JPH04249770A - Cancer diagnostic agent - Google Patents
Cancer diagnostic agentInfo
- Publication number
- JPH04249770A JPH04249770A JP41854490A JP41854490A JPH04249770A JP H04249770 A JPH04249770 A JP H04249770A JP 41854490 A JP41854490 A JP 41854490A JP 41854490 A JP41854490 A JP 41854490A JP H04249770 A JPH04249770 A JP H04249770A
- Authority
- JP
- Japan
- Prior art keywords
- cea
- tracer
- cancer
- autoantibody
- diagnostic agent
- 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.)
- Granted
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 47
- 201000011510 cancer Diseases 0.000 title claims abstract description 37
- 239000000032 diagnostic agent Substances 0.000 title claims abstract description 15
- 229940039227 diagnostic agent Drugs 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 47
- 239000000700 radioactive tracer Substances 0.000 claims abstract description 35
- 230000002494 anti-cea effect Effects 0.000 claims abstract description 28
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 5
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 claims description 44
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- 229940079593 drug Drugs 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 108060003951 Immunoglobulin Proteins 0.000 claims description 5
- 229960002685 biotin Drugs 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- 102000018358 immunoglobulin Human genes 0.000 claims description 5
- 108090001008 Avidin Proteins 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 239000007790 solid phase Substances 0.000 abstract description 16
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 238000003018 immunoassay Methods 0.000 abstract description 7
- 238000005259 measurement Methods 0.000 abstract description 6
- 239000004793 Polystyrene Substances 0.000 abstract description 4
- 238000002372 labelling Methods 0.000 abstract description 4
- 229920002223 polystyrene Polymers 0.000 abstract description 4
- 230000003100 immobilizing effect Effects 0.000 abstract description 3
- -1 plate Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000011324 bead Substances 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 239000004816 latex Substances 0.000 abstract description 2
- 229920000126 latex Polymers 0.000 abstract description 2
- 239000002245 particle Substances 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 abstract 4
- 239000013043 chemical agent Substances 0.000 abstract 1
- 239000010419 fine particle Substances 0.000 abstract 1
- 238000004062 sedimentation Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 18
- 239000000243 solution Substances 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000003745 diagnosis Methods 0.000 description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 230000000405 serological effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 201000011591 microinvasive gastric cancer Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 108010043671 prostatic acid phosphatase Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000009589 serological test Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【0001】0001
【産業上の利用分野】本発明は、癌の新規な診断剤に関
するものであり、特に早期癌の発見を可能とするもので
あって、医学上の検査、診断分野において大きな貢献が
期待できるものである。[Industrial Application Field] The present invention relates to a novel diagnostic agent for cancer, and in particular, it is capable of detecting early cancer, and is expected to make a significant contribution to the field of medical testing and diagnosis. It is.
【0002】0002
【従来の技術】従来癌の血清学的診断は、主として血中
の腫瘍マーカーと総称される物質を測定することによっ
てなされていた。腫瘍マーカーとしては、癌胎児性抗原
(Carcinoembryonic Antige
n,CEA)、α−フェトプロテイン(AFP)、CA
19−9、前立腺酸性フォスファターゼ等が知られてい
る。これら腫瘍マーカーは、癌種による陽性率に差はあ
るものの一般的に腫瘍の増大とともに血中濃度が上昇す
る。従って癌の治療効果のモニタリングや予後の診断な
どに利用されてきた。しかしこれらの腫瘍マーカーが血
中に検出されるのは癌がかなり成長した段階であり、癌
発生の初期段階、いわゆる早期癌においてこれらの腫瘍
マーカーを検出することは全く困難であった。すなわち
治癒確率の極めて高い早期癌のスクリーニングには役に
立たなかったのである。BACKGROUND OF THE INVENTION Conventionally, serological diagnosis of cancer has mainly been made by measuring substances collectively called tumor markers in the blood. As a tumor marker, carcinoembryonic antigen (Carcinoembryonic antigen)
n, CEA), α-fetoprotein (AFP), CA
19-9, prostatic acid phosphatase, etc. are known. Although the positive rate of these tumor markers differs depending on the type of cancer, the blood concentration of these tumor markers generally increases as the tumor grows. Therefore, it has been used for monitoring the effects of cancer treatment and diagnosing prognosis. However, these tumor markers are detected in the blood only when the cancer has grown considerably, and it has been completely difficult to detect these tumor markers in the early stages of cancer development, so-called early cancer. In other words, it was not useful for screening for early stage cancers, which have an extremely high chance of being cured.
