JPH0743364B2 - Analysis method for cardiac glycosides by immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for analyzing cardiac glycosides by immunoassay.

[Prior Art] The immunoassay method, which is characterized by high-sensitivity analysis, is an important technology in the fields of medicine and clinical examination. Conventional immunoassay detection methods for cardiac glycosides include RIA and ELI.
The SA method has been commonly used. In addition, the conventionally known RIA
Regarding the law, The Journal of Clinical Investigation
(Vol 47,1968, p1035-1042, George C. Oliver et al.)
And Phytochemistry (vol 15,1976, p1537-1545, Elmar W.
Weiler), and regarding the ELISA method, 1
Ikeda et al. Presented at the Japan Pharmaceutical Society in 989 (Abstract: 5J, 1
0-3). Such an analysis technique using an immunoassay for a cardiac glycoside is generally used because it has higher sensitivity than ordinary instrumental analysis and can be analyzed without further purification. However, such an immunoassay also cross-reacts with similar compounds, and when identification of a substance is required, fractionation or concentration from a large amount of sample is performed, followed by identification by thin layer chromatography or liquid chromatography. Had to do. Therefore, a complicated operation and time are required to distinguish between similar cardiac glycosides.

[Problems to be Solved by the Invention]

Therefore, an object of the present invention is not only to detect cardiac glycosides in an analysis sample, but even if cardiac glycosides similar to each other are mixed, the cardiac glycosides can be accurately and easily identified and identified. It aims to provide a new method for analyzing glycosides.

[Means for Solving the Problems]

In order to solve the above-mentioned problems, the present inventors first develop a sample containing a cardiac glycoside or a similar compound thereof by thin layer chromatography or paper chromatography, and if it can be detected by an immunoassay, the cardiac glycoside or its analogues can be detected. Research was carried out on the assumption that compounds could be easily identified and identified. As a result, it was difficult to carry out the immunoassay directly on thin layer chromatography or paper chromatography, but once the development pattern on these chromatographies was transferred onto a support membrane, the immunoassay was performed on the support membrane. The present inventors have completed the present invention by discovering that cardiac glycosides can be detected and that cardiac glycosides can be identified and identified from the detected mobility of spots.

Hereinafter, the present invention will be described in more detail.

The cardiac glycoside to be analyzed in the present invention is a compound that directly acts on the myocardium and exhibits a cardiotonic action, and a general structure is a compound in which various sugars are glycosylated in a compound having a steroid nucleus as a mother, Representative compounds include digitoxin, digoxin, gitoxin, strophanthin and the like. Since all of these compounds are glycosides having a steroid skeleton, even if an antibody is prepared using these compounds as an antigen, digitoxin, digoxin, gitoxin,
It is difficult to produce an antibody that specifically identifies strophanthin and the like, and it is generally known to cross-react with other cardiac glycosides. Therefore, even if the total amount of cardiac glycosides can be estimated by the conventionally used ELISA method or RIA method, it was difficult to identify the components of the cardiac glycoside contained.

However, according to the analysis method of the present invention, after the cardiac glycoside-containing sample is developed by thin layer chromatography or paper chromatography, it is transferred to the development pattern and a supporting membrane such as a nitrocellulose membrane to perform an immunoassay. Thus, not only the cardiac glycoside can be detected, but also the cardiac glycoside can be identified and identified by the mobility of the detected spot.

That is, in the present invention, first, after developing a cardiac glycoside-containing sample by thin layer chromatography or paper chromatography, preferably, a solution such as physiological saline containing a protein and a cross-linking agent such as periodate. The developed pattern is transferred to the nitrocellulose membrane and fixed by adhering the nitrocellulose membrane soaked in the.

Examples of the protein to be used include bovine serum albumin, immunoglobulin, thyroglobulin, synthetic polylysine, albumin, etc., and low molecular weight compounds such as cardiac glycosides in the above sample are bound to these proteins by a crosslinking agent, It is indirectly adsorbed on the membrane by nitrocellulose, which enables: the above-mentioned low molecular weight compounds such as cardiac glycosides to be immobilized more stably than directly adsorbed on the supporting membrane.

