JPH0291572A - Method of measuring anti-dna antibody value in biological liquid by using virus dna and kit for measurement - Google Patents
Method of measuring anti-dna antibody value in biological liquid by using virus dna and kit for measurementInfo
- Publication number
- JPH0291572A JPH0291572A JP22552888A JP22552888A JPH0291572A JP H0291572 A JPH0291572 A JP H0291572A JP 22552888 A JP22552888 A JP 22552888A JP 22552888 A JP22552888 A JP 22552888A JP H0291572 A JPH0291572 A JP H0291572A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- tracer
- enzyme
- measuring
- antibody titer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000003172 anti-dna Effects 0.000 title claims abstract description 24
- 238000005259 measurement Methods 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims description 28
- 239000007788 liquid Substances 0.000 title claims 2
- 241000700605 Viruses Species 0.000 title abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 239000000700 radioactive tracer Substances 0.000 claims abstract description 17
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 claims abstract description 6
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 claims abstract description 6
- 238000000691 measurement method Methods 0.000 claims abstract description 3
- 108020004414 DNA Proteins 0.000 claims description 47
- 108020005202 Viral DNA Proteins 0.000 claims description 11
- 239000013060 biological fluid Substances 0.000 claims description 11
- 238000002372 labelling Methods 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 230000027455 binding Effects 0.000 claims description 3
- 230000001900 immune effect Effects 0.000 claims 2
- 239000000941 radioactive substance Substances 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 1
- 241001515965 unidentified phage Species 0.000 abstract description 4
- 206010025135 lupus erythematosus Diseases 0.000 abstract description 2
- 241000193830 Bacillus <bacterium> Species 0.000 abstract 1
- 102000004357 Transferases Human genes 0.000 abstract 1
- 108090000992 Transferases Proteins 0.000 abstract 1
- 206010033675 panniculitis Diseases 0.000 abstract 1
- 230000009897 systematic effect Effects 0.000 abstract 1
- 102000053602 DNA Human genes 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- 241000473945 Theria <moth genus> Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000014447 Complement C1q Human genes 0.000 description 1
- 108010078043 Complement C1q Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 241001400590 Richia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 208000018631 connective tissue disease Diseases 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
「産業上の利用分野」
本発明は、生物学的液体試料中の抗ＤＮＡ抗体価を測定
する方法および測定用キットに関するものである。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.
「従来の技術」
周知のように、全身性紅斑性狼１（Ｓ Ｌ Ｅ ； ５
ｙ−ｓｔｅＩｌｉｃ Ｌｕｐｕｓ Ｅｒｙｔｈｅｍａｔ
ｏｓｕｓ）の診断には、抗核抗体（ＡＮＡ）の検出によ
る検査法があるが、この検査法には、検出方法の煩雑さ
や、ＳＬＥ以外の結合組織疾患においても高率に陽性と
なる、いわゆる“ｆａｌｓｅ ｐｏｓｉｔｉｖｅ”（診
断において、検査結果が誤って陽性を呈すること）が多
くみられることがあり、鑑別診断には特異性に欠ける問
題がある。“Prior Art” As is well known, systemic erythematous erythematosus 1 (SLE; 5
y-steIlic Lupus Erythemat
osus), there is a test method that detects antinuclear antibodies (ANA), but this test method is complicated and has a high rate of positive results even in connective tissue diseases other than SLE. "False positives" (false positive test results in diagnosis) are often observed, and differential diagnosis has the problem of lacking specificity.
これに対し、ＳＬＥ患者の血中にＤＮＡに対する抗体が
特異的に見出だされるという事実が明らかにされて以来
、抗ＤＮＡ抗体の測定の臨床的意義が高く評価されるよ
うになり、血清または血漿等の生物学的液体中の抗ＤＮ
Ａ抗体価の測定は、ＳＬＥの診断に極めて有用なものと
なっている。On the other hand, since it was revealed that antibodies against DNA are specifically found in the blood of SLE patients, the clinical significance of measuring anti-DNA antibodies has been highly valued, and serum or anti-DN in biological fluids such as plasma.
Measurement of A antibody titer has become extremely useful in diagnosing SLE.
このような抗ＤＮＡ抗体価の測定法としては、赤血球凝
集反応法、放射免疫測定法（ＲＩ Ａ法）、酵素免疫測
定法（ＥＩＡ法）がある。Methods for measuring such anti-DNA antibody titers include hemagglutination, radioimmunoassay (RIA), and enzyme immunoassay (EIA).
