-binding protein and other antibodies.

[0018]

[Means to Solve the Problems]
The inventors of the present invention have made extensive studies and found that the DNA trace
In a measurement system in which a standard substance that binds to a saccharide is used for measurement, a linear double-stranded DNA obtained by cleaving a double-stranded circular DNA such as plasmid DNA with a restriction enzyme is used as the DNA tracer to label at the end. It has been found that the following effects can be achieved by using the DNA tracer consisting of the following.

(B) Management by Molar Concentration of Antigen As described above, in the measuring system for specific antibody, the amount of antigen in the measuring system must be controlled by the molar concentration.
In order to solve this, it is necessary to control the number of antigen molecules contained in a unit weight of the antigen at a constant level when the antigen is produced. For this purpose, the present inventor used a double-stranded circular DNA such as a recombinant DNA plasmid or a natural plasmid DNA cleaved with a restriction enzyme. According to this method, the number of antigen molecules per unit weight of antigen DNA can be kept within a constant range. For example, Example 1 yields a 1: 1 mixture of DNA with lengths of 1442 bp and 989 bp.

As a method for preparing a DNA having a constant molecular weight, it is theoretically possible to use a molecular weight fractionation method in which high molecular weight DNA is cleaved by ultrasonic waves or an enzyme, and then electrophoresis, gel filtration or ultracentrifugation is used. However, in these methods, even a DNA molecule obtained by separating in the most ideal state has a certain molecular weight distribution, and like the DNA used in the present invention, a constant number of antigen molecules is always present in a unit weight. Cannot be included. Furthermore, these molecular weight fractionation methods can fractionate only a very small amount of antigen DNA, and the method is complicated, which is a very difficult method in terms of production of a measurement kit on a commercial scale. Therefore, as a method for producing DNA containing a constant number of molecules per unit weight, it is best to use a double-stranded circular DNA such as a recombinant or natural plasmid as a material and cut it with a restriction enzyme.

(B) Single-stranded DNA contamination Double-stranded circular DNA such as recombinant or natural plasmid DNA can also be used as a method for minimizing single-stranded DNA contamination.
(Double-stranded closed circular DNA) is optimal. Double-stranded closed ring D
NA (ccc-DNA) is a molecule containing no single-stranded DNA from the beginning. ccc-DNA is a chromosome or c
Since physical properties such as buoyant density are greatly different from linear DNA derived from the decomposition of cc-DNA, it may be purified by a method often used in gene recombination such as CsCl density gradient ultracentrifugation, electrophoresis, gel filtration and the like. For example, it is possible to obtain an extremely high-purity double-stranded circular DNA. Further, when this is used as a material and cleaved with a restriction enzyme such as BanI, CfrI, and AvaI listed in the examples, a single-stranded portion of 4 bases at the maximum is generated. Such a short single-stranded DNA portion can be formed by Klenow enzyme or the like used in Examples described later.
It can be easily restored to complete double-stranded DNA.

Furthermore, the use of such a DNA molecule makes it extremely clear to control the amount of single-stranded DNA or other DNA molecules mixed in. Double-stranded circular DNA such as recombinant or natural plasmids can be easily and clearly separated and analyzed from other DNAs by physical techniques such as electrophoresis, ultracentrifugation, gel filtration and the like. In electrophoresis, double-stranded cyclic D
Not only NA itself but also a fragment obtained by cutting it with a restriction enzyme can be distinguished from other DNA. Therefore, after labeling the DNA, electrophoresis is performed and the radioactivity of the band of the DNA is measured to obtain D other than the target DNA tracer.
It becomes possible to accurately control the degree of contamination of NA (including single-stranded DNA).

As described above, by using a DNA of a certain length derived from a double-stranded circular DNA such as a recombinant or natural type plasmid, the single-stranded DNA in the anti-double-stranded DNA measuring system can be We have found that the problem of contamination can be overcome.