【0003】例えば現在知られているサンドイッチ法C
EA測定キットでは、CEA 0.5ng/ml〜1
000ng/mlの測定しか可能でなく(日本臨▲しょ
う▼、34〔6〕(1976)、p1274−1279
)、極低濃度のCEAを測定することはできず、早期癌
の発見には至らなかったのである。For example, the currently known sandwich method C
In the EA measurement kit, CEA 0.5ng/ml to 1
It is possible to measure only 000 ng/ml (Nippon Rinsho, 34 [6] (1976), p1274-1279).
), it was not possible to measure extremely low concentrations of CEA, and it was not possible to detect early cancer.
【0004】0004
【発明が解決しようとする課題】本発明の目的は、早期
癌の発見につながる真に有効な癌の血清学的診断に有用
なシステムを新たに開発することである。SUMMARY OF THE INVENTION An object of the present invention is to develop a new system that is useful for truly effective serological diagnosis of cancer, which leads to early detection of cancer.
【0005】[0005]
【課題を解決するための手段】このような現状に鑑み、
本発明者らは、早期癌が発見できる血清学的診断システ
ムを新たに開発するために鋭意研究した結果、腫瘍マー
カーの一つであるCEAに対する自己抗体が早期癌に於
て出現することを見いだし、そして更にその抗CEA自
己抗体を効率的且つ正確に測定する方法も確立し、ここ
に本発明を完成させるに至った。[Means to solve the problem] In view of the current situation,
As a result of intensive research to develop a new serological diagnostic system that can detect early cancer, the present inventors discovered that autoantibodies against CEA, one of the tumor markers, appear in early cancer. Furthermore, we have established a method for efficiently and accurately measuring the anti-CEA autoantibody, and have now completed the present invention.
【0006】すなわち本発明は、CEAに対する自己抗
体を測定しもって癌の早期診断を行うことをその基本的
技術思想とするものであって、具体的には癌診断のため
の抗CEA自己抗体測定用試薬、つまり癌診断剤に関す
るものである。以下、本発明を詳細に説明する。[0006] That is, the basic technical idea of the present invention is to perform early diagnosis of cancer by measuring autoantibodies against CEA. The present invention relates to reagents for cancer diagnosis, that is, cancer diagnostic agents. The present invention will be explained in detail below.
【0007】本発明に係る癌診断剤は、抗CEA自己抗
体を測定しうる薬剤(系)をすべて包含するものである
が、CEAに対する自己抗体の測定は、酵素免疫測定法
(EIA)、ラジオイムノアッセイ(RIA)、蛍光イ
ムノアッセイ(FIA)、発光イムノアッセイ等の既存
の高感度イムノアッセイに用いられている技術がすべて
応用でき、したがってこれらのアッセイに使用される薬
剤(系)は、すべて本発明に係る癌診断剤として利用す
ることができる。[0007] The cancer diagnostic agent according to the present invention includes all drugs (systems) capable of measuring anti-CEA autoantibodies, but autoantibodies to CEA can be measured by enzyme immunoassay (EIA), radio All the techniques used in existing highly sensitive immunoassays such as immunoassay (RIA), fluorescence immunoassay (FIA), and luminescence immunoassay can be applied, and therefore all the drugs (systems) used in these assays are applicable to the present invention. It can be used as a cancer diagnostic agent.
【0008】抗CEA自己抗体を測定する方法としては
、例えば次の方法が挙げられる。[0008] Examples of methods for measuring anti-CEA autoantibodies include the following method.
