JPH03218462A - Method of measuring anti-dna value in biological liquid and measuring kit - Google Patents

Abstract

PURPOSE: To make stable supply of a reagent easier and to reduce error by using a DNA tracer wherein a linear double-strand DNA obtained by cutting a double-strand annular TNA with restriction enzyme is given a label. CONSTITUTION: An organism, such as Escherichia coli, which contains a double-strand annular DNA such as recombination type or natural type plasmid DNA is cultured in an appropriate medium, and bacteria is collected by such method as centrifugation, filtering, etc., then bacteriolisis and protein-removal are performed for extracting nucleic acid, and RAN is resolved with RAN resolution enzime, thus pair DNA fraction is obtained. Then, the plasmide DNA is separated by electrophoresis, etc., and the plasmide DNA is cut with such restriction enzime as Eco RI, and a DNA fragment is labeled with in vitro. Then, a labeled DNA tracer and an anti DNA antibody in a patient specimen are reacted each other in the appropriate buffer liquid, and after the reaction, the antibody which was combined with antibody is separated from the tracer which was not conbined. Then, with the use of the DNA tracer, an anti DNA antibody value in a sample is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial Application Field" The present invention relates to a method and kit for measuring anti-DNA antibody titer in a biological fluid sample.

``Prior Art'' The measurement of anti-DNA antibodies in biological fluids such as serum or plasma is a
cLupus Erythesatosus) has become an extremely important test for diagnosis. Known anti-DNA antibodies include 1) antibodies that react only with double-stranded DNA, 2) antibodies that react with both double-stranded and single-stranded DNA, and 3) antibodies that react only with single #tI DNA. Of these, l)
and 2) antibodies are specifically found in the blood of SLE patients,
Although it is important for diagnosis, the antibody in 3) also appears in other diseases (rheumatoid arthritis, Sjögren's syndrome, etc.), so its usefulness for diagnosis is low. two! 1 If DNA is used as an antigen, ■
) and 2) react, and when one IDNA is used as an antigen, antibodies 2) and 3) react. Therefore, in order to diagnose SLE with high specificity, it is necessary to use pure double-stranded DNA.
An anti-DNA antibody measurement system using A as an antigen is important.

Methods for measuring such anti-DNA antibody titers include hemagglutination, radioimmunoassay (RIA), and enzyme immunoassay (EIA).

The blush sphere agglutination reaction method is widely used because it is cheaper than the RIA method and EIA method, but the sensitivity is low. The drawback is that it lacks quantitative properties. On the other hand, It l A method, EI
Method A has good sensitivity and quantitative properties, and correlates well with the severity or degree of recovery of SLE patients, so it is extremely effective clinically.

Among these methods, a method using labeled DNA called the Farr method is currently widely used as the most sensitive and accurate method. In the Farr method, a radioactively labeled DNA tracer is reacted with an anti-DNA antibody in a patient specimen, and then ammonium sulfate (ammonium sulfate) is added so that the final concentration is 50% saturation.
After adding and mixing an aqueous solution (ammonium sulfate), the mixture is centrifuged to precipitate Immunoguchi purine, and the antibody titer is calculated from the radioactivity (amount of DNA) precipitated by binding with Immunoguchi purine.

This is because DNA does not precipitate in a 50% saturated ammonium sulfate aqueous solution.
It is based on the principle that DNA bound to antibodies precipitates.

"Problems to be Solved by the Invention" The anti-DNA antibody measurement systems that have been used so far have had the following problems.

(b) Control of the number of antigen molecules in the measurement system (1) Contamination of single-stranded DNA into the anti-double-stranded DNA antibody measurement system (c) Linearity of antibody titer and antigen binding amount Regarding each of these points, The conventional technology will be explained.

[Prior art]

(b) Regarding the management of the number of antigen molecules in the measurement system, conventional anti-DNA antibody measurement systems have used bovine thymus DNA (Japanese Unexamined Patent Publication No. 58-56694) and human cultured cell DNA (Pincus et al. , The New
England Journal of Medicine
ine. (1969) 2gI P 701), C
Purified network DNA of the genus rithfdia (Japanese Unexamined Patent Publication No. 60-78349) and bovine thymus DNA
DNAs obtained by cutting A with ultrasonic waves (Japanese Unexamined Patent Publication No. 57-42632) have been used. All of these DNAs are composed of DNA molecules of non-uniform length (molecular weight).