(C) Linearity between antibody titer and antigen binding amount As described above, when a long DNA molecule is used as an antigen,
The probability that a plurality of antibodies will bind to one antigen increases, which causes the linearity between the antibody titer and the amount of antigen binding to be deteriorated by the conventional method, which in turn lowers the accuracy of measurement. Furthermore, DNA
It has been found that when the length is very long, the binding to nonspecific DNA binding proteins and the like also increases.

The present inventor can obtain DNAs of various lengths by arbitrarily cutting double-stranded circular DNAs of recombinant or natural type plasmids with a restriction enzyme, and thus DNAs of various lengths can be obtained. Using the antigen, the linearity between the antibody titer and the amount of bound antigen was examined. As a result, if the length of the DNA antigen is sufficiently short, the probability that the antigen and the antibody will bind one to one will increase.
That is, the region showing good linearity becomes long. Furthermore, it has been found that the use of sufficiently short DNA also reduces non-specific binding to DNA binding proteins. And
Such DNA has a length of 0.1 to 6.6 kb.
p is preferably 2.3 kbp or less, and above all,
It was found that the one of 0.1 to 1.4 kbp is the most preferable.

As described above, the present invention provides an antigen D
As a NA tracer, D which is obtained by labeling a linear double-stranded DNA obtained by cleaving the double-stranded circular DNA with a restriction enzyme.
The use of NA tracer was used as a means for solving the problem.

In the present invention, first, an organism containing double-stranded circular DNA such as recombinant or natural plasmid DNA is selected.

Such organisms include Escherichia coli (Esch
erichia coli), Bacillus su
btilis, Pseudomonas putida
Bacteria such as Saccharomyces cere
Yeasts such as Viciae are available. Here, an Escherichia coli plasmid will be described as an example, but a double-stranded circular DNA similar to a plasmid, such as a replicative form of bacteriophage, can naturally be used. Escherichia coli harboring the plasmid is cultured in an appropriate medium, and the bacterial cells are collected by a method such as centrifugation or filtration. Next, the bacterial cells are lysed with a lytic enzyme such as lysozyme, a surfactant such as sodium dodecyl sulfate, a protein denaturant such as phenol, etc., and deproteinized by phenol extraction or the like to extract nucleic acids. Since this nucleic acid contains DNA and RNA, RN
RNA is degraded with A-degrading enzyme to obtain a crude DNA fraction. This crude DNA fraction contains plasmid DNA and chromosome-derived D
Since it contains fragmented DNA derived from NA and a plasmid, ccc-D can be obtained by a method such as CsCl equilibrium density gradient ultracentrifugation, gel filtration, hydroxyapatite column, and electrophoresis.
NA (plasmid DNA) is isolated. The ccc-DNA thus obtained is an extremely high-purity double-stranded DNA.
This can be confirmed by agarose gel electrophoresis, HPLC using gel filtration or the like.

Subsequently, the ccc-DNA is cleaved with an appropriate restriction enzyme, preferably 0.1 to 6.6 kbp, particularly 2.3 kbp or less, most preferably 0.1 to 1.
It gives rise to a 4 kbp DNA fragment. The restriction enzymes used at this time were EcoRI, HindIII, BamHI, and B.
Although any restriction enzyme such as an I can be used, it must be carefully selected to yield a DNA fragment of the desired length. The ends of the obtained DNA fragments are exogenously labeled in vitro. Previously 125I-Citizen, 14C-
So-called invitro labeling of DNA by incorporating thymidine, 3H-thymidine, etc. into human cultured cells, Escherichia coli cells, etc.
The endogenous labeling of the vo labeling method was often used. This i
The nvivo labeling method is also applicable to the present invention, but DNA
The method of labeling the ends of the fragments in vitro is extremely effective in controlling the number of molecules or the number of DNA fragments,
Large stocks of unlabeled antigen are possible. For this reason,
There are various advantages as compared with the in vivo labeling method, such that the labor of manufacturing is largely omitted, the problem of error between antigen kits can be reduced, and the work time for handling radioactivity is shortened.