赤血球凝集反応法は、ＲＩＡ法、ＥＩＡ法に比べ、定量
性に欠けるという欠点があるが、安価なために広く用い
られている。これに対し、ＲＩＡ法、ＥＩＡ法は定量性
がよく、ＳＬＥ患者の重篤度または回復度とよく相関す
るために臨床上極めて有効である。Although the hemagglutination reaction method has the disadvantage of lacking quantitative properties compared to the RIA method and the EIA method, it is widely used because it is inexpensive. On the other hand, the RIA method and the EIA method have good quantitative properties and correlate well with the severity or degree of recovery of SLE patients, so they are extremely effective clinically.
「発明が解決しようとする課題Ｊ
しかし、現在広く用いられているＲＩＡ法による測定用
キットには、下記のような欠点があることが指摘されて
いる。``Problems to be Solved by the Invention J'' However, it has been pointed out that the currently widely used measurement kits based on the RIA method have the following drawbacks.
（イ）従来のＤＮＡ、ｌ製法では、平均２０キロベース
ベア（ｋｂｐ）程度の長さが様々なりＮＡが得られ、こ
れらには多くの一本鎖のＤ Ｎ Ａ （ｓｓＤ Ｎ Ａ
）が含まれている。−本鎖ＤＮＡは、血中の補体ＣＩ
ｑと結合する。このため、従来の測定用キットでは、Ｃ
１ｑを不活性化するために、血清を５６℃で３０分間も
処理する必要があるものもある。(b) With the conventional DNA production method, NAs with various lengths of about 20 kilobase bears (kbp) on average are obtained, and these include many single-stranded DNAs (ssDNAs).
)It is included. - Double-stranded DNA is complement CI in the blood
Combines with q. For this reason, with conventional measurement kits, C
Some require treatment of serum at 56°C for as long as 30 minutes to inactivate 1q.
このように、従来の測定法および測定用キットでは、使
用する二本鎖ＤＮＡ トレーサーに一本鎖ＤＮＡが混入
してしまっており、これが測定の特異性を下げる原因と
なっている。As described above, in conventional measurement methods and measurement kits, the double-stranded DNA tracer used is contaminated with single-stranded DNA, which causes a decrease in the specificity of the measurement.
（ロ）抗原とするＤＮＡとしてヒトＤＮＡまたは牛胸腺
ＤＮＡをそのままの形で用いているため、分子量が大き
すぎ、非特異的結合を起こしやすくなっている。さらに
抗体価とトレーサー結合量の直線性を悪くしている。(b) Since human DNA or bovine thymus DNA is used as the antigen DNA, the molecular weight is too large and non-specific binding is likely to occur. Furthermore, the linearity between antibody titer and tracer binding amount is worsened.
このように、従来の生物学的液体中の抗ＤＮＡ抗体価の
測定法および測定用キットには、測定誤差が大きくなる
という問題点があり、これらの問題点を解決することが
課題となっている。As described above, conventional methods and kits for measuring anti-DNA antibody titers in biological fluids have the problem of large measurement errors, and solving these problems has become an issue. There is.
「課題を解決するための手段」
上記従来の課題に対し、本願発明者らは鋭意研究を重ね
たところ、次のような知見を得るに至った。"Means for Solving the Problems" The inventors of the present application have conducted intensive research to solve the above-mentioned conventional problems, and have come to the following knowledge.
近年、分子生物学の劇的な進歩により任意の一定の長さ
のウィルスＤＮＡを大量に調製することが可能となった
。天然または組み換え体ウィルスは、宿主由来のＤＮＡ
との分離が容易であり、これらの精製ウィルスからＤＮ
Ａを調製すれば、高純度の一定の長さのＤＮＡが製造可
能となる。従来のＤＮＡ調製法では、前記したように、
平均２０キロベースペア（ｋｂｐ）程度の長さが様々な
りＮＡが得られ、これらには多（の−本鎖のＤ Ｎ Ａ
（ｓｓＤＮＡ）が含まれていた。長い２本鎖ＤＮＡは
血中の非特異的なタンパクと結合することが多く、−本
鎖ＤＮＡは、血中の補体Ｃ１ｑと結合する。このため、
従来の測定用キットでは、ｃｒｑを不活性化するために
、血清を５６℃で３０分間も処理する必要があるものも
あった。In recent years, dramatic advances in molecular biology have made it possible to prepare large quantities of viral DNA of any given length. Natural or recombinant viruses contain DNA derived from the host.