Since this heterogeneity (what length and what proportion of DNA is included) varies depending on the prepared lot, it is virtually impossible to control the molar concentration of DNA.

In antibody measurement systems, the amount of antigen in the system is very important for the measured value. In particular, in a measurement system such as the Farr method, in which the antibody titer is determined from the amount of bound antigen, the amount of antigen must always be controlled to be constant based on the molar concentration. Usual antibody measurement systems, such as rheumatoid factor (denatured 1gG
In the measurement system for antibodies against antibodies), since the antigen has a certain molecular layer, if the antigen is managed by weight concentration, it is also possible to manage it by molar concentration. On the other hand, the above-mentioned non-uniform D
NA (DNA that exists with a wide distribution of molecular weights)
In the case of antigens such as, weight concentration and molar concentration do not show a fixed relationship.

The inventor of the present application has realized that a major cause of deterioration in measurement accuracy in conventional anti-DNA antibody measurement methods is that the amount of antigen in the measurement system is not controlled as a constant number of moles. It is.

(Note) Regarding the contamination of single-stranded DNA, as already mentioned, antibodies against single-stranded DNA are
Since it also appears in other diseases, double-stranded DNA with as little single-stranded DNA as possible must be used as an antigen in anti-double-stranded DNA antibody measurement systems. For this purpose, JP-A No. 57-42632 discloses Sl nuclease treatment, and JP-A No. 58-56694 discloses treatment with Sl nuclease.
After treatment with l nuclease, purification using hydroxyabatite is performed. Although Sl nuclease is known as an enzyme that specifically degrades single-stranded mRNA, it actually creates a single-stranded DNA cleavage (Nick) in a double-stranded DNA molecule. (Shishido et al., Protein Nucleic Acid Enzyme, 30
, p. 981 (1985)) Double-stranded DNA with such nicks may degrade into single-stranded DNA during storage, and the instability of such antigens has already been pointed out. . (Tojo et al., Japan Clinical 1985
Autumn Special Issue (Part 2), pl97) Also, S! It is also known that double-stranded DNA treated with nuclease contains some single-stranded DNA portions at the ends (Shishido et al., supra). It's not necessarily perfect.

In addition, purification using hydroxyabatite is suitable for separating completely double-stranded DNA molecules from completely single-stranded DNA molecules, but some single-stranded DNA parts may be present at the ends or the center of double-stranded DNA. Complete two molecules containing @D
It is difficult to separate it from NA.

A more serious problem is that when DNA molecules with non-uniform molecular weights are used as antigens, it is extremely difficult to evaluate the amount of single-stranded DNA contamination. Heterogeneous double strand D
NA and single-stranded DNA derived from it or double-stranded DNA with a partially single-stranded portion can be processed using conventional DNA techniques such as electrophoresis, gel filtration, ultracentrifugation, and noxoapatite columns.
Separation and analysis cannot be performed using the techniques used for NA analysis. Therefore, in the conventional method, it was only possible to assume that single-stranded DNA does not exist after processing to remove single-stranded DNA. For this reason, single-stranded DNA may be mixed into the antigen or one strand during storage! The possibility of IDNA generation has always been pointed out (Tojo et al., supra), and no clear refutation has been made to deny it.

(c) Regarding the linearity of antibody titer and antigen binding amount Far
In the r method, there was also a problem with the linearity of the antibody titer and the amount of antigen binding in the conventional method. As mentioned above, Fa r r
The method is to measure the amount of anti-DNA antibody of the antigen bound to the antibody! (radioactivity). Therefore, in order to accurately determine the antibody titer, it is most ideal for the antigen and antibody to bind in a 1:1 ratio, and in such a state the number of bound antigen molecules becomes equal to the number of bound antibody molecules. In the anti-DNA antibody measurement system, antigen DNA
When the amount of antibodies against an antigen is sufficiently low, the antigen and antibody bind together in a controlled manner, but when the concentration of antibodies becomes high, multiple antibodies bind to one antigen.