As an in vitro labeling method for DNA,
DN represented by so-called "nick translation method"
There are a method of labeling not the end of the A molecule but the middle thereof, and a method of labeling only the end of one DNA molecule. Of these, the nick translation method is a good labeling method that can obtain a DNA with the highest specific activity because it can increase the number of labels, and is usually most often used. However, this labeling method results in a single-strand break (so-called nick) in the middle of the DNA molecule, resulting in double-stranded DNA.
Stability will deteriorate. On the other hand, in the method of labeling only the ends, the double stranded DNA is labeled in the most stable state because the DNA molecule is not scratched by nicks or the like. As such an end labeling method, a method using polynucleotide kinase, a method using DNA polymerase large fragment (Klenow enzyme), T4-DNA
A method using a polymerase and a method using terminal deoxytransferase (TdT) can be used. Particularly, in the method using Kleno-enzyme, a short single-stranded portion at the end can be restored to a complete double-stranded DNA.

The labeling substances are 125 I, 14C, 3H,
It can be carried out using radioactive substances such as 32P, enzymes such as peroxidase, alkaline phosphatase, β-galactosidase, fluorescent substances, deoxyribonucleotide phosphates labeled with chemiluminescent substances, and the like. Alternatively, biotinylated deoxyribonucleotide phosphate or the like may be used for indirect labeling with avidin labeled with a radioactive substance, an enzyme, a fluorescent substance, or a chemiluminescent substance. Of these, 125I is most practical as a clinical test reagent.

The DNA tracer thus labeled is bound to an appropriately set standard substance (standard serum),
The relationship between the antigen binding rate and the number of antibodies in the standard substance (antibody titer) is determined as a standard curve, and then the same DNA tracer is contained in the human body fluid, particularly in the serum or plasma of the patient. The antigen binding rate is determined by reacting with an anti-double-stranded DNA antibody, and the antibody titer of serum or the like is determined from the antigen binding rate using the standard curve previously determined.

The antigen binding rate is determined as follows.

After the reaction, the tracer bound to the antibody (bound tracer) and the tracer not bound to the antibody (free tracer) are separated. In the Farr method, only the bound tracer is precipitated with an ammonium sulfate aqueous solution having a final concentration of 50%. The amount of label (for example, radioactivity) in the precipitate is measured to determine the antigen binding rate corresponding to each sample. A standard curve is prepared with the antibody titer of each standard serum on the horizontal axis and the antigen binding rate on the vertical axis, and the anti-double-stranded DNA antibody titer in the sample is calculated from this standard curve and the antigen binding rate of the sample to be measured. .

The thus obtained anti-double-stranded DNA
From the antibody titer, systemic lupus erythematosus, rheumatoid arthritis, systemic progressive sclerosis, dermatomyositis, polymyositis, Sjögren's syndrome, mixed connective tissue disease, Behcet's disease, selected from collagen diseases selected from The detection is performed, and is particularly useful for detection of systemic lupus erythematosus (SLE).

As the standard substance, SLE patient serum is usually used, and it is used after dilution with horse serum or the like. However, as an antibody peculiar to SLE disease, double-chain D
Although NA antibody appears, this monoclonal antibody cannot be used as a standard substance. The reason for this is that the properties are different from those in serum and the influence of other binding components contained in serum and the like cannot be considered.

[0037]

EXAMPLES Next, specific examples will be described.