DNA can be easily isolated from these purified viruses.
If A is prepared, highly purified DNA of a certain length can be produced. In the conventional DNA preparation method, as mentioned above,
NAs with various lengths of about 20 kilobase pairs (kbp) on average are obtained, and these include many (long-stranded DNAs).
(ssDNA) was included. Long double-stranded DNA often binds to non-specific proteins in the blood; - double-stranded DNA binds to complement C1q in the blood. For this reason,
Some conventional measurement kits require that serum be treated at 56° C. for as long as 30 minutes in order to inactivate crq.
一方、抗体価と、抗体に結合するトレーサー量がよい直
線性を有するためには、トレーサー分子に対し、−分子
のイムノグロブリンＧ（ＩｇＧ）が結合することが望ま
しい。この目的のためには、充分に短い長さの二本鎖Ｄ
ＮＡが最もよい。On the other hand, in order to have good linearity between the antibody titer and the amount of tracer that binds to the antibody, it is desirable that -molecules of immunoglobulin G (IgG) bind to the tracer molecule. For this purpose, a sufficiently short length of the duplex D
NA is best.
また、分子生物学に付励してＤＮＡの標識法も大変進歩
した。これまでの測定用キットにはＨｅｒａ細胞にＩＩ
Ｊ−デオキシシチヂンを取り込ませるようなｉｎ ｖｉ
ｖｏ標識法標識−たものがあるが、このような方法では
、純度の高い二本鎖ＤＮＡの調製が難しい。１ｎｖｉｔ
ｒｏ標識法としては、ニックトランレーション法、ポリ
ヌクレオチドキナーゼを用いる方法、ＤＮＡポリメラー
ゼ・ラージフラグメン＋（クレノー酵素）を用いる方法
、Ｔ４−ＤＮＡポリメラーゼを用いる方法、ターミナル
デオ牛シトランスフェラーゼ（Ｔ ｄＴ ）を用いる方
法等がある。In addition, with the help of molecular biology, great advances have been made in DNA labeling methods. Previous measurement kits include Hera cells and II.
In vitro that incorporates J-deoxycytidine
Although there is a vo labeling method, it is difficult to prepare double-stranded DNA with high purity using such a method. 1nvit
Examples of the ro labeling method include the nick translation method, a method using polynucleotide kinase, a method using DNA polymerase large fragment + (Klenow enzyme), a method using T4-DNA polymerase, and a method using terminal deobovine citransferase (T dT ). There are various methods to use.
前記ニックトランスレーション法は、最も比活性の高い
ＤＮＡを得る良い方法であるが、標識後、二本鎖ＤＮＡ
中に多数の一本鎖切断を生じるため、−本鎖ＤＮＡの混
入が起こり、このために、抗ＤＮＡ抗体価の測定には向
かない。The above-mentioned nick translation method is a good method for obtaining DNA with the highest specific activity, but after labeling, double-stranded DNA
Since a large number of single-stranded breaks occur in the DNA, contamination with -single-stranded DNA occurs, and therefore it is not suitable for measuring anti-DNA antibody titers.
一方、ポリヌクレオチドキナーゼ、クレノー酵素、Ｔ４
−ＤＮＡポリメラーゼあるいはＴｄＴを用いるようなり
ＮＡの末端のみを標識する方法は、ニックトランレーシ
ョン法はど高比活性のＤＮＡを得ることはできないが、
−本鎖ＤＮＡを生じさ。On the other hand, polynucleotide kinase, Klenow enzyme, T4
- Methods that label only the ends of NA, such as using DNA polymerase or TdT, cannot obtain DNA with high specific activity, as can the nick translation method.
- Generates double-stranded DNA.
せないため、生物学的液体中の抗ＤＮＡ抗体価の測定に
は合った方法である。This method is suitable for measuring anti-DNA antibody titers in biological fluids.
本願発明者らは、上記知見に基づいて本発明をなすに至
ったものである。The inventors of the present application have accomplished the present invention based on the above findings.