In this situation, even if the amount of anti-DNA antibody increases, the amount of bound antigen does not increase significantly, and the linearity between the antibody titer and the amount of antigen binding deteriorates. This is a so-called "standard curve sleeping" condition, and is a major cause of deteriorating measurement accuracy. The inventor of the present application has realized that this is because the length of the DNA used for the antigen is too long, which increases the probability that multiple antibodies will bind to one antigen.

"Means to Solve the Problem" In order to solve the above-mentioned conventional problems, the inventors of the present application have conducted extensive research and have found that plasmid DNA has been developed as a DNA tracer.
By using a DNA tracer made by labeling linear double-stranded DNA obtained by cutting double-stranded circular DNA such as A with a restriction enzyme, the following effects can be achieved in solving the above-mentioned problems. I found out something.

(a) Control by molar concentration of antigen As mentioned above, in a specific antibody measurement system, the amount of antigen in the measurement system must be controlled by molar concentration.

In order to solve this problem, it is necessary to constantly control the number of antigen molecules contained in a unit weight of antigen during antigen production. For this purpose, the present inventors used double-stranded circular DNA, such as genetically recombinant or natural plasmid DNA, cut with restriction enzymes. According to this method, the number of antigen molecules per unit weight of antigen DNA can be kept constant.

For example, in Example 1, a mixture of DNA tits having a length of 1442b and 989 bp is obtained.

Polymeric DNA is a method for preparing DNA with a certain molecular weight.
It is theoretically conceivable to cut the protein using ultrasound or enzymes and then perform molecular weight fractionation using electrophoresis, gel filtration, ultracentrifugation, or the like. However, in these methods, even DNA molecules obtained by separation under the most ideal conditions have a certain degree of molecular weight distribution, and DNA molecules that always contain a constant number of molecules per unit weight, such as the DNA used in the present invention, have a certain degree of molecular weight distribution. It cannot be. Furthermore, with these molecular weight fractionation methods, extremely small amounts of antigen DNA can be detected.
, cannot be fractionated, and the method is complicated, making it extremely difficult to manufacture measurement kits on a commercial scale. Therefore, DNA that always contains a constant number of molecules per unit weight
The best method for producing this is to use double-stranded circular DNA such as recombinant or natural plasmid as the material.

(Note) Single-stranded DNA contamination One way to minimize single-stranded DNA contamination is to use double-stranded circular DNA (such as recombinant or natural plasmid DNA).
Double-stranded closed circular DNA) is optimal.

Double-stranded closed circular DNA (ccc-DNA) is a molecule that does not contain any single-stranded DNA from the beginning. cc
cDNA is often used in genetic recombination processes such as CsCC density gradient ultracentrifugation, electrophoresis, and gel filtration because it has very different physical properties such as buoyant density from linear DNA derived from chromosome or ccc-DNA degradation. If purified using a method described above, extremely pure double-stranded circular DNA can be obtained. In addition, Ban
When cut with restriction enzymes such as Cfr1, Cfr1, and Aval, a single-stranded portion of up to 4 bases is generated at the end. Such a short single-stranded DNA portion can be easily repaired into a complete double-stranded DNA using Klenow enzyme or the like used in the Examples described later.

Furthermore, by using such DNA molecules, single-stranded D
The amount of contamination of NA or other DNA molecules can be controlled very clearly. Double-stranded circular DNA such as a recombinant or natural plasmid can be easily and clearly analyzed separately from other DNA by physical methods such as electrophoresis, ultracentrifugation, and gel filtration. Moreover, in electrophoresis, not only double-stranded circular DNA itself but also fragments thereof cut with restriction enzymes can be distinguished from other DNA. Therefore, by performing electrophoresis after labeling the DNA and measuring the radioactivity of the DNA band, it is possible to accurately determine the degree of contamination by DNA other than the target DNA tracer (single-stranded DNA is also included). It becomes possible to manage.