Example 1 (I) Preparation of Reagent (i) Preparation of DNA Tracer 1. E. coli carrying the plasmid pNDPC1. coli K12
The JM109 strain is cultured in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl), and the plasmid is purified by a usual method. A restriction map of pNDPC1 is shown in FIG. That is, as shown in this map, the plasmid pNDPC1 was cloned into pUC18 (Yanisch).
Perron, C.I. et al. , Gene 33:
103 (1985)) is partially cleaved with CfrI and then directly ligated with T4-DNA ligase. The selection marker in this case is ApR
And lac-. 2. Add pNDPC1 to [10 mM Tris.HCl (pH
8.5), 7 mM MgCl2, 20 mM NaCl,
Cleavage with the restriction enzyme CfrI in an aqueous solution containing 7 mM 2 mercaptoethanol, 0.01% bovine serum albumin]. 3. This DNA fragment was added to [50 mM Tris.HCl.
(PH 7.2), 10 mM MgSO ₄ , 1 mM dithiothreitol, 500 μg / m bovine serum albumin 1 mM
dGTP] in an aqueous solution containing ¹²⁵ I-dCTP
And DNA polymerase I large fragment (Klenow enzyme) are added, and one end of the DNA is labeled. 4. The labeled DNA is separated from unreacted ¹²⁵ I-dCTP of this reaction solution by a Sephadex G25 column. Add this to [50 mM sodium borate, 15 mM EDTA,
0.01% NaN ₃ ] in an aqueous solution containing a final concentration of 0.1 μC
Dissolve to i / m. This is a tracer solution.

(Ii) Preparation of standard solution The amount of protein bound to 1 pmole of DNA was 100
It was defined as 0U.

By the hemagglutination method, the sera of patients with anti-double-stranded DNA antibody titer ++++ were respectively 0, 6, 2, 45 and 8
It was diluted with horse serum to a standard solution of 0,178 U / m.

(Iii) Preparation of Ammonium Sulfate Aqueous Solution 390 g of ammonium sulfate is dissolved in 1 liter of distilled water.

(II) Measurement operation (i) Add 20 standard solutions or serum (sample) to a test tube.
Add μl. (Ii) Add 200 μL of tracer solution,
Incubate at ℃ for 2 hours. (Iii) Add 1 ml of ammonium sulfate solution,
Mix well. (Iv) Centrifuge at 1500 G for 15 minutes, and aspirate the supernatant. (V) Using a well scintillation counter, measure the radioactivity of each test tube. (Vi) The standard curve shown in FIG. 2 is prepared, and the anti-double-stranded DNA antibody titer is read based on the obtained serum antigen binding rate using the standard curve.

FIG. 2 is a standard curve of the anti-double-stranded DNA antibody titer thus obtained.

Table 1 shows the values actually measured by this measurement method and the values measured by the conventional anti-double-stranded DNA antibody titer measuring kit (anti-double-stranded DNA antibody titer measuring kit manufactured by Ameram).
Shown in

[0045]

[Table 1]

Example 2 In this example, 1.1 kbp and 1.2 kbp DNA fragments were labeled with ¹²⁵ I and used as a tracer, S
In this example, LE patient serum was used as a standard substance.

(I) Preparation of Reagent (i) Preparation of DNA Tracer 1. The plasmid pNDPC1 used in the above Example 1 was [10 mM Tris.HCl (pH 8.5), 7 mM
MgCl2, 7 mM 2-mercaptoethanol,
Cleavage with the restriction enzyme BanI in an aqueous solution containing 0.1% bovine serum albumin (pH 8.0). By this cleavage, pNDPC1 becomes two DNA fragments of 1.1 kbp and 1.2 kbp. 2. These DNA fragments were respectively labeled with [50 mM Tri
s · HCl (pH 7.2), 10 mM MgSO 4,
1 mM dithiothreitol, 500 μg / ml bovine serum albumin, 1 mM dGTP, 1 mM dAT
P, 1 mM dTTP (pH 7.2)], and ¹²⁵ I-dCTP and DNA polymerase I large fragment (Klenow enzyme) are added to label the ends. 3. Labeled DNA is separated from unreacted ¹²⁵ I-dCTP from these reaction solutions by a Sephadex G25 column.
Add this to [50 mM sodium borate, 15 mM EDT
A, 0.01% NaN ₃ ] in an aqueous solution containing a final concentration of 0.1
Dissolve to give μCi / ml. These are used as tracer solutions.