本発明においては、まず、大腸菌に感染する２本ＭＤＮ
Ａウィルス（バクテリオファージ）を宿主菌白米ＤＮＡ
を含まぬように調製し、これよりウィルスＤＮＡを抽出
し、適当な制限酵素で切断してトレーサーの材料とした
。ここに用いるウィルスＤＮＡは、大腸菌（Ｅｓｃｈｅ
ｒｉｃｈｉａ ｃｏｌｉ）に感染するバクテリオファー
ジより調製したが、Ｂａａ ｉ １１ｕｓ ５ｕｂｔｉ
ｌｉｓ、 Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａＳＳ
ａｃｃｈａｒａｍ−ｙｃｅｓ ｃｅｒｅｖｉｃｉａｅ等
ゐバクテリア、酵母や動植物に感染するあらゆるウィル
スが利用可能である。In the present invention, first, two MDNs infected with E. coli are prepared.
A virus (bacteriophage) is host bacteria with white rice DNA
Viral DNA was extracted from this and cut with an appropriate restriction enzyme to be used as a tracer material. The viral DNA used here was derived from Escherichia coli (Esche.
Richia coli) was prepared from bacteriophage that infects Baa i 11us 5ubti.
lis, Pseudomonas putida SS
Accharam-yces cerevisiae and other bacteria, yeast, and any viruses that infect animals and plants can be used.
さらに、本発明では、このように大腸菌等に感染するウ
ィルスから調製したＤＮＡを適当な制限酵素で充分に短
い長さに切断した後、その末端を前記ポリヌクレオチド
キナーゼ、クレノー酵素、Ｔ４−ＤＮＡポリメラーゼあ
るいはＴｄＴ等の酵素により末端を標識して抗ＤＮＡ抗
体価測定用のトレーサーとした。Furthermore, in the present invention, the DNA prepared from the virus that infects E. coli, etc. is cut into a sufficiently short length with an appropriate restriction enzyme, and then the ends are digested with the polynucleotide kinase, Klenow enzyme, or T4-DNA polymerase. Alternatively, the ends were labeled with an enzyme such as TdT to use as a tracer for anti-DNA antibody titer measurement.
「実施例」
この実施例は、５．４ｋｂｐのＤＮＡ断片を１１５１に
より標識して、これを抗原トレーサーとし、ＳＬＥ患者
の血清を標準物質としたＤＮＡ抗体価の測定系の例であ
る。"Example" This example is an example of a DNA antibody titer measurement system in which a 5.4 kbp DNA fragment is labeled with 1151 and used as an antigen tracer, and SLE patient serum is used as a standard substance.
（Ｉ） 試薬の調製
い） ＤＮＡトレーサーの作製
■ 大腸菌由来のウィルスであるφＸ１７４ＲＦバクテ
リオファージＤ Ｎ Ａ （Ｋｏｍａｎｏ、Ｔ、ｅｔ
ａｌ。(I) Preparation of reagents) Preparation of DNA tracer■ φX174RF bacteriophage DNA, a virus derived from Escherichia coli (Komano, T, et al.
al.
Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙｓ、 Ａｃｔａ、、１
５５２９５−２９８（１９６ｇ））を［１０ｚＭ Ｔ
ｒｉｓ−ＨＣｉ２．７ ｍＭ Ｍ９Ｃ（Ｉｔ、 ＩＯｍ
ＭＮａＣσ、７ｘＭ２−メルカプトエタノール（ｐＨ７
，３）コを含む水溶液中で、制ｗｉ酵素Ａｖａｌにより
切断する。この切断によりＤＮＡは、５．４ｋｂｌ）の
断片となる。Biochem, Biophys, Acta, 1
55295-298 (196g)) to [10zMT
ris-HCi2.7mM M9C (It, IOm
MNaCσ, 7xM2-mercaptoethanol (pH 7
, 3) is cleaved by anti-widget enzyme Aval in an aqueous solution containing . Through this cleavage, the DNA becomes a 5.4 kbl) fragment.
■ このＤＮＡ断片を、［５０ｘＭ Ｔ ｒｉｓ−ＨＣ
Ｃ（ｐＨ７，２）、１．ＯｗＭ ＭｇＳ Ｏ４＋ ｌ
ｘＭジチオスレイトール、５００ｕ ９／Ｍ（ｌ牛血清
アルブミシ、１ｍＭ ｄＧＴ Ｐ ％ １ ｍＭ
ｄＡＴ Ｐ、１ｍＭ ｄＴＴ Ｐ（ｐ Ｈ
７，２）コを含む水溶液中に溶解し、”’Ｉ −ｄＣＴ
ＰとＤＮＡポリメラーゼＩ・ラージフラグメント（ク
レノー酵素）を加え、標識を行なう。■ This DNA fragment was added to [50xM Tris-HC
C (pH 7,2), 1. OwM MgS O4+ l
xM Dithiothreitol, 500u 9/M (l Bovine Serum Albumis, 1mM dGT P % 1mM
dATTP, 1mM dTTP (p H
7,2) Dissolved in an aqueous solution containing
Labeling is performed by adding P and DNA polymerase I large fragment (Klenow enzyme).