As described above, by using DNA of a certain length derived from double-stranded circular DNA such as recombinant or natural plasmids, single-stranded D
It has been found that the problem of NA contamination can be overcome.

(c) Linearity between antibody titer and antigen binding amount As mentioned above, when a long DNA molecule is used as an antigen,
The probability of multiple antibodies binding to one antigen increases, which causes poor linearity between the antibody titer and the amount of antigen binding in conventional methods, and ultimately reduces measurement accuracy.

The inventor of the present application noticed that DNA of any given length can be obtained by cleaving double-stranded circular DNA such as recombinant or natural plasmid with restriction enzymes, and discovered that DNA of various lengths can be obtained.
Using the antigen, we examined the linearity between the antibody titer and the amount of bound antigen. As a result, if the length of the DNA antigen is sufficiently short, the probability that the antigen and antibody will bind in a ratio of 1 to 1 increases. (In other words, the region exhibiting good linearity becomes longer.) Such DNA has a length of 0.1 to 6. 6
kbp is preferable, and 0.1 to 1. 4kbp
It has been found that the most preferable is

As described above, the present invention aims to use, as a DNA tracer for an antigen, a DNA tracer obtained by labeling linear double-stranded DNA obtained by cutting double-stranded circular DNA with a restriction enzyme. It was used as a solution.

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

Such an organism is Escherichia coli (Escherichia coli).
acoli), Bacillus subtilis,
Bacteria like Pseudoionas putida, Saccharoe + yces cerevisia
Yeasts such as Y. e. Here, we will explain using Escherichia coli plasmid as an example. E. coli containing plasmid is cultured in an appropriate medium, and bacterial cells are collected by centrifugation, filtration, or other methods. Next, the bacterial cells are lysed using a lytic enzyme such as lysozyme, a surfactant such as sodium dodecyl sulfate, a protein denaturant such as phenol, etc., and the proteins are removed by phenol extraction or the like, and the nucleic acids are extracted. Since this nucleic acid contains DNA and RNA, the RNA is degraded with RNA degrading enzyme to obtain a crude DNA fraction. This crude DNA fraction contains plasmid DNA, chromosomal DNA, and plasmid-derived fragmented DNA, so it was analyzed using methods such as CsCl2 equilibrium density gradient ultracentrifugation, gel filtration, neuroquinoapatite force ram, and electrophoresis. -DNA (Blasmid D
NA) is separated.

The CCC-DNA thus obtained is double-stranded DNA of extremely high purity, and this can be confirmed by agarose gel electrophoresis, HPLC using gel filtration, or the like.

Subsequently, this CCC-DNA is digested with an appropriate restriction enzyme, preferably 0.1 to 6. Generates DNA fragments of 6 kbp, most preferably 0.1-1.4 kbp.

The restriction enzymes at this time were EcoRI, I{indl[l,
Any restriction enzyme can be used, such as Bag+I{I, Ban I, etc., but must be carefully selected to yield a DNA fragment of the desired length. The obtained DNA fragment is
Label in vitro. Conventionally, a so-called in vivo labeling method was often used in which DNA was labeled by incorporating l''St cytidine, 10-thymidine, 3H-thymidine, etc. into cultured human cells or E. coli cells.This in vivo labeling method is also described in this book. Although it can be applied to inventions, the in vitro labeling method allows a large amount of unlabeled antigen to be stocked, which greatly reduces the manufacturing effort.
In addition, it has various advantages over in vivo labeling methods, such as reducing the problem of errors between antigen lots and shortening the time required to handle radioactivity.

In vitro labeling methods for DNA include a method of labeling the middle of the DNA molecule, typified by the so-called nick translation method, and a method of labeling only the ends of the DNA. Among these, the nick transrayon method is a good labeling method that yields DNA with the highest specific activity and is usually used most often. However, with this labeling method, single-strand breaks (so-called nicks) occur in the middle of the DNA molecule.
As a result, the stability of the double-stranded DNA deteriorates. In contrast, the method of labeling only the ends of DN
Since there is no nick or other damage to the A molecule, double-stranded D
NA is labeled in the most stable state. Such end-labeling methods include methods using polynucleotide kinase, methods using DNA polymerase large fragment (Klenow enzyme), methods using T4-DNA polymerase, and terminal deoquinotransferase (T dT). method is possible.