【0009】〔サンドイッチ法〕固相固定化CEAに検
体中の抗CEA自己抗体を反応せしめ、固相上に生成し
た複合体に、更にトレーサー標識CEAを反応させるか
、またはビオチン結合CEAとアビジン結合トレーサー
を同時にまたは時間をずらして反応させ、固相CEA−
自己抗体−トレーサー標識CEAのサンドイッチ複合体
を形成させ、固相上のトレーサーを測定し、抗CEA自
己抗体を測定する方法。[Sandwich method] Solid phase-immobilized CEA is reacted with anti-CEA autoantibody in the sample, and the complex formed on the solid phase is further reacted with tracer-labeled CEA, or biotin-bound CEA and avidin-bound The tracers can be reacted simultaneously or staggered and solid-phase CEA-
A method of forming an autoantibody-tracer-labeled CEA sandwich complex, measuring the tracer on a solid phase, and measuring anti-CEA autoantibodies.
【0010】〔第二抗体法〕固相固定化CEA及び検体
中の抗CEA自己抗体を反応せしめ、固相上に生成した
複合体に、更にトレーサー標識抗ヒトイムノグロブリン
抗体(標識第二抗体)を反応させ、固相上にCEA−自
己抗体−第二抗体の複合物を形成させ、固相上のトレー
サーを測定し、抗CEA自己抗体を測定する方法。[Second antibody method] CEA immobilized on a solid phase is reacted with anti-CEA autoantibody in the sample, and a tracer-labeled anti-human immunoglobulin antibody (labeled second antibody) is added to the complex formed on the solid phase. A method of reacting to form a CEA-autoantibody-second antibody complex on a solid phase, measuring a tracer on the solid phase, and measuring anti-CEA autoantibodies.
【0011】〔競合法〕固相固定化CEAに対し検体中
の抗CEA自己抗体とトレーサー標識抗CEA抗体を競
合的に反応させ、固相上のトレーサーを測定し、抗CE
A自己抗体を測定する方法。[Competitive method] Anti-CEA autoantibodies in the sample and tracer-labeled anti-CEA antibodies are competitively reacted with solid phase-immobilized CEA, and the tracer on the solid phase is measured.
A. Method for measuring autoantibodies.
【0012】〔沈澱法〕トレーサー標識CEAと検体中
の抗CEA自己抗体を反応させた後、ポリエチレングリ
コールを用いて生成した複合体を沈澱させ、沈澱物中又
は上清中のトレーサーを測定し、抗CEA自己抗体を測
定する方法。[Precipitation method] After reacting the tracer-labeled CEA with the anti-CEA autoantibody in the sample, the resulting complex is precipitated using polyethylene glycol, and the tracer in the precipitate or supernatant is measured, Method for measuring anti-CEA autoantibodies.
【0013】上記した各方法において、トレーサーとし
ては、酵素(ペルオキシダーゼ、β−ガラクトシダーゼ
、グルコースオキシダーゼ、アルカリホスファターゼ、
G−6−Pデヒドロゲナーゼ等)、アイソトープ(12
5I、131I等)、蛍光物質(フルオレセイン、FI
TC、ローダミン等)、発光物質(フェリチン、ルシフ
ェリン、ルミノール等)、その他常用される標識剤がす
べて自由に使用できる。[0013] In each of the above-mentioned methods, tracers include enzymes (peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase,
G-6-P dehydrogenase, etc.), isotope (12
5I, 131I, etc.), fluorescent substances (fluorescein, FI
TC, rhodamine, etc.), luminescent substances (ferritin, luciferin, luminol, etc.), and other commonly used labeling agents can all be freely used.