(Ii) Preparation of standard solution SLE patient serum having an anti-double-stranded DNA antibody titer of 600 U / ml was equine serum at 0, 5, 10, 25, 5 respectively.
It was dissolved so as to be 0,100 U / ml.

(Iii) Preparation of Ammonium Sulfate Aqueous Solution 390 g of ammonium sulfate is dissolved in 1 liter of distilled water.

(II) Measuring operation (i) 25 standard solution or serum (sample) was placed in a test tube.
Add μl. (Ii) Add 200 μL of tracer solution,
Incubate at ℃ for 2 hours. (Iii) Add 1 ml of ammonium sulfate solution,
Mix well. (Iv) Centrifuge at 1500 G for 15 minutes, and aspirate the supernatant. (V) Using a well scintillation counter, measure the radioactivity of each test tube. (Vi) A standard curve is prepared and the anti-double-stranded DNA antibody titer of serum (sample) is read. FIG. 3 is a standard curve of the anti-double-stranded DNA antibody titer thus obtained. As can be seen in FIG. 3, a good standard curve is obtained over the area. (Vii) The binding rates obtained from the standard serum and the sample (serum) of one patient at this time and the measured values of the patient sample read from the binding rate are shown in Tables 1 and 2. Further, FIG. 7 is a graph in which the change with time of the measured value is plotted and compared with the result of the Amersham method.

[0051]

[Table 2]

[0052]

[Table 3]

Example 3 In this example, a 6.6 kbp DNA fragment prepared from naturally occurring plasmid DNA was labeled with ¹²⁵ I, and this was used as a tracer, and the serum of an SLE patient was used as a standard substance. is there.

(I) Preparation of reagent (i) Preparation of DNA tracer 1. E. coli-derived ColEl, DNA (Chan P.
T. et al, J .; Bid. Chem. 260 89
25-8935, (1985)) [10 mM Tris.
-HCl, 7 mM MgCl2, 10 mM NaC
1, 7 mM 2-mercaptoethanol (pH 7.
3)] is contained in an aqueous solution containing the restriction enzyme AvaI. By this cleavage, the DNA becomes a 6.6 kbp fragment. 2. This DNA fragment was added to [50 mM Tris.HCl.
(PH 7.2), 10 mM MgSO ₄ , 1 mM dithiothreitol, 500 μg / ml bovine serum albumin, 1 mM dGTP, 1 mM dATP, 1 mM d
TTP (pH 7.2)] in an aqueous solution containing ¹²⁵
I-dCTP and DNA polymerase I large fragment (Klenow enzyme) are added to carry out end labeling.
3. The labeled DNA is separated from unreacted ¹²⁵ I-dCTP of this reaction solution by a Sephadex G25 column. Add this to [50 mM sodium borate, 15 mM EDTA,
0.01% NaN ₃ ] in an aqueous solution containing a final concentration of 0.1 μC
Dissolve to i / ml. This is a tracer solution.

(Ii) Preparation of standard solution SLE patient serum having an anti-double-stranded DNA antibody titer of 600 U / ml was used as horse serum and 0, 5, 10, 25, 5 respectively.
It was dissolved so as to be 0,100 U / ml.

(Iii) Preparation of Ammonium Sulfate Aqueous Solution 390 g of ammonium sulfate is dissolved in 1 liter of distilled water.

(II) Measurement operation (i) 25 standard solution or serum (sample) was placed in a test tube.
Add μl. (Ii) Add 200 μL of tracer solution,
Incubate at ℃ for 2 hours. (Iii) Add 1 ml of ammonium sulfate solution,
Mix well. (Iv) Centrifuge at 1500 G for 15 minutes, and aspirate the supernatant. (V) Using a well scintillation counter, measure the radioactivity of each test tube. (Vi) A standard curve is prepared and the anti-double-stranded DNA antibody titer of serum (sample) is read. FIG. 4 is a standard curve of the anti-double-stranded DNA antibody titer thus obtained. As shown in FIG. 4, the standard curve becomes gentle in the region where the anti-double-stranded DNA antibody titer is 50 U / ml or more, but shows good linearity up to 50 U / ml. Accurate measurement is possible in such a region with good linearity.

