JPH03273163A - Analysis of cardiac glycoside by immunoassay - Google Patents
Analysis of cardiac glycoside by immunoassayInfo
- Publication number
- JPH03273163A JPH03273163A JP7181190A JP7181190A JPH03273163A JP H03273163 A JPH03273163 A JP H03273163A JP 7181190 A JP7181190 A JP 7181190A JP 7181190 A JP7181190 A JP 7181190A JP H03273163 A JPH03273163 A JP H03273163A
- Authority
- JP
- Japan
- Prior art keywords
- cardiac glycosides
- sample
- immunoassay
- cardiac
- chromatography
- 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
- 229940097217 cardiac glycoside Drugs 0.000 title claims abstract description 42
- 239000002368 cardiac glycoside Substances 0.000 title claims abstract description 42
- 229930002534 steroid glycoside Natural products 0.000 title claims abstract description 42
- 238000003018 immunoassay Methods 0.000 title claims abstract description 19
- 238000004458 analytical method Methods 0.000 title claims description 10
- LPMXVESGRSUGHW-HBYQJFLCSA-N ouabain Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1C[C@@]2(O)CC[C@H]3[C@@]4(O)CC[C@H](C=5COC(=O)C=5)[C@@]4(C)C[C@@H](O)[C@@H]3[C@@]2(CO)[C@H](O)C1 LPMXVESGRSUGHW-HBYQJFLCSA-N 0.000 title claims description 8
- 150000008143 steroidal glycosides Chemical class 0.000 claims abstract description 34
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 11
- 238000004816 paper chromatography Methods 0.000 claims abstract description 7
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- 239000012528 membrane Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 claims description 4
- 229920001220 nitrocellulos Polymers 0.000 abstract description 18
- 239000003431 cross linking reagent Substances 0.000 abstract description 3
- 230000000747 cardiac effect Effects 0.000 abstract 1
- 230000037029 cross reaction Effects 0.000 abstract 1
- 150000003839 salts Chemical class 0.000 abstract 1
- 239000000020 Nitrocellulose Substances 0.000 description 14
- 239000000523 sample Substances 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- WDJUZGPOPHTGOT-OAXVISGBSA-N Digitoxin Natural products O([C@H]1[C@@H](C)O[C@@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@@](C)([C@H](C6=CC(=O)OC6)CC5)CC4)CC3)CC2)C[C@H]1O)[C@H]1O[C@@H](C)[C@H](O[C@H]2O[C@@H](C)[C@@H](O)[C@@H](O)C2)[C@@H](O)C1 WDJUZGPOPHTGOT-OAXVISGBSA-N 0.000 description 7
- 229940098773 bovine serum albumin Drugs 0.000 description 7
- 229960000648 digitoxin Drugs 0.000 description 7
- WDJUZGPOPHTGOT-XUDUSOBPSA-N digitoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)CC5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O WDJUZGPOPHTGOT-XUDUSOBPSA-N 0.000 description 7
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 5
- 229960005156 digoxin Drugs 0.000 description 5
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 5
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000037230 mobility Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- CTNPHHZPAJYPFO-PDXBGNJTSA-N (3s,5s,8r,9s,10s,13r,14s,17r)-3-[(2r,4s,5r,6r)-5-[(2r,3s,4s,5r,6s)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-4-methoxy-6-methyloxan-2-yl]oxy-5,14-dihydroxy-13-methyl-17-(5-oxo-2h-furan Chemical compound C1([C@@H]2[C@@]3(C)CC[C@@H]4[C@@]5(C=O)CC[C@@H](C[C@@]5(O)CC[C@H]4[C@@]3(O)CC2)O[C@H]2C[C@@H]([C@@H]([C@@H](C)O2)O[C@@H]2[C@H]([C@H](O)[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)[C@H](CO)O2)O)OC)=CC(=O)OC1 CTNPHHZPAJYPFO-PDXBGNJTSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- OCEDEAQHBIGPTE-UHFFFAOYSA-N Gitoxin Natural products CC1OC(CC(O)C1O)OC2C(O)CC(OC3C(O)CC(OC4CCC5(C)C(CCC6C5CCC7(C)C(C(O)CC67O)C8=CCOC8=O)C4)OC3C)OC2C OCEDEAQHBIGPTE-UHFFFAOYSA-N 0.000 description 2
- FHIREUBIEIPPMC-UHFFFAOYSA-N K-Strophanthin-beta Natural products O1C(C)C(OC2C(C(O)C(O)C(CO)O2)O)C(OC)CC1OC(CC1(O)CCC2C3(O)CC4)CCC1(C=O)C2CCC3(C)C4C1=CC(=O)OC1 FHIREUBIEIPPMC-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- LKRDZKPBAOKJBT-CNPIRKNPSA-N gitoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(C[C@H](O)[C@@H]([C@@]6(C)CC5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LKRDZKPBAOKJBT-CNPIRKNPSA-N 0.000 description 2
- 229950000974 gitoxin Drugs 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- TZYVRXZQAWPIAB-FCLHUMLKSA-N 5-amino-3-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4h-[1,3]thiazolo[4,5-d]pyrimidine-2,7-dione Chemical compound O=C1SC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O TZYVRXZQAWPIAB-FCLHUMLKSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 239000000538 analytical sample Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003177 cardiotonic effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 229940082663 other cardiac glycosides in atc Drugs 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は強心配糖体を免疫測定法によって分析する方法
に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for analyzing cardiac glycosides by immunoassay.