【0014】CEAを固定する固相としては、従来用い
られているポリスチレンビーズ、プレート、ラテックス
粒子、磁性微粒子などが使用可能であり、材質や形状に
は何等制限はない。固定する方法も物理吸着法、共有結
合法、ビオチン−アビジン結合利用、抗原抗体反応利用
することなど既知の方法をすべて利用することが可能で
ある。また、トレーサー標識CEAやトレーサー標識抗
体などの調製もアミノ基やチオール基を利用した化学結
合法やビオチン−アビジン結合を利用した標識法などの
既存の技術を適宜自由に利用することができる。As the solid phase for immobilizing CEA, conventionally used polystyrene beads, plates, latex particles, magnetic particles, etc. can be used, and there are no restrictions on the material or shape. All known methods for immobilization can be used, including physical adsorption, covalent bonding, biotin-avidin binding, and antigen-antibody reaction. Further, for the preparation of tracer-labeled CEA, tracer-labeled antibodies, etc., existing techniques such as chemical bonding methods using amino groups or thiol groups and labeling methods using biotin-avidin bonding can be used as appropriate.
【0015】サンドイッチ法では、固相固定化CEAと
検体及びトレーサー標識CEAを同時にまたは時間をず
らし、インキュベーションする。この反応時間は用いる
固相や、トレーサーの種類に依存するが、通常数分から
数時間を要する。またトレーサーはCEAに直接標識し
てもよいが、トレーサーが酵素などの場合は標識操作の
簡単なビオチンで標識したビオチン−CEAを用いるこ
ともできる。この場合固相固定化CEA、検体、ビオチ
ン結合CEA及びアビジン結合トレーサー(酵素)を同
時にまたは時間をずらしてインキュベーションする。時
間をずらす場合は、途中に未反応物を除去するいわゆる
BF分離操作を行なうことが好ましい。最終的に、固相
CEA−自己抗体−ビオチン標識CEA−アビジン結合
トレーサー(酵素)複合体が得られる。トレーサーの測
定法はその種類によって異なるが、既存の方法を利用で
き、活性や強度の程度は検体中の自己抗体の存在量に比
例するので、適当なコントロール検体や標準液を同時測
定することによって、自己抗体を測定できる。本法は比
較的高感度で自己抗体が測定でき、最も好ましい方法で
ある。In the sandwich method, CEA immobilized on a solid phase and the sample and tracer-labeled CEA are incubated simultaneously or at different times. The reaction time depends on the solid phase used and the type of tracer, but it usually takes several minutes to several hours. Further, the tracer may be directly labeled with CEA, but if the tracer is an enzyme, biotin-CEA labeled with biotin, which is easy to label, can also be used. In this case, solid phase-immobilized CEA, specimen, biotin-bound CEA, and avidin-bound tracer (enzyme) are incubated simultaneously or at staggered times. When changing the time, it is preferable to perform a so-called BF separation operation to remove unreacted substances during the reaction. Finally, a solid phase CEA-autoantibody-biotinylated CEA-avidin-bound tracer (enzyme) complex is obtained. The method for measuring tracers varies depending on the type, but existing methods can be used, and the degree of activity and intensity is proportional to the amount of autoantibodies in the sample, so by simultaneously measuring an appropriate control sample or standard solution. , autoantibodies can be measured. This method allows autoantibodies to be measured with relatively high sensitivity and is the most preferred method.
【0016】第二抗体法では、固相固定化CEAと検体
をインキュベーションし、洗浄によるBF分離後、トレ
ーサー標識抗ヒトイムノグロブリン抗体を反応させる。
このとき用いる抗ヒトイムノグロブリン抗体は種々動物
由来のポリクローナル抗体かまたはマウスその他動物の
モノクローナル抗体を使用する。反応時間やトレーサー
の測定法はサンドイッチ法と同様である。In the second antibody method, a sample is incubated with CEA immobilized on a solid phase, and after BF separation by washing, a tracer-labeled anti-human immunoglobulin antibody is reacted. The anti-human immunoglobulin antibodies used in this case are polyclonal antibodies derived from various animals or monoclonal antibodies from mice and other animals. The reaction time and tracer measurement method are the same as the sandwich method.
【0017】競合法では固相固定化CEAと検体及びト
レーサー標識抗CEA抗体を同時にまたは時間をずらし
てインキュベーションし、反応させる。トレーサー標識
抗体は検体中の自己抗体と競合的に固定化CEAと反応
し、その固相に結合する量は検体中の自己抗体量に反比
例する。トレーサー標識に使用する抗体は動物由来のポ
リクローナル抗体が好ましい。In the competitive method, CEA immobilized on a solid phase, a specimen, and a tracer-labeled anti-CEA antibody are incubated at the same time or at different times to react. The tracer-labeled antibody reacts with the immobilized CEA competitively with the autoantibody in the sample, and the amount bound to the solid phase is inversely proportional to the amount of autoantibody in the sample. The antibody used for tracer labeling is preferably an animal-derived polyclonal antibody.
