EA015275B1 - 28-norbrassinolide horseradish peroxidase conjugate as labeled antigen in enzyme immunoassay of 7-oxa-6-oxobrassinosteroids - Google Patents

Abstract

The invention relates to immune-enzyme analysis of brassinosteroid phytohormones, namely to a conjugate of 28-norbrassinolide with horseradish peroxidase which may be used as labeled antigen. The task of the present invention is 28-norbrassinolide-horseradish peroxidase conjugate which may be used as labeled antigen in enzyme immunoassay of 7-oxa-6-oxobrassinosteroids - brassinolide, 24-epibrassinolide, 28-homobrassinolide, 28-norbrassinolide et al. The problem is solved by a conjugate of 28-norbrassinolid with bovine-serum albumin which was prepared by reaction of N-oxysuccinimide ester of 22R,23R)-2α,3α,22,23-tetrahydroxy-26-(hemisuccinat)-B-homo-7-oxa-5α-cholestan-6-one (hapten) with horseradish peroxidase in solution of dioxane-bicarbonate buffer (pH 8.5) followed by chromatographic purification of product on Sephadex and freezing in glycerin.

Description

The invention relates to the field of immunochemical analysis of phytohormones of brassinosteroids, namely, a conjugate of 28-norbrassinolide with horseradish peroxidase of the formula 1

where HRP is horseradish peroxidase, which can be used as a labeled antigen in an analytical test system for enzyme-linked immunosorbent assay for the determination of 7-oxa-6-oxobrassinosteroids - brassinolide, 24-epibrassinolide, 28 homocastasterone, 28-norbrassinolide, etc.

Brassinosteroids (BS) are natural low molecular weight bioregulators that are plant hormones [1, 2]. They are present in all plant objects and have growth modulating and adaptogenic effects. The content of brassinosteroids in plants is very low - less than 10 ^-5 %. In very small concentrations, they also exhibit their biological effect. It has been established, for example, that when processing various crops in doses of 5-20 mg per hectare of crops, a noticeable growth-stimulating and adaptogenic effect is observed, leading to an increase in yield, an increase in the quality of products and plant resistance to adverse conditions and diseases [3-8]. These properties of brassinosteroids make them attractive as a basis for agricultural products. One of the most famous is the epin drug, the active ingredient of which is the phytohormone 24-epibrassinolide. The most active are the brassinosteroids, which have 7-oxa-6-oxo grouping in their structure (the so-called brassinosteroid lactones) [1]. The low BS content in natural objects and the vanishingly low doses that cause their effect make it necessary to develop methods for the analysis of brassinosteroids, in particular brassinosteroid lactones.

All of the above puts on the agenda the development of methods for the analysis of brassinosteroids, which should have high sensitivity, selectivity, ease of execution and be accessible. The recent development of methods based on the use of antibodies, in particular enzyme immunoassay, requires both an immunogen and an enzyme immunoassay, which can be a substrate labeled with horseradish peroxidase.

Currently, a conjugate of 24-epicastasterone with horseradish peroxidase 2 is known, which was obtained and used in immunoanalytic systems to determine only 24 Kmethylbrassinosteroids (24-epicastasterone and 24-epibrassinolide, 24-epi-6-deoxocastasterone) [9]. Its use to determine other BS is problematic due to low sensitivity.

The aim of the invention is a previously unknown conjugate of 28-norbrassinolide and horseradish peroxidase, which can be used as a labeled antigen in an enzyme-linked immunosorbent assay for 7-oxa-6-oxobrassinosteroids (brassinosteroid lactones).

This goal is achieved by the claimed conjugate 1, which is obtained by the interaction of Ν oxysuccinimide ester (22K, 23K) -2a, 3a, 22,23-tetrahydroxy-26- (hemisuccinate) -B-homo-7-oxa-5-cholestan-6-one (hapten) with horseradish peroxidase in a dioxane medium in the presence of potassium hydrogen carbonate (pH 8.5) and purification of the resulting product on Sephadex, followed by freezing of the conjugate solution in glycerol.

The invention is confirmed by examples of specific performance.

Example 1

30 mg (52 mmol) (22K, 23K) -2a, 3a, 22,23-tetrahydroxy-26- (hemisuccinate) -B-homo-7ox-5a-cholestan-6-one (1) are dissolved in 6 ml of abs. dioxane, add 8 mg of dry Ν-oxisuccinimide (70 mmol) and dropwise at 1 = 8-10 ° С 13 mg (63 mmol) of dicyclohexyl carbodiimide in 4 ml of abs. dioxane. The reaction mixture was stirred for 30 minutes at 5 ° C and 20 hours at room temperature. The precipitated dicyclohexylcarbourea is filtered off and the filtrate is evaporated. The residue was dissolved in ethyl acetate, washed with water, dried with anhydrous α ₂ 8 ₄ , and evaporated. The resulting ether is used without up to

- 1 015275 additional cleaning.