Labels include radioactive substances such as ItJ, "C, 31-1, 311), enzymes such as peroxidase, alkaline phosphatase, and β-galactosidase, fluorescent substances, and deoxyribonucleodiphosphate labeled with chemiluminescent substances. Alternatively, indirect labeling can be performed using biotinylated deoxyribonucleotide phosphate, etc., and avidin labeled with a radioactive substance, an enzyme, a fluorescent substance, or a chemiluminescent substance.

The measurement is performed using a labeled DNA tracer and anti-D in the patient sample.
The NA antibody is reacted in an appropriate buffer.

After reaction, tracer bound to antibody (bound tracer)
and the tracer that did not bind to the antibody (free tracer). In the Farr method, only the bound tracer is precipitated using an ammonium sulfate aqueous solution with a final concentration of 50% saturation. It is also possible to precipitate only the bound tracer by a method that combines two antibody methods.Also, any method that can separate bound and free tracers, such as columns such as gel filtration, ultracentrifugation, and electrophoresis, can be used. be.

Next, a specific explanation will be given using examples.

“Example 1” (I) Preparation of reagents (i) Preparation of DNA tracer ■ E coli K1 with plasmid pNDPcI
2JMl09 strain was grown in LB medium (1% tributone, 0.5%
Yeast extract, 0.5% NaCg) and purify the plasmid using a conventional method. The restriction enzyme map of pNDPcl is shown in FIG. That is, as shown in this map, plasmid pNDPcl is pU C 1
B (Yanisch Perron, C. et a
l. , Gene 33:103 (1985)) was partially cleaved by Cfrl and then directly converted to T4.
- It is formed by joining with DNA ligase. The selection markers in this case were ApR and Iac-.

■ pNDPcI [10mM Tris HC
(! (pH 8.5), 7mM M9CL, 20i
M NaCl, 7 nM 2-mercaptoethanol,
o. oi% bovine serum albumin] and cleaved with the restriction enzyme Ctrl.

■ This DNA fragment was transformed into [5hM Tris-HC
(!(p}{7.2), 10mM M9SO.
．． ImM dithiothreitol, 500 μ9/x (l
Bovine serum albumin, I-dCTP and DNA polymerase I.
Add large fragment (Klenow enzyme) and perform labeling.

■ Separate the labeled DNA from unreacted 1''51-dCTP using a Sephadex G25 column.
A, 0.01% NaN3] at a final concentration of 0.
Dissolve to a concentration of 1 μCi/mi2. This is used as a tracer solution.

(II) Preparation of standard solution The amount of protein that binds to I p mole DNA is 10
It was defined as 00U.

Sera from patients with anti-DNA antibody titers of 10+ by hemagglutination were collected at 0, 6, 12, 45, 87, and 178 U/m, respectively.
It was diluted with horse serum to give a standard solution.

(Mountain) Preparation of ammonium sulfate aqueous solution Ammonium sulfate 3909 is dissolved in Iff distilled water.

(II) Measurement procedure (i) Put 20 μg of standard solution or serum (sample) into a test tube.

(ii) Add 200 μg of tracer solution and heat at 37°C.
, keep warm for 2 hours.

(iii) Add ammonium sulfate solution to llIQ,
Mix well.

(iv) Centrifuge at 1500G for 15 minutes and remove the supernatant by suction.

(V) Measure the radioactivity in each test tube using a well-type scintillation counter.

(v1) Create a standard curve and compare serum (sample) anti-DN
Read the A antibody titer. FIG. 2 is a standard curve of anti-DNA antibody titers thus obtained.

Table 1 shows the values actually measured using this measurement method and the values measured using a conventional anti-DNA antibody titer measurement kit (anti-DNA antibody titer measurement kit manufactured by Amersham).