【0018】沈澱法においては、検体とトレーサー標識
CEAをインキュベーションし、自己抗体−トレーサー
標識CEA複合体を生成せしめ、その後ポリエチレング
リコール(PEG)その他既知の沈澱剤を反応系に加え
、生成した複合体を沈澱させる。上清と沈澱物を遠心分
離し、沈澱物中または上清中のトレーサーを測定する。
沈澱物中のトレーサー量は検体中の自己抗体に比例する
。In the precipitation method, a sample and tracer-labeled CEA are incubated to generate an autoantibody-tracer-labeled CEA complex, and then polyethylene glycol (PEG) or other known precipitating agent is added to the reaction system to separate the resulting complex. precipitate. The supernatant and precipitate are centrifuged, and the tracer in the precipitate or supernatant is measured. The amount of tracer in the precipitate is proportional to the autoantibody in the sample.
【0019】本発明に係る診断剤は、イムノアッセイ用
キット調製の常法にしたがい、適宜容易に各薬剤を分注
したり、プレートや試験管を用意したり、バッファー液
や酵素基質液を更に用意したり、用時調製用の場合は凍
結乾燥した所定薬剤に希釈ないし溶解用の水(バッファ
ー、生理的食塩水等)を更に用意し、更に対照血清(血
漿)等を用意して、所望するキットにしておくと実用上
特に有効である。The diagnostic agent according to the present invention can be easily prepared by dispensing each drug appropriately, preparing plates and test tubes, and further preparing a buffer solution and an enzyme substrate solution according to the conventional method for preparing an immunoassay kit. Or, in the case of preparation at the time of use, additionally prepare water (buffer, physiological saline, etc.) for diluting or dissolving the lyophilized prescribed drug, and further prepare control serum (plasma), etc., and prepare as desired. It is particularly useful in practice if you prepare it as a kit.
【0020】例えばサンドイッチ法による診断剤キット
の内容としては、CEA固定化ポリスチレンボールを収
容した試験管、リン酸バッファー、血清希釈剤、トレー
サー標識CEA溶液、酵素結合体、酵素基質等が挙げら
れ、これらを適宜包装してキットとして販売すればよい
。For example, the contents of a diagnostic kit using the sandwich method include a test tube containing CEA-immobilized polystyrene balls, a phosphate buffer, a serum diluent, a tracer-labeled CEA solution, an enzyme conjugate, an enzyme substrate, etc. These may be appropriately packaged and sold as a kit.
【0021】[0021]
【実施例】以下に、本発明に係る診断剤を用いて癌の診
断を目的とした血清、血漿その他体外にとり出した各種
検体中の抗CEA自己抗体の測定についての実施例及び
参考例について述べるが、これらは、本発明を更に詳細
に説明することを目的とするものであって、本発明はこ
れらの実施例のみに限定されるものではない。[Example] Examples and reference examples of measuring anti-CEA autoantibodies in serum, plasma, and other various specimens taken outside the body for the purpose of cancer diagnosis using the diagnostic agent according to the present invention will be described below. However, these examples are intended to explain the present invention in more detail, and the present invention is not limited to these examples.
【0022】[0022]
【実施例1 サンドイッチ法による抗CEA自己抗体
測定】検体25μl、リン酸緩衝液(pH7.3、0.