In addition, cardiac glycosides, etc.
The basic method of immobilizing on a support membrane has been developed by the present inventor and has already been filed (Japanese Patent Application No. 63-107510).
After this, an immunoassay is performed on the nitrocellulose membrane. At this time, even if cardiac glycosides similar to each other are mixed in the sample and the antibody used cross-reacts with these compounds and spots of both are detected, these spots have different mobilities by the chromatography. Cage,
Since this is directly transferred to the nitrocellulose membrane, it is possible to identify each of these cardiac glycosides and also identify the cardiac glycoside.

Therefore, it is possible to analyze the components of the cardiac glycoside extremely simply and accurately.

The immunoassay may be performed by a conventional method, for example, P. Tijsen's "Enzyme Immunoassay" translated by Eiji Ishikawa (Tokyo Kagaku Dojin) or Keiichi Watanabe, "Revised Enzyme Antibody Method" edited by Kazuho Nakane (interdisciplinary project). The immunoassay etc. described in ()) can be used.

〔Example〕

 Hereinafter, the method of the present invention will be described more specifically.

Example 1 The cardiac glycosides digitoxin and digoxin were detected. The steps will be described below in order.

(1) Preparation of antibody Method by TWS Smith (J.Pharmacol.Exp.Ther., Vol 175, p
352 (1970)), and immunized rabbits with digitoxin conjugated with bovine serum albumin every two weeks four times,
Two weeks after the final immunization, whole blood was collected. The obtained rabbit serum was diluted 500 times with physiological saline, and then 5% bovine serum albumin boiled and solidified in an amount equal to that of the diluted serum was added and homogenized, and the mixture was allowed to stand at 4 ° C. for 2 days.

Then, centrifugation was performed to remove insolubilized bovine serum albumin and antibodies against bovine serum albumin. The centrifuged supernatant was used in the immunoassay as a digitoxin antibody.

(2) Thin layer chromatography Kieselge 160F as a plate for thin layer chromatography
254Art5715 was used and developed with a solvent of chloroform / pyridine (6: 1). The analytical samples used at this time were 10 μg each of digitoxin and digoxin dissolved in ethanol.

(3) Transfer from Thin Layer Chromatography Plate to Support Membrane The thin layer chromatography plate on which the sample was developed was dried at room temperature for 30 minutes, and then 1% bovine serum albumin and 2 mg / ml sodium metaperiodate were added. A nitrocellulose membrane moistened with a physiological saline containing was adhered to a thin layer chromatography plate and left at room temperature. After 30 minutes, the nitrocellulose membrane was removed from the thin layer chromatography plate and dried for 30 minutes at room temperature to immobilize the cardiac glycosides of the sample on the nitrocellulose membrane.

(4) Antibody treatment of supporting membrane The method described in "Enzyme Immunoassay" was mainly followed. After washing the nitrocellulose membrane with immobilized cardiac glycosides in physiological saline containing 0.1% Tween 20,
It was immersed in a physiological saline solution containing 1% bovine serum albumin at 4 ° C. overnight. Then, the nitrocellulose membrane was dipped in the digitoxin antibody, reacted at room temperature for 2 hours, and washed again to remove unreacted antibody.

Next, the nitrocellulose membrane is immersed in a peroxidase-labeled secondary antibody that reacts with rabbit antibody (manufactured by Wako, diluted 1000 times), reacted at room temperature for 2 hours, and then washed again to remove unreacted secondary antibody. Removed.

(5) Coloring Reaction of Cardiac Glycoside on Supporting Film Color was developed using Konica Immunostain Kit.
The result of color development is shown in FIG.

〔The invention's effect〕

According to the present invention, as described above, even if cardiac glycosides that are extremely similar to each other are mixed in the analysis sample, each cardiac glycoside can be accurately and easily identified and identified. Thus, it has been possible to provide an extremely useful and effective method for analyzing cardiac glycosides.

[Brief description of drawings]

FIG. 1 is a diagram showing the positions of spots corresponding to digitoxin and digoxin colored on the nitrocellulose membrane.

Claims (2)

[Claims] 1. After developing a sample containing a cardiac glycoside by thin layer chromatography or paper chromatography,
Transfer these chromatographic development patterns to the support membrane, then perform an immunoassay on the support membrane,
An analytical method for analyzing cardiac glycosides based on the mobility of detected spots. 2. The method for analyzing cardiac glycosides according to claim 1, wherein the supporting membrane is treated with a solution containing protein and periodate to fix the cardiac glycoside to the supporting membrane.