[Table 1] "Example 2" This example consists of 1. In this example, a DNA fragment of 1 kbP and 1.2 kbl was labeled with +Isr and used as a tracer, and serum from patients with SL and E was used as a standard substance.

(1) Preparation of reagents (i) Preparation of DNA tracer ■ Plasmid pNDPCl used in Example 1 above was converted to [1
0mM Tris-}{C (!(PH 8.5)
, 7IIIMM9Cat1 7IIIM 2-mercaptoethanol, 0.1% bovine serum albumin (pH 8.
0)] in an aqueous solution containing restriction enzyme B an r. Due to this cutting, ρNDPC l becomes 1. lkb
pSl. This results in two 2kbp DNA fragments.

■ Each of these DNA fragments [50s+MT
ris-}{C ((pfl 7.2), 10mM
M9SO. , lmM dithiothreitol, 5
00μ9/IIIQ bovine serum albumin, lmM-dGT
P, 1mM dATPS 1mM dTT p (p
i-+ 7.2)] and dissolved in an aqueous solution containing
Labeling is performed by adding dCTP and DNA polymerase 1 large fragment (Klenow enzyme).

■ Separate the labeled DNA from unreacted 1''51-dCTP from these reaction solutions using a Sephadex G25 column.
Dissolve in an aqueous solution containing MEDTA, 0.01% NaNs to a final concentration of 0.1 μCi/wIQ. These are used as a tracer solution.

(11) Preparation of standard solution SLE patient serum with anti-DNA antibody titer 600 U/ml was added to horse serum at 0, 5, IO, 25, 50, and 100 U, respectively.
/ml2.

(B1) Preparation of ammonium sulfate aqueous solution 390 g of ammonium sulfate is dissolved in tC distilled water.

(II) Measurement procedure (1) Add 25 ml of standard solution or serum (sample) to a test tube.
Add μg.

(ii) Add 200 μQ of tracer solution and heat at 37°C.
Keep warm for 2 hours.

(iii) Add ammonium sulfate solution (1x) and mix well.

(iv) Centrifuge at 1500G for 15 minutes and remove the supernatant by suction.

(v) Measure the radioactivity in each test tube using a well-type scintillation counter.

(v1) Create a standard curve and compare serum (sample) anti-DN
Read the A antibody titer. FIG. 3 is a standard curve of anti-DNA antibody titers thus obtained. As shown in the figure, a good standard curve is obtained over the entire region.

Example 3 This example was prepared from naturally occurring plasmid DNA 6. This is an example in which a DNA fragment of 6 itbp was labeled with 5L, and this was used as a tracer, and the serum of an SLE patient was used as a standard substance.

(1) Preparation of reagents (i) Preparation of DNA tracer■ E. coli-derived ColEI, DNA (Cha
nP. ■． etall. Bid. Chew. 260
8925-8935, (1985)) [1. Oi
MTris-HCl2, 7mM MgC Q*S
Cleavage is performed with the restriction enzyme Ava I in an aqueous solution containing IOmM NaC (!-7sM2-mercabutoethanol (pH 7.3)).

Through this cleavage, the DNA becomes 6. It becomes a 6 kbp fragment.

■ This DNA fragment was dissolved in [50mM Tris・
H CO (p1-1 7.2), 10a+M MgS
0 4+ I mM dithiothreitol, 500 μg
/ x (l bovine serum albumin, 1mM dGTP, l
mM dATP, I n+M dT 'l'' P (p
H 7.2)] and dissolved in an aqueous solution containing
Labeling is performed by adding dCTP and DNA polymerase 1 large fragment (Klenow enzyme).

■ Separate the labeled DNA from unreacted I-dCTP using a Sephadex G25 column.
Dissolve in an aqueous solution containing EDTA, 0.01% NaN3 to a final concentration of 0.1 μCi/*12. This is used as a tracer solution.

(+1) Preparation of standard solution Anti-DNA antibody titer 600 U/pass of SLE patient serum to horse serum, respectively 0, 5, 10, 25, 50, +
000/jlc.

(■) Preparation of ammonium sulfate aqueous solution Ammonium sulfate 3909 is dissolved in Iff distilled water.