高感度に分析できる特徴を持つ免疫測定法は医薬、臨床
検査の分野では重要な技術である。強心配糖体の従来か
らのイムノアッセイによる検出法としては、ＲＩＡ法や
ＥＬ Ｉ ＳＡ法が一般的に用いられてきた。なお、従
来公知のＲＩＡ法に関して、Ｔｈｅ Ｊｏｕｒｎａｌ
ｏｆ Ｃ１１ｎｉｃａｌ Ｉｎｖｅｓｔｉｇａｔｉｏｎ
（ｖｏｌ ４７．１９６８． ｐ１０３５−１０４２．
Ｇｅｏｒｇｅ Ｃ，０１ｉｖｅｒｅｔ ａｌ、）やＰ
ｈｙＬｏｃｈｅｍｉｓｔｒｙ（ｖｏｌ １５．１９７６
、 ｐ１５３７１５４５ Ｅｌｍａｒ Ｗ、 Ｗｅｔ
ｌｅｒ）に記載されており、また、ＥＬＩＳＡ法に関し
て、１９８９年の日本薬学会で池田らが発表している（
要旨： ５Ｊ、 １Ｏ−３）。このような強心配糖体の
イムノアッセイによる分析技術は通常の機器分析よりも
高感度であり、さらに精製しなくても分析が可能である
ことから一般的に利用されている。しかし、このような
免疫測定法は、類似化合物とも交差反応し、物質の同定
が必要な場合は、多量のサンプルから分画や濃縮を行な
った後、薄層クロマトグラフィーや液体クロマトグラフ
ィーなどによって識別する必要があった。このため、各
々類似する強心配糖体間の識別には煩雑な操作と時間を
要していた。Immunoassay methods, which have the characteristics of highly sensitive analysis, are important technologies in the fields of medicine and clinical testing. As conventional immunoassay detection methods for cardiac glycosides, the RIA method and the ELISA method have been generally used. Regarding the conventionally known RIA method, The Journal
of C11nical Investigation
(vol 47.1968. p1035-1042.
George C, 01ivere et al.) and P
hyLochemistry (vol 15.1976
, p15371545 Elmar W, Wet
ler), and Ikeda et al. presented the ELISA method at the Pharmaceutical Society of Japan in 1989 (
Abstract: 5J, 1O-3). Such analytical techniques for cardiac glycosides by immunoassay are more sensitive than ordinary instrumental analysis, and are commonly used because they enable analysis without further purification. However, such immunoassays also cross-react with similar compounds, and when it is necessary to identify a substance, it is necessary to fractionate and concentrate a large amount of sample and then identify it by thin layer chromatography or liquid chromatography. I needed to. Therefore, distinguishing between similar cardiac glycosides requires complicated operations and time.
そこで本発明の課題は、分析試料中の強心配糖体を単に
検出するのみでなく、互いにＲ似する強心配糖体が混在
していたとしてもその各々を正確かつ簡便に識別、同定
できる強心配糖体の新たな分析方法を提供しようとする
ものである。Therefore, the object of the present invention is not only to simply detect cardiac glycosides in an analytical sample, but also to be able to accurately and easily identify and identify each cardiac glycoside even if there are a mixture of cardiac glycosides that resemble each other. The aim is to provide a new method for analyzing glycosides of concern.