■ この反応液をセファデックスＧ２５カラムにより標
識ＤＮＡを未反応”５ｒ−ｄＣＴＰから分離する。これ
を［５０ｚＭホウ酸ナトリウム、１５Ｊ！ＭＥＤＴＡ、
０．０１％ＮａＮ＊］を含む水溶液に終濃度０．２μＣ
ｉ／ｘρとなるように溶解する。これをトレーサー溶液
とする。■ Separate the labeled DNA from unreacted 5r-dCTP using a Sephadex G25 column.
A final concentration of 0.2 μC in an aqueous solution containing 0.01% NaN*]
It is dissolved so that i/xρ. This is used as a tracer solution.
（１１）標準溶液の作製
抗ＤＮＡ抗体価６００Ｕ ／ｍＱのＳＬＥ患者血清を馬
血清に、それぞれ０，５．１０．２５．５０．１００Ｕ
＃ｇとなるように溶解した。(11) Preparation of standard solutions SLE patient serum with anti-DNA antibody titer 600 U/mQ was added to horse serum at 0, 5, 10, 25, 50, and 100 U, respectively.
#g was dissolved.
（ｉｉｉ） Ｗｔ酸アンモニウム水溶液の作製硫酸ア
ンモニウム３９０ｆＩを１１２の蒸留水に溶解する。(iii) Preparation of ammonium Wt acid aqueous solution 390 fI of ammonium sulfate is dissolved in 112 ml of distilled water.
（ＩＩ） 測定操作
（ｉ） 試験管に標準溶液または血清（サンプル）を
２５μｇ入れる。(II) Measurement procedure (i) Put 25 μg of standard solution or serum (sample) into a test tube.
（ｉｉ） トレーサー溶液を２００μρ加え、３７℃
２時間保温する。(ii) Add 200 μρ of tracer solution and heat at 37°C.
Keep warm for 2 hours.
（ｉｉｉ ） 硫酸アンモニウム溶液をＩｊｌ（加え
、よく混和する。(iii) Add ammonium sulfate solution to Ijl and mix well.
（ｉｖ） １５００９１５分遠心し、上澄みを吸引除
去する。(iv) Centrifuge for 1500915 minutes and remove the supernatant by suction.
（ｖ） 井戸型シンチレーションカウンターを用いて
、各試験管の放射能を測定する。(v) Measure the radioactivity in each test tube using a well-type scintillation counter.
（ｖｉ） 標準曲線を作製し、血清（サンプル）の抗
ＤＮＡ抗体価を読み取る。第１図は、このようにして得
た抗ＤＮＡ抗体価の標準曲線である。(vi) Prepare a standard curve and read the anti-DNA antibody titer of the serum (sample). FIG. 1 is a standard curve of anti-DNA antibody titers obtained in this manner.
「発明の効果」
以上説明したように、本願発明に係る生物学的液体中の
抗ＤＮＡ抗体価の測定法および測定用キットによれば、
測定誤差が少なく、正確な測定が可能となる。"Effects of the Invention" As explained above, according to the method and kit for measuring anti-DNA antibody titer in biological fluid according to the present invention,
There are few measurement errors and accurate measurements are possible.
第１図は本発明の実施例で得た抗ＤＮＡ抗体の標準曲線
である。
出願人 ニラポン・デイ−ビーシー・コーポレ−ショ
ンFIG. 1 is a standard curve of anti-DNA antibodies obtained in Examples of the present invention. Applicant Nirapon DC Corporation
Claims (6)
して結合し得る標準物質とを用い、免疫学的方法により
生物学的液体中の抗ＤＮＡ抗体価を測定する測定法であ
って、 前記ＤＮＡトレーサーとしてウィルスＤＮＡを制限酵素
で切断して調製した一定の長さのＤＮＡを用いることを
特徴とするウィルスＤＮＡを用いた生物学的液体中の抗
ＤＮＡ抗体価の測定法。(1) A measurement 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 this DNA, the method comprising: A method for measuring an anti-DNA antibody titer in a biological fluid using viral DNA, which is characterized in that a DNA of a certain length prepared by cutting viral DNA with a restriction enzyme is used as a tracer.