To a solution of 6 mg horseradish peroxidase in 600 ml dist. water in the presence of 2-3 drops of a 0.1 M solution of sodium bicarbonate (pH 8.35) add a solution of 3 mg (4.4 mmol) of Ν-succinimide ether, mix for 30 min at 10 ° C and at room temperature for 1.5 h. Purify on a column with Sephadex, eluent - dist. water. Collect the colored fraction (tan), then dilute with glycerol (1: 2) and store in the freezer (at -18 ° C).

The synthesized conjugate is used as a labeled antigen in the immunoassay system for the quantitative determination of 7-oxa-6-oxobrassinosteroids, which is confirmed by an example of a specific implementation.

Example 2

For analysis, 50 μl of calibration samples or test solutions of brassinosteroids and 100 μl of a working solution of horseradish 28-norbrassinolide peroxidase conjugate were added to the wells of an immobilized antibody plate. The plates were incubated for 2 hours at 37 ° C without shaking, then the liquid was removed from the wells and washed with a 1% solution of No. C1 containing 0.02% Teteya 20. To conduct a color reaction, 150 μl of solution 3 was immediately added to the wells , 3 ', 5.5' tetramethylbenzidine in substrate buffer and the plate was incubated for 15 min at 37 ° C without access of light. The reaction was stopped by adding 50 μl of a stop reagent (5% solution of Н ₂ 80 ₄ ) to each well and, not later than 30 min, the optical density was measured on an ELISA reader at a wavelength of 450 nm.

The analysis results were calculated by interpolation using a calibration curve [9].

The concentration of the native antigen at which substitution of half of the labeled antigen in the antibody-antigen complex is achieved was 0.3 nmol / L or 7 pcg in 50 μl of the sample.

The inventive conjugate of 28-norbrassinolide with horseradish peroxidase is obtained in high yield, is stable, provides the necessary immunoreactivity of the label, and allows the quantification of 7-oxa-6-oxobrassinosteroids (brassinolide, 24-epibrassinolide, 28 homobrassinolide, 28-imnorbodromide irbrazenoferbide). analysis.

Literature

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2. Kyprasy ν.Α., ΖΙιαόίηδΚίί ν.Ν., be Sgoo! Aye. All in all - A Νονν С1а§8 о £ Р1аи1 Ногтопек. 8ai P1edo: Asab. Rhekk. 1999, 456 p.

3. Patent of the Republic of Belarus No. 2806 of 11.23.1995. Means for reducing the accumulation of radionuclides by plants and the method of its application./ Khripach V.A., Zhabinsky V.N., Litvinovskaya R.P., Zavadskaya M.I., Deeva V.P., Vedeneev A.N.//Of . Bull. Belgospat., 2000, No. 2 (26). S. 159.

4. Patent of the Republic of Belarus No. 3488 dated July 4, 1996. A way to increase the nutritional value of potatoes. / Rattler

B. A., Zhabinsky V.N., Litvinovskaya R.P., Zavadskaya M.I., Savelyeva E.A., Karas I.I. (Belarus), Vakulenko V.V. (Russia) .// Of. Bull. Belgospat., 2000, No. 3 (27). S. 74.

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C. N. // Of. Bull. Belgospat., 2000, No. 2 (25). S. 75.

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7. Patent RB 5212. A way to increase the yield of flax and improve the quality of flax fiber. / Voskresenskaya L.G., Khripach V.A., Zhabinsky V.N., Zavadskaya M.I., Litvinovskaya R.P. // Of. Bull. NCIS RB, 2003, No. 2 (37). S. 93.

8. Patent of the Republic of Belarus No. 5698. The method of propagation of healthy seed material of potatoes. / Bobrik A.O., Khripach V.A., Zhabinsky V.N., Zavadskaya M.I., Litvinovskaya R.P. // Of. Bull. NCIS RB, 2003 No. 4 (39).

9. Patent of the Republic of Belarus No. 9771 (C2 C071 41/00). Conjugate of 24-epicastasterone with horseradish peroxidase as a labeled antigen for enzyme-linked immunosorbent assay for 24K.-methylbrassinsteroids./ Khripach V.A., Sviridov O.V., Litvinovskaya R.P., Pryadko A.G., Drach S.V., Matveentsev V.D., Novik T.V.//Of. Bull. invented. NCIS, 2007, No. 5, p. 94.

10. Khripach V.A., Sviridov O.V., Pryadko A.G., Litvinovskaya R.P., Drach S.V., Matveentsev V.D., Novik T.V., Mikhailopulo K.I., Zhabinsky V.N., Zavadskaya M.I., Averkova M.A., Drachenova O.A., Chashchina N.M. Enzyme-linked immunosorbent assay (24V) -brassinosteroids.// Bioorg. chemistry. - 2007 - vol. 33, No. 3. S. 371378.

11. Egorov A.M. Theory and practice of enzyme immunoassay. M .: Higher. school., 1991.

Claims (1)

CLAIM Conjugate 28-norbrassinolide with horseradish peroxidase of the formula - 2,015,275 where HRP is horseradish peroxidase, as labeled oxabrassynosteroids. antigen for immunoassay 7-oxa-6-