［課題を解決するための手段］
上記課題を解決するため本発明者等は、まず強心配糖体
やその類似化合物を含む試料を薄層クロマトグラフィー
やペーパークロマトグラフィーで展開した後、イムノア
ッセイにより検出できれば、強心配糖体やその類似化合
物の識別や同定が容易に行なえると考え、研究を行なっ
た。その結果、薄層クロマトグラフィーやペーパークロ
マトグラフィー上で直接、イムノアッセイを行なうこと
は困難であったが、これらクロマトグラフィー上の展開
パターンを一旦支持膜上に転写した後、該支持膜上でイ
ムノアッセイを行なうことにより、強心配糖体を検出す
ることができるとともに、その検出されたスポットの移
動度から強心配糖体を識別、同定できることを見い出し
本発明を完成するに至ったものである。[Means for Solving the Problems] In order to solve the above problems, the present inventors first developed a sample containing cardiac glycosides and their similar compounds by thin layer chromatography or paper chromatography, and then detected them by immunoassay. We conducted this research with the idea that if possible, cardiac glycosides and their similar compounds could be easily identified and identified. As a result, it has been difficult to perform immunoassays directly on thin-layer chromatography or paper chromatography; however, after transferring the development pattern on these chromatography onto a support membrane, immunoassays can be performed on the support membrane. The present invention was completed by discovering that by carrying out this method, cardiac glycosides can be detected, and also cardiac glycosides can be identified and identified from the mobility of the detected spots.
以下、本発明を更に詳述する。The present invention will be explained in more detail below.
本発明の分析対象となる強心配糖体とは、心筋に直接作
用して、強心作用を呈する成分で一般的構造はステロイ
ド核を母体とした化合物に様々な糖が配糖化した化合物
であり、代表的な化合物として、ジギトキシン、ジゴキ
シン、ギトキシン、ストロファンチンなどが例示できる
。これらの化合物はいずれもステロイド骨格を持った配
糖体であるため、これらの化合物を抗原とした抗体を作
製しても、ジギトキシン、ジゴキシン、ギトキシン、ス
トロファンチンなどを特異的に識別する抗体を作製する
ことは困難であり、−船釣に他の強心配糖体と交差反応
することが知られている。そのため、従来用いられてい
るＥＬＩＳＡ法やＲＩＡ法では強心配糖体の総量を推定
出来ても、含まれる強心配糖体の成分を同定するのは困
難であった。Cardiac glycosides, which are the subject of analysis in the present invention, are components that act directly on the cardiac muscle and exhibit cardiotonic effects, and their general structure is a compound in which various sugars are glycosylated to a compound with a steroid nucleus as its base. Typical compounds include digitoxin, digoxin, gitoxin, and strophanthin. All of these compounds are glycosides with a steroid skeleton, so even if antibodies are made using these compounds as antigens, it will not be possible to develop antibodies that specifically identify digitoxin, digoxin, gitoxin, strophanthin, etc. It is difficult to make and - is known to cross-react with other cardiac glycosides. Therefore, even if the total amount of cardiac glycosides can be estimated using conventionally used ELISA and RIA methods, it is difficult to identify the components of cardiac glycosides contained.
しかしながら、本発明の分析法によれば、強心配糖体含
有試料を薄層クロマトグラフィーやペーパークロマトグ
ラフィーで展開した後、該展開パターンとニトロセルロ
ース膜等の支持膜に転写し、イムノアッセイを行なうこ
とにより強心配糖体を検出すのみでなく、検出されるス
ポットの移動度によってｘ強心配糖体を識別、同定する
ことができる。However, according to the analytical method of the present invention, after a cardiac glycoside-containing sample is developed by thin layer chromatography or paper chromatography, the developed pattern and the developed pattern are transferred to a support membrane such as a nitrocellulose membrane, and an immunoassay is performed. Not only can cardiac glycosides be detected by this method, but also cardiac glycosides can be identified and identified based on the mobility of the detected spot.