Example 4 In this example, 141, 313, 315, 368 and 47 are used.
This is an example in which the 5, 1307 and 1444 bp DNA fragments were labeled with ¹²⁵ I, and this was used as a tracer, and SLE patient serum was used as a standard substance.

(I) Preparation of Reagent (i) Preparation of DNA Tracer 1. Plasmid pBR322 (Sanger. F. et.
al, J .; Mol. Biol. 125 225-24
6, (1978)) [10 mM Tris.HCl,
10 mM MgCl2, 100 mM NaCl, 10
Cleavage with the restriction enzyme TthHB8I in an aqueous solution containing mM2-mercaptoethanol (pH 7.3)].
Due to this cleavage, the DNA becomes 141, 313, 315,
There are 7 fragments of 368, 475, 1307 and 1444 bp. 2. These DNA fragments were treated with [50 mM Tris.H
Cl (pH 7.2), 10 mM MgSO ₄ , 1 mM dithiothreitol, 500 μg / ml bovine serum albumin, 1 mM dGTP, 1 mM dATP, 1 mM
dTTP (pH 7.2)] in an aqueous solution containing
¹²⁵ I-dCTP and DNA polymerase I large fragment (Klenow enzyme) are added to carry out end labeling. 3. Labeled DNA is separated from unreacted ¹²⁵ I-dCTP from these reaction solutions by a Sephadex G25 column.
Add this to [50 mM sodium borate, 15 mM EDT
A, 0.01% NaN ₃ ] in an aqueous solution containing a final concentration of 0.1
Dissolve to give μCi / ml. This is a tracer solution.

(Ii) Preparation of standard solution SLE patient serum having an anti-double-stranded DNA antibody titer of 600 U / ml was used as horse serum and 0, 5, 10, 25, 5 respectively.
It was dissolved so as to be 0,100 U / ml.

(Iii) Preparation of Ammonium Sulfate Aqueous Solution 390 g of ammonium sulfate is dissolved in 1 liter of distilled water.

(II) Measurement operation (i) 25 standard solution or serum (sample) was placed in a test tube.
Add μl. (Ii) Add 200 μL of tracer solution,
Incubate at ℃ for 2 hours. (Iii) Add 1 ml of ammonium sulfate solution,
Mix well. (Iv) Centrifuge at 1500 G for 15 minutes, and aspirate the supernatant. (V) Using a well scintillation counter, measure the radioactivity of each test tube. (Vi) A standard curve is prepared and the anti-double-stranded DNA antibody titer of serum (sample) is read. FIG. 5 is a standard curve of anti-double-stranded DNA antibody titers thus obtained. As shown in FIG. 5, the standard curve becomes gentle in the region where the double-stranded DNA antibody titer is 10 U / ml or less.

As described above, the first and second embodiments of the present invention are
In Nos. 3 and 5, the DNA derived from the recombinant plasmid was 0.
1 to 1.4 kbp is shown, and in Example 4, it is shown that 6.6 kbp is possible. As a result, in the present invention, the DNA length is 0.1
It was judged that the one of ˜6.6 kbp was possible. As is apparent to those skilled in the art, in the technical field of the present invention, DNA derived from a natural plasmid is used as an antigen substance.
Also, the recombinant DNA can be used equally.

Example 5 In this example, the effect of complement CIq was investigated. The effect of complement on the serum of normal subjects was examined. Serum of 10 normal individuals was (1), as it was (complement was active) (2), after inactivation by treatment at 56 ° C for 30 minutes (complement inactivated). The binding to the DNA tracer was investigated. The DNA tracer is (a) the tracer used in Example 3 (double-stranded DNA tracer, dsDN
A) and (b) a single-stranded DNA tracer (ssDNA) prepared by treating the tracer of Example 3 at 95 ° C. for 1 minute and then rapidly cooling in ice were used.