、化学的発光物質などの標識物質で行なうことを特徴と
する請求項１記載のウィルスＤＮＡを用いた生物学的液
体中の抗ＤＮＡ抗体価の測定法。(2) The anti-DNA antibody in a biological fluid using viral DNA according to claim 1, wherein the DNA is labeled with a labeling substance such as a radioactive substance, an enzyme, a fluorescent substance, or a chemiluminescent substance. How to measure valence.
ラージフラグメント（クレノー酵素）、ポリヌクレオチ
ドキナーゼ、Ｔ４−ＤＮＡポリメラーゼ、ターミナルデ
オキシトランスフェラーゼ等の酵素を用い、ＤＮＡ分子
の末端のみを標識することを特徴とする請求項１記載の
ウィルスＤＮＡを用いた生物学的液体中の抗ＤＮＡ抗体
価の測定法。(3) When labeling the DNA, use DNA polymerase.
Biology using viral DNA according to claim 1, characterized in that only the ends of DNA molecules are labeled using enzymes such as large fragment (Klenow enzyme), polynucleotide kinase, T4-DNA polymerase, terminal deoxytransferase, etc. Method for measuring anti-DNA antibody titer in target fluid.
して結合し得る標準物質とを有し、免疫学的方法により
生物学的液体中の抗ＤＮＡ抗体価を測定するための測定
用キットであって、 前記ＤＮＡトレーサーとしてウィルスＤＮＡを制限酵素
で切断して調製した一定の長さのＤＮＡを用いたことを
特徴とするウィルスＤＮＡを用いた生物学的液体中の抗
ＤＮＡ抗体価の測定用キット。(4) A measurement kit for measuring 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 this DNA. A kit for measuring an anti-DNA antibody titer in a biological fluid using viral DNA, characterized in that the DNA tracer is a DNA of a certain length prepared by cutting viral DNA with a restriction enzyme. .
、化学的発光物質などの標識物質で行なったことを特徴
とする請求項４記載のウィルスＤＮＡを用いた生物学的
液体中の抗ＤＮＡ抗体価の測定用キット。(5) Anti-DNA in a biological fluid using viral DNA according to claim 4, wherein the DNA is labeled with a labeling substance such as a radioactive substance, an enzyme, a fluorescent substance, or a chemiluminescent substance. Kit for measuring antibody titer.
ラージフラグメント（クレノー酵素）、ポリヌクレオチ
ドキナーゼ、Ｔ４−ＤＮＡポリメラーゼ、ターミナルデ
オキシトランスフェラーゼ等の酵素を用い、ＤＮＡ分子
の末端のみを標識したことを特徴とする請求項４記載の
ウィルスＤＮＡを用いた生物学的液体中の抗ＤＮＡ抗体
価の測定用キット。(6) DNA polymerase when labeling the DNA,
Biology using viral DNA according to claim 4, characterized in that only the ends of the DNA molecules are labeled using enzymes such as large fragment (Klenow enzyme), polynucleotide kinase, T4-DNA polymerase, terminal deoxytransferase, etc. Kit for measuring anti-DNA antibody titer in target liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22552888A JPH0291572A (en) | 1988-09-08 | 1988-09-08 | Method of measuring anti-dna antibody value in biological liquid by using virus dna and kit for measurement |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22552888A JPH0291572A (en) | 1988-09-08 | 1988-09-08 | Method of measuring anti-dna antibody value in biological liquid by using virus dna and kit for measurement |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0291572A true JPH0291572A (en) | 1990-03-30 |
Family
ID=16830718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22552888A Pending JPH0291572A (en) | 1988-09-08 | 1988-09-08 | Method of measuring anti-dna antibody value in biological liquid by using virus dna and kit for measurement |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0291572A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007000832A1 (en) * | 2005-06-29 | 2007-01-04 | Shibayagi Co., Ltd. | Method of assaying endocrine substance in specimen |
-
1988
- 1988-09-08 JP JP22552888A patent/JPH0291572A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007000832A1 (en) * | 2005-06-29 | 2007-01-04 | Shibayagi Co., Ltd. | Method of assaying endocrine substance in specimen |
| US8058011B2 (en) | 2005-06-29 | 2011-11-15 | Shibayagi Co., Ltd. | Method for the measurement of endocrine substances in an analyte |
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