すなわち、本発明においては、まず、薄層クロマトグラ
フィーやペーパークロマトグラフィーによって、強心配
糖体含有試料を展開した後、好ましくは、タンパク質と
過ヨウ素酸等の架橋剤を含む生理食塩水等の溶液に浸し
たニトロセルロース膜を密着させることによって、展開
パターンをニトロセルロース膜に転写させ、固定化させ
る。That is, in the present invention, first, a cardiac glycoside-containing sample is developed by thin layer chromatography or paper chromatography, and then a solution such as physiological saline containing protein and a crosslinking agent such as periodic acid is preferably added. By bringing a nitrocellulose membrane soaked in the nitrocellulose membrane into close contact with the nitrocellulose membrane, the developed pattern is transferred and fixed onto the nitrocellulose membrane.
使用するタンパク質としては、牛血清アルブミン、イム
ノグロブリン、サイログロブリン、台底ポリリジン、ア
ルブミン等を挙げることができ、上記試料中の強心配糖
体等の低分子化合物は、架橋剤によりこれらタンパク質
と結合し、間接的にニトロセルロースで膜上に吸着され
、これにより；上記強心配糖体等の低分子化合物を支持
膜上に直接吸着させるよりも安定して固定化することが
できる。Examples of proteins used include bovine serum albumin, immunoglobulin, thyroglobulin, basal polylysine, albumin, etc., and low molecular weight compounds such as cardiac glycosides in the above sample are bonded to these proteins by a cross-linking agent. , is indirectly adsorbed onto the membrane with nitrocellulose, thereby making it possible to immobilize low molecular weight compounds such as the cardiac glycosides more stably than when directly adsorbed onto the support membrane.
なお、強心配糖体等をタンパク質と過ヨウ素酸により、
支持膜へ固定化する基本的な方法は本発明者により開発
され、すでに出願されている （特ＩＰＪＩ昭（３３−
１０７５１０号〉。そしてこの後、二ｉ・ロセルロース
膜上でイムノアッセイを行なう。この際、試料中に互い
に類似する強心配糖体が混在し、使用抗体がこれら化合
物と交差反応して両者のスポットが検出されたとしても
これらスポットは前記クロマトグラフィーによる移動度
に差異が生じており、これがニトロセルロース膜にその
まま転写されていることから、これら強心配糖体を各々
識別できるとともに強心配糖体の同定も行なうことがで
きる。In addition, cardiac glycosides, etc. are treated with protein and periodic acid.
The basic method of immobilization on a supporting membrane was developed by the present inventor and has already been applied for (Special IPJI Akira (33-
No. 107510>. After this, an immunoassay is performed on a dicellulose membrane. At this time, even if cardiac glycosides that are similar to each other are mixed in the sample and the antibodies used cross-react with these compounds and both spots are detected, these spots will have different mobilities due to the chromatography. Since this is directly transferred to the nitrocellulose membrane, each of these cardiac glycosides can be identified, and the cardiac glycoside can also be identified.
したがって、強心配糖体の成分の分析を極めて簡便、正
確に行なうことが可能である。Therefore, it is possible to analyze components of cardiac glycosides extremely easily and accurately.
イムノアッセイの手法については通常の手法を用いれば
よく、例えばＰ、Ｔｉｊｓｅｎ著、石川栄治監訳の「エ
ンザイムイムノアッセイ」（東京化学同人）や渡辺慶−
１中根−穂編集の「改訂版 酵素抗体法」 （学際企画
）に記載されているイムノアッセイ等を用いることがで
きる。For immunoassay methods, conventional methods may be used, such as "Enzyme Immunoassay" by P. Tijsen, supervised translation by Eiji Ishikawa (Tokyo Kagaku Doujin), and by Kei Watanabe.
Immunoassays and the like described in ``Revised Edition of Enzyme-Antibody Methods'' (Interdisciplinary Project) edited by Ho Nakane 1 can be used.
［実施例］ 以下、本発明方法を更に具体的に説明する。[Example] The method of the present invention will be explained in more detail below.
実施例１
強心配糖体であるジギトキシンとジゴキシンの検出を行
なった。以下工程順に説明する。Example 1 Cardiac glycosides digitoxin and digoxin were detected. The steps will be explained below in order.
（１） 抗体の作製
Ｔ、Ｗ、Ｓｍ１ｔｈ らの方法（Ｊ、ｒ’ｈａｒｍａｃ
ｏ１． ＥＸ９． Ｔｈｅｒ、。(1) Preparation of antibodies The method of T, W, Sm1th et al. (J, r'harmac
o1. EX9. Ther.