Binding rate to each tracer DNA (B / T)
Was expressed by (count in 50% saturated ammonium sulfate precipitation) / (total count). This is shown in Table 4. The B / T values in this table are (Mean ± SD) values, and the unit is%.

As is clear from Table 4, in the double-stranded DNA (dsDNA) of the present invention, no effect of complement inactivation (inactivation) was observed, and the measurement method using this tracer was CI.
It can be seen that it is not affected by complements such as q.

On the other hand, for single-stranded DNA (ssDNA),
A large difference was observed in the binding rate before and after inactivation, which clearly shows that the complement component in the serum sample influences the measurement.

[0068]

[Table 4]

Example 6 Clinical Examination In this example, a total of 213 healthy subjects and patients with various diseases including SLE were measured by the method described in Example 3 above. The result is shown in FIG.

A normal value of 6 was obtained by measuring 140 healthy subjects.
When set below U / ml, 96.4% of active SLE (out of 27/28 cases), 62.5% of inactive SLE (out of 30/48 cases), other collagen diseases 4.4% (2/46 cases) were positive.

[0071]

As described above, according to the method for measuring an anti-double-stranded DNA antibody titer in human body fluid and the measuring kit according to the present invention, stable supply of the reagent is easy and the measurement error is small. Accurate measurement is possible.

[Brief description of drawings]

FIG. 1 is a plasmid pNDPC used in the examples of the present invention.
It is explanatory drawing which shows the restriction enzyme map of 1.

FIG. 2 is an anti-duplex D prepared in Example 1 of the present invention.
It is a graph which shows the standard curve of NA antibody.

FIG. 3 is an anti-double-stranded DNA prepared in an example according to the present invention.
It is a graph which shows the standard curve of an antibody.

FIG. 4 is an anti-double-stranded DN prepared in Example 3 according to the present invention.
It is a graph which shows the standard curve of A antibody.

FIG. 5: Anti-double-stranded DNA prepared in Examples according to the present invention
It is a graph which shows the standard curve of an antibody.

FIG. 6 shows the results of Example 6 carried out to confirm the clinical effect of the present invention, and is a graph plotting anti-double-stranded DNA antibody titers in various diseases.

FIG. 7: Anti-D of the same patient comparing the present invention with a comparative example
It is a graph which shows a time-dependent change of NA antibody titer.

Claims (6)

[Claims] 1. A double-stranded circular DNA is a linear double-stranded DNA cut into a certain length by restriction oxygen, and the end of the linear double-stranded DNA formed by the cutting is labeled. A probe for detecting an anti-double-stranded DNA antibody in a body fluid such as SLE serum. 2. The label is a polynucleotide kinase,
A probe for detecting an anti-double-stranded DNA antibody in a body fluid such as SLE serum according to claim 1, which is labeled with Klenow enzyme, T4-DNA polymerase, and terminal deoxytransferase. 3. The linear double-stranded DNA is added to 0.1-6.
The SLE according to claim 1, wherein the SLE is 6 kbp.
A probe for detecting an anti-double-stranded DNA antibody in body fluid such as serum. 4. The anti-duplex in a body fluid such as SLE serum according to claim 1, 2 or 3, wherein the end of the linear double-stranded DNA is labeled with iodine 125 [ ¹²⁵ I]. Detection probe for chain DNA antibody. 5. The detection probe according to claim 1, which is a detection probe for measuring an anti-double-stranded DNA antibody titer in a body fluid such as SLE serum.
A probe for detecting anti-double-stranded DNA in body fluid such as LE serum. 6. The detection probe according to claim 1, which is a detection probe for measuring an antibody titer of an anti-double-stranded DNA antibody in a body fluid such as SLE serum by the Farr method. A probe for detecting an antibody double-stranded DNA antibody in a body fluid such as SLE.