1M NaCl、0.5%ウシ血清アルブミン含有)
200μl、CEA固定化ポリスチレンボール(PSB
)1個を試験管中にて、37℃、2hrインキュベーシ
ョンした。PSBを3mlの蒸留水で3回洗浄した後、
ビオチン標識CEA溶液200μlを加え37℃、1時
間反応させた。蒸留水で3回洗浄後、アビジン結合ペル
オキシダーゼ200μlを加え更に37℃、1hr反応
させ、前回と同様に洗浄した。2,2’−アジノービス
−3−エチルベンゾチアゾリン−6−スルホン酸(AB
TS)と過酸化水素からなる発色液500μlを加え、
室温で1hr酵素反応を行なった後、1%蓚酸を加え反
応を停止させた。420nmに於ける吸光度を測定し、
検体中の抗CEA自己抗体を測定した。結果を表1に示
す。[Example 1 Anti-CEA autoantibody measurement by sandwich method] 25 μl of sample, phosphate buffer (pH 7.3, 0.5 μl)
(contains 1M NaCl, 0.5% bovine serum albumin)
200 μl, CEA-immobilized polystyrene balls (PSB
) was incubated in a test tube at 37°C for 2 hours. After washing the PSB three times with 3 ml of distilled water,
200 μl of biotin-labeled CEA solution was added and reacted at 37° C. for 1 hour. After washing three times with distilled water, 200 μl of avidin-conjugated peroxidase was added, the reaction was further carried out at 37° C. for 1 hr, and the cells were washed in the same manner as before. 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (AB
Add 500 μl of a coloring solution consisting of TS) and hydrogen peroxide,
After carrying out the enzymatic reaction for 1 hour at room temperature, 1% oxalic acid was added to stop the reaction. Measure the absorbance at 420 nm,
Anti-CEA autoantibodies in the specimens were measured. The results are shown in Table 1.
【0023】[0023]
【表1】[Table 1]
【0024】[0024]
【実施例2 第二抗体法による抗CEA自己抗体測定
】検体25μl及びCEA固定化PSB1個を37℃、
2hr反応させ、3mlの蒸留水を用いて洗浄を3回行
なった。ペルオキシダーゼ標識ウサギ抗ヒトイムノグロ
ブリン抗体液200μlを加え、37℃、1hr反応さ
せた後、同様に洗浄した。実施例1と同様に酵素反応を
行い、吸光度を測定して、検体中の自己抗体を測定した
。
結果を表1に示す。[Example 2 Anti-CEA autoantibody measurement by second antibody method] 25 μl of the sample and 1 piece of CEA-immobilized PSB were heated at 37°C.
The reaction was carried out for 2 hours, and washing was performed three times using 3 ml of distilled water. 200 μl of peroxidase-labeled rabbit anti-human immunoglobulin antibody solution was added, and the mixture was reacted at 37° C. for 1 hour, and then washed in the same manner. An enzymatic reaction was performed in the same manner as in Example 1, and the absorbance was measured to determine the autoantibody in the sample. The results are shown in Table 1.
【0025】[0025]
【実施例3 競合法による抗CEA自己抗体測定法】
適切に希釈した検体100μl、ペルオキシダーゼ標識
ウサギ抗CEA抗体300μl及びCEA固定化PSB
1個を37℃、2hrインキュベーションし、洗浄後実
施例1と同様の操作で検体中の抗CEA自己抗体を測定
した。結果を表1に示す。[Example 3 Anti-CEA autoantibody measurement method using competitive method]
100 μl of appropriately diluted specimen, 300 μl of peroxidase-labeled rabbit anti-CEA antibody, and CEA-immobilized PSB
One sample was incubated at 37°C for 2 hours, and after washing, the anti-CEA autoantibody in the sample was measured in the same manner as in Example 1. The results are shown in Table 1.
【0026】[0026]
【実施例4 沈澱法による抗CEA自己抗体測定】検
体50μl、125I標識CEA溶液50μl及び0.
01M酢酸アンモニウム(pH6.8)150μlを4
℃、一夜インキュベーションした。25%ポリエチレン
グリコール250μl加え、3000rpm、20分遠
心分離し、総放射活性に対する沈澱の放射能活性比(%
)を測定した。結果を表1に示す。[Example 4 Anti-CEA autoantibody measurement by precipitation method] 50 μl of specimen, 50 μl of 125I-labeled CEA solution, and 0.5 μl of 125I-labeled CEA solution.