(II) Measurement procedure (i) Put 25 μQ of standard solution or serum (sample) into a test tube.

(ii) Add 200 μC of tracer solution and heat at 37°C.
Keep warm for 2 hours.

(iii) Add 11 parts of ammonium sulfate solution and mix well.

(iv) Centrifuge at 1500G for 15 minutes and remove the supernatant by suction.

(v) Measure the radioactivity in each test tube using a well-type scintillation counter.

(v1) Create a standard curve and compare serum (sample) anti-DN
Read the A antibody titer. FIG. 4 is a standard curve of anti-DNA antibody titers thus obtained. As shown in the figure, anti-D
NA antibody titer 500/x (The standard curve becomes gentle in the region above.

“Example 4” This example includes 141, 3!3, 315, 368, 47
5, l307, 1 4 4 4 bp DNA fragment
181 and used it as a tracer to detect SLE.
This is an example in which each patient's serum was used as a standard substance.

(!) Preparation of reagents (i) Preparation of DNA tracer ■ Plasmid pB R 322 (Sanger. F, et al.
, Mol. Bio1.125 225-246. (19
7g)) to [lomM Tris-HC(l,
lOmM M9C (1*% lOhM NaCI2, l
OmM 2-mercabutoethanol (pl-1 7.3
) ] in an aqueous solution containing the restriction enzyme T thHB8 [
Cut by. This cleavage makes the DNA 141,3
13, 315, 368, 475, 1307, I 4 4
This results in 7 fragments of 4 bp each.

■These DNA fragments were treated with [50mM Tris-
HCC (pH 7.2), lhM M9SO. ．．
1 g + M dithiothreitol, 500μ9/Shoulder Q bovine serum albumin, lmMdGTP, mM
dATP, la+M dTTP (pH 7.2)
], and labeling is performed by adding 51-dCTP and DNA polymerase I large fragment (Klenow enzyme).

■ Separate the labeled DNA from unreacted I-dCTP using a Sephadex 025 column.
Dissolve in an aqueous solution containing IM EDTA, 0.01% NaNs to a final concentration of 0.1 uCi/x (2).

This is used as a tracer solution.

(ii) Preparation of standard solutions SLE patient serum with anti-DNA antibody titer 600U/z& was added to horse serum at 0, 5, IO, 25, 50, 100U/z, respectively.
It was dissolved so that it became j112.

(Yama) Preparation of ammonium sulfate aqueous solution Dissolve ammonium sulfate 3909 in distilled water of Ig.

(II) Measurement procedure (-) Add 25 liters of standard solution or serum (sample) to a test tube.
Add μQ.

(ii) Add 200μff of }L/-Sur solution,
Keep warm at 7℃ for 2 hours.

(iii) Add ammonium sulfate solution to IR (l,
Mix well.

(iv) Centrifuge at 1500G for 15 minutes and remove the supernatant by suction.

(v) Measure the radioactivity in each test tube using a well-type nondilation counter.

(vi) Prepare a standard curve and read the anti-DNA antibody titer of the serum (sample). FIG. 5 is a standard curve of anti-DNA antibody titers thus obtained. As you can see in the figure,
The standard curve becomes gentle in the region where the anti-DNA antibody titer is 10 U/il2 or less.

As mentioned above, in Examples 1, 2, 3, and 5 of the present invention,
0.1 to 1.0 l. with DNA derived from a recombinant plasmid. 4k
bp up to 6 bp, and in Example 4, 6. 6
It was shown that kbp is possible.

As a result, in the present invention, the length of DNA is
0.1-6. 6 kbl) was determined to be possible. This is because, as is clear to those skilled in the art, in the technical field of the present invention, both natural plasmid-derived DNA and genetically recombinant DNA can be equally used as antigenic substances.

"Example 5" In this example, the influence of complement CIQ was investigated.

The effect of the capture body on the serum of normal subjects was investigated.