ｖｏｌ １７５． ｐ３５２（１９７０））に従って牛
血清アルブミンを結合させたジギトキシンをウサギに対
して２週間ごと４回免疫し、最終免疫の２週間後、全採
血を行った。得られたウサギ血清を生理食塩水で５００
倍に希釈した後、希釈した血清と等量の煮沸固化させた
５％牛血清アルブミンを加えホモゲナイズし、２日間４
°Ｃで放置した。vol 175. Rabbits were immunized four times every two weeks with digitoxin conjugated to bovine serum albumin according to p.352 (1970)), and whole blood was collected two weeks after the final immunization. The obtained rabbit serum was diluted with physiological saline for 500 min.
After diluting the serum, 5% bovine serum albumin that had been solidified by boiling was added in an equal amount to the diluted serum, and the mixture was homogenized for 2 days.
It was left at °C.
その後、不溶化させた牛血清アルブミンと牛血清アルブ
ミンに対する抗体を除去するために遠心した。遠心した
−に清をジギトキシン抗体として、イムノアッセイに用
いた。Thereafter, the mixture was centrifuged to remove insolubilized bovine serum albumin and antibodies against bovine serum albumin. The centrifuged supernatant was used as a digitoxin antibody for immunoassay.
（２） 薄層クロマトグラフィー
薄層クロマトグラフィーのプレートとしてＫｉｅｓｅ１
ｇｅ１６０Ｆ２５４Ａｒｔ５７１５を用い、クロロホル
ム／ピリジン（６： １）の溶媒で展開した。なお、こ
の時の分析サンプルは、エタノールに溶解させたジギ１
−キシンとジゴキシンをそれぞれ１０ｕｇとした。(2) Thin layer chromatographyKiese1 as a plate for thin layer chromatography
Using gel160F254Art5715, it was developed with a solvent of chloroform/pyridine (6:1). The analysis sample at this time was Jigi 1 dissolved in ethanol.
-Xin and digoxin were each 10 ug.
（３）薄層クロマトグラフィープレートから支持膜への
転写
サンプルを展開した薄層クロマトグラフィーのプレート
を３０分間室温で乾燥させた後、１％の牛血清アルブミ
ンと２■／ｍβのメタ過ヨウ素酸ナトリウムを含む生理
食塩水で濡らしたニトロセルロース膜を薄層クロマトグ
ラフィーのプレートに密着させ、室温で放置した。３０
分後、ニトロセルロース膜を薄層クロマトグラフィーの
プレートから剥がし、３０分間室温で乾燥させ、サンプ
ルの強心配糖体をニトロセルロース膜に固定化させた。(3) Transfer from thin layer chromatography plate to supporting membrane After drying the thin layer chromatography plate on which the sample was developed at room temperature for 30 minutes, 1% bovine serum albumin and 2 μ/mβ metaperiodic acid were added. A nitrocellulose membrane wetted with physiological saline containing sodium was brought into close contact with a thin layer chromatography plate and left at room temperature. 30
After minutes, the nitrocellulose membrane was peeled off from the thin layer chromatography plate and dried for 30 minutes at room temperature to immobilize the sample cardiac glycosides on the nitrocellulose membrane.
（４）支持膜の抗体処理
「エンザイムイムノアッセイ」に記載されている方法に
主に従った。強心配糖体を固定化させたニトロセルロー
ス膜を０．１％のＴｗｅｅｎ２０を含む生理食塩水で洗
浄した後、１％牛血清アルブミンを含む生理食塩水に４
°Ｃで一晩浸した。その後、ニトロセルロース膜をジギ
トキシン抗体に浸し、室温で２時間反応させ、再び洗浄
し、未反応の抗体を除いた。(4) Antibody treatment of support membrane The method described in "Enzyme Immunoassay" was mainly followed. After washing the nitrocellulose membrane on which cardiac glycosides were immobilized with physiological saline containing 0.1% Tween 20, it was washed with physiological saline containing 1% bovine serum albumin for 4 hours.
Soaked overnight at °C. Thereafter, the nitrocellulose membrane was immersed in digitoxin antibody, reacted at room temperature for 2 hours, and washed again to remove unreacted antibody.
次にウサギ抗体に反応するパーオキシダーゼを標識した
２次抗体（和光型、１０００倍希釈）にニトロセルロー
ス膜を浸し、室温で２時間反応させた後、再び洗浄し、
未反応の２次抗体を除去した。Next, the nitrocellulose membrane was immersed in a secondary antibody labeled with peroxidase that reacts with rabbit antibodies (Wako type, 1000-fold diluted), allowed to react at room temperature for 2 hours, and then washed again.