Add 150 μl of 01M ammonium acetate (pH 6.8) to 4
℃, overnight incubation. Add 250 μl of 25% polyethylene glycol, centrifuge at 3000 rpm for 20 minutes, and calculate the radioactivity ratio of the precipitate to the total radioactivity (%
) was measured. The results are shown in Table 1.
【0027】[0027]
【参考例1 サンドイッチEIA法によるCEAの測
定】検体50μl、ペルオキシダーゼ標識ウサギ抗CE
A抗体液300μl及び抗CEAモノクローナル抗体固
定化PSB1個を37℃、2hrインキュベーションし
、蒸留水2mlを用いて3回洗浄した。実施例1と同様
に発色液を加え酵素反応を行い、吸光度を測定した。
標準CEAによる検量線から検体のCEA濃度を読みと
った。結果を表1に示す。[Reference Example 1 Measurement of CEA by sandwich EIA method] 50 μl of sample, peroxidase-labeled rabbit anti-CE
300 μl of antibody solution A and one PSB immobilized with anti-CEA monoclonal antibody were incubated at 37° C. for 2 hours, and washed three times with 2 ml of distilled water. A coloring solution was added in the same manner as in Example 1, an enzyme reaction was performed, and the absorbance was measured. The CEA concentration of the sample was read from the standard CEA calibration curve. The results are shown in Table 1.
【0028】表1の結果から明らかなように、本発明を
利用する癌診断法によれば従来広く用いられているCE
A濃度が正常値レベルの早期胃癌例を感度良く検出出来
ることが確認された。As is clear from the results shown in Table 1, the cancer diagnostic method using the present invention is effective against the conventionally widely used CE.
It was confirmed that early gastric cancer cases with normal A concentration can be detected with high sensitivity.
【0029】[0029]
【発明の効果】従来の腫瘍マーカーは、かなりステージ
の進んだ癌患者血中でしか検出できず、早期癌の発見に
は無力であった。しかし本発明によれば、血中のCEA
に対する自己抗体を測定することによって比較的早期の
癌の存在を発見することが可能となった。従来の生理学
的検査や血清学的検査で困難であった早期癌の発見が本
発明によってなされることの意義は極めて大きい。[Effects of the Invention] Conventional tumor markers can only be detected in the blood of cancer patients at a fairly advanced stage, and are powerless in detecting early cancer. However, according to the present invention, CEA in blood
By measuring autoantibodies against cancer, it has become possible to detect the presence of cancer at a relatively early stage. It is extremely significant that the present invention enables the detection of early cancer, which is difficult to detect using conventional physiological tests and serological tests.
Claims (7)
抗体を測定する薬剤からなることを特徴とする癌診断剤
。1. A cancer diagnostic agent comprising a drug for measuring autoantibodies against carcinoembryonic antigen (CEA).
標識CEAからなり、サンドイッチ法にしたがって抗C
EA自己抗体を測定するものであることを特徴とする請
求項1の癌診断剤。2. The drug consists of immobilized CEA and tracer-labeled CEA, and anti-CEA is prepared according to the sandwich method.
2. The cancer diagnostic agent according to claim 1, which measures EA autoantibodies.
標識抗CEA抗体からなり、競合法にしたがって抗CE
A自己抗体を測定するものであることを特徴とする請求
項1の癌診断剤。3. The drug consists of immobilized CEA and a tracer-labeled anti-CEA antibody, and the anti-CEA
2. The cancer diagnostic agent according to claim 1, which measures A autoantibody.
ン結合CEAとアビジン結合トレーサーを用いてなるこ
とを特徴とする請求項2の癌診断剤。4. The cancer diagnostic agent according to claim 2, which uses biotin-bound CEA and an avidin-bound tracer instead of tracer-labeled CEA.
標識抗ヒトイムノグロブリン抗体からなり、第二抗体法
にしたがって抗CEA自己抗体を測定するものであるこ
とを特徴とする請求項1の癌診断剤。5. The cancer diagnostic agent according to claim 1, wherein the agent comprises immobilized CEA and a tracer-labeled anti-human immunoglobulin antibody, and is for measuring anti-CEA autoantibodies according to a second antibody method. .