Normal serum from a case of 1O is left as is (with complement active) at 56℃3
After inactivation by treatment for 0 minutes (in a state in which complement was inactivated), binding to the DNA tracer was examined. DNA
l-racer is (a) the tracer used in Example 3 (double-stranded DNA tracer, dsDNA), (b)
Two types of single-stranded DNA tracers (SSDNA) were used, which were prepared by treating the tracer of Example 3 with 95° Cl for a minute and then rapidly cooling it in water.

The binding rate (B/T) to valley tracer DNA is (5
It was expressed as (counts in 0% saturated ammonium sulfate precipitation)/(total counts). This is shown in Table 2. The B/T value in this table is (M
ean±SD) value, and the unit is %.

As is clear from the table, the double-stranded DNA (dsD
With NA), no effect of complement deactivation (inactivation) was observed, indicating that the measurement method using this tracer is not affected by complements such as Clq.

On the other hand, with one I'iD N A (ssD N A),
A large difference was observed in the binding rate before and after inactivation, clearly indicating that the complement components in the serum sample affected the measurement.

[Table 21 B/T (Mean+SD) "Example 6" Clinical study 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. did. The results are shown in FIG.

Healthy person! After measuring 40 cases, normal values were found. When set to 6 U/mQ or less, 96.4% (2
7 cases (out of 728 cases), and 62.5% (3 out of 728 cases) in inactive SLE.
0 cases/48 cases), and 4.4% (2 cases/46 cases) of other collagen diseases were positive.

[-Effects of the Invention] As explained above, according to the method and kit for measuring anti-DNA antibody titer in biological fluids according to the present invention, stable supply of reagents is easy and measurement errors are reduced. This makes it possible to make accurate measurements.

[Brief explanation of drawings]

Figure 1 shows the plasmid pNDPC used in the examples of the present invention.
FIG. 2 shows a restriction enzyme map of No. I of the present invention.
Figure 3 shows the standard curve of the anti-DNA antibody obtained in Example 2 of the present invention.
The figure shows the standard curve of the anti-DNA antibody obtained in the third example of the present invention, Figure 5 shows the standard curve of the anti-DNA antibody obtained in the fourth example of the present invention, and Figure 6 shows the standard curve of the anti-DNA antibody obtained in the fourth example of the present invention. This is a graph plotting anti-DNA antibody titers in various diseased areas, showing the results of the sixth example conducted to confirm the clinical effect. Figure 2 (com) Anti-DNA antibody titer (U/ml?) (%) Figure 3 Anti-DNA antibody titer (Ll/rrl) Figure 4 (%) Anti-DNA antibody titer (u/mQ) Figure 6 Anti-DNA antibody titer in various diseases

Claims (6)

[Claims] (1) An assay method for measuring the anti-DNA antibody titer in a biological fluid by an immunological method using a DNA tracer as an antigen and a standard substance capable of binding to the DNA tracer, comprising: Measurement of anti-DNA antibody titer in a biological fluid, characterized by using a DNA tracer that is obtained by labeling linear double-stranded DNA obtained by cleaving double-stranded circular DNA with a restriction enzyme. Law. (2) The double-stranded circular DNA is derived from bacteria or yeast and prepared by a genetic recombination method.
The method for measuring an anti-DNA antibody titer in a biological fluid according to claim 1. (3) The method for measuring an anti-DNA antibody titer in a biological fluid according to claim 1, wherein the double-stranded circular DNA is a naturally occurring double-stranded circular DNA derived from bacteria or yeast. (4) A measurement kit for measuring the anti-DNA antibody titer in a biological fluid by an immunological method, which includes a DNA tracer as an antigen and a standard substance capable of binding to the DNA tracer. The present invention is characterized in that the DNA tracer is a DNA tracer obtained by labeling linear double-stranded DNA obtained by cleaving double-stranded circular DNA with a restriction enzyme. Kit for measuring DNA antibody titer. (5) The double-stranded circular DNA is derived from bacteria or yeast and prepared by a genetic recombination method.
The kit for measuring an anti-DNA antibody titer in a biological fluid according to claim 4. (6) The kit for measuring an anti-DNA antibody titer in a biological fluid according to claim 4, wherein the double-stranded circular DNA is a naturally occurring double-stranded circular DNA derived from bacteria or yeast.