Unreacted secondary antibody was removed.
（５）支持膜上での強心配糖体の発色反応コニカ製イム
ノスナインキットを用いて、発色させた。発色させた結
果を第１図に示す。(5) Color development reaction of cardiac glycoside on support membrane Color was developed using Konica's ImmunoSine kit. The results of color development are shown in FIG.
本発明によれば、以上述べた様に分析試料中に互いに極
めて類似する強心配糖体が混在していたとしても、その
各々の強心配糖体を正確かつ簡便に識別、同定すること
ができ、極めてｈ用かつｆ１効な強心配糖体の分析手法
を提供することができた。According to the present invention, as described above, even if cardiac glycosides that are extremely similar to each other are present in an analysis sample, each cardiac glycoside can be identified and identified accurately and easily. We were able to provide a method for analyzing cardiac glycosides that is extremely effective for h and f1.
第１図はニトロセルロース膜−ににおいて発色したジギ
トキシンとジゴキシンに対応するスポットの位置を示す
図である。FIG. 1 is a diagram showing the positions of spots corresponding to digitoxin and digoxin that developed colors on a nitrocellulose membrane.
Claims (1)
はペーパークロマトグラフィーで展開した後、支持膜に
これらクロマトグラフィー上の展開パターンを転写し、
その後、支持膜上でイムノアッセイを行ない、検出され
るスポットの移動度により強心配糖体の分析を行なう分
析方法。 ２、強心配糖体を支持膜に固定するために支持膜をタン
パク質および、過ヨウ素酸を含む溶液で処理することを
特徴とする請求項１の強心配糖体の分析方法。[Claims] 1. After developing a sample containing a cardiac glycoside by thin layer chromatography or paper chromatography, transferring the developed pattern on the chromatography to a support membrane,
This is an analytical method in which cardiac glycosides are then analyzed by immunoassay on a support membrane and the mobility of the detected spots. 2. The method for analyzing cardiac glycosides according to claim 1, wherein the support membrane is treated with a solution containing protein and periodic acid in order to fix the cardiac glycosides on the support membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2071811A JPH0743364B2 (en) | 1990-03-23 | 1990-03-23 | Analysis method for cardiac glycosides by immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2071811A JPH0743364B2 (en) | 1990-03-23 | 1990-03-23 | Analysis method for cardiac glycosides by immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03273163A true JPH03273163A (en) | 1991-12-04 |
| JPH0743364B2 JPH0743364B2 (en) | 1995-05-15 |
Family
ID=13471326
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2071811A Expired - Lifetime JPH0743364B2 (en) | 1990-03-23 | 1990-03-23 | Analysis method for cardiac glycosides by immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0743364B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2013005642A1 (en) * | 2011-07-01 | 2015-02-23 | 株式会社ダイセル | Spot detection set, spot detection method, and transferred sheet |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100942988B1 (en) * | 2008-02-19 | 2010-02-17 | 주식회사 에이치브이엘에스 | Analysis method for glycosides using reversed-phase high-performance liquid chromatography and pulsed amperometric detection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59166866A (en) * | 1983-02-24 | 1984-09-20 | アボット・ラボラトリーズ | Analysis method |
| JPS61142463A (en) * | 1984-11-15 | 1986-06-30 | シンテツクス（ユ−・エス・エイ）インコ−ポレイテツド | Device for chromatography |
| JPS62187254A (en) * | 1986-02-14 | 1987-08-15 | Shinotesuto Kenkyusho:Kk | Analysis of ganglioside |
-
1990
- 1990-03-23 JP JP2071811A patent/JPH0743364B2/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59166866A (en) * | 1983-02-24 | 1984-09-20 | アボット・ラボラトリーズ | Analysis method |
| JPS61142463A (en) * | 1984-11-15 | 1986-06-30 | シンテツクス（ユ−・エス・エイ）インコ−ポレイテツド | Device for chromatography |
| JPS62187254A (en) * | 1986-02-14 | 1987-08-15 | Shinotesuto Kenkyusho:Kk | Analysis of ganglioside |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2013005642A1 (en) * | 2011-07-01 | 2015-02-23 | 株式会社ダイセル | Spot detection set, spot detection method, and transferred sheet |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0743364B2 (en) | 1995-05-15 |
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