澱剤からなり、沈澱法にしたがって抗CEA自己抗体を
測定するものであることを特徴とする請求項1の癌診断
剤。6. The cancer diagnostic agent according to claim 1, wherein the agent comprises tracer-labeled CEA and a precipitant, and is used to measure anti-CEA autoantibodies according to a precipitation method.
光物質、発光物質から選択されるものであること、を特
徴とする請求項1〜5のいずれか1項に記載の癌診断剤
。7. The cancer diagnostic agent according to claim 1, wherein the tracer is selected from enzymes, isotopes, fluorescent substances, and luminescent substances.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP41854490A JP2931111B2 (en) | 1990-12-28 | 1990-12-28 | Cancer diagnostics |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP41854490A JP2931111B2 (en) | 1990-12-28 | 1990-12-28 | Cancer diagnostics |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH04249770A true JPH04249770A (en) | 1992-09-04 |
JP2931111B2 JP2931111B2 (en) | 1999-08-09 |
Family
ID=18526372
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP41854490A Expired - Lifetime JP2931111B2 (en) | 1990-12-28 | 1990-12-28 | Cancer diagnostics |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP2931111B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103472229A (en) * | 2013-09-17 | 2013-12-25 | 武汉生之源生物科技有限公司 | Magnetic particle chemiluminescence immune assay detection kit for carcino-embryonic antigen and detection method of detection kit |
-
1990
- 1990-12-28 JP JP41854490A patent/JP2931111B2/en not_active Expired - Lifetime
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103472229A (en) * | 2013-09-17 | 2013-12-25 | 武汉生之源生物科技有限公司 | Magnetic particle chemiluminescence immune assay detection kit for carcino-embryonic antigen and detection method of detection kit |
Also Published As
Publication number | Publication date |
---|---|
JP2931111B2 (en) | 1999-08-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4469787A (en) | Immunoassay involving soluble complex of second antibody and labeled binding protein | |
Yolken | Enzyme-linked immunosorbent assay (ELISA): a practical tool for rapid diagnosis of viruses and other infectious agents. | |
KR920005963B1 (en) | Method for the determination of a specific binding substance | |
US4201763A (en) | Solid phase immunofluorescent assay method | |
KR920000056B1 (en) | Process for the determination of a specifically bindable substance | |
WO2006024239A1 (en) | A method an a kit for detecting multiple tumor specimens s multaneously and indicating interference | |
JP2636331B2 (en) | One-step assay for antigen-specific antibodies and suitable reagents | |
KR920000057B1 (en) | Process and reagent for the determination of a specifically bindable substance | |
WO1985000663A1 (en) | Immunometric assay using polyclonal and monoclonal antibodies and a kit for use therein | |
JPS60259964A (en) | Assay of immunologically active substance in analysis sample | |
CN104034892A (en) | Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof | |
EP0124366B1 (en) | Method of measuring biological ligands | |
CN103969447A (en) | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof | |
JPH09504094A (en) | Method for assaying immunological substances using magnetic latex particles and non-magnetic particles | |
JPH03229153A (en) | Detection of existence of specific anti- body or antigen useful for diagnosis of rheumatism and test kit used therefor | |
CN106645756A (en) | Kit for detecting NMP22 (Nuclear Matrix Protein 22) and preparation method thereof | |
CA1124642A (en) | Antibody adsorbed support method for carcinoembryonic antigen assay | |
JPH02228561A (en) | Method and reagent for measuring substance detectable immunologically | |
JPH02124462A (en) | Improved immunity measuring method | |
JPH0421819B2 (en) | ||
USRE32696E (en) | Enzymatic immunological method for determination of antigens and antibodies | |
CN108872594A (en) | A kind of alpha-fetoprotein detection kit and preparation method thereof | |
JPH04249770A (en) | Cancer diagnostic agent | |
US5436132A (en) | Quantitative determination of tenascin as glioma marker | |
JPH03503566A (en) | Immunoassay using monoclonal antibodies against natural binding proteins |