RU1832122C - Method of preparing of conjugated barbituric acid derivatives for immunochemical analysis of amytal - Google Patents

Abstract

Usage: as a reagent for immunochemical analysis of barbamil. SUMMARY OF THE INVENTION: The product horse south is 5-zoam and l-5 - (5-a box and pentyl) barbituric acid and bovine serum albumin. Reagent 1: 5- and z o a m and l - 5- (5-a carboxy and pententyl) barbituric acid. Reagent 2: isobutyl chloroformate. Reagent 3: bovine serum albumin. Process conditions: in dioxane at a pH of 8-9 and a temperature of 0-10 ° C. Yo

Description

The invention relates to a method for producing new conjugated derivatives of barbituric acid having the properties of reagents for the immunochemical analysis of barbamil, which may find application in the immunochemical analysis.

An object of the invention is to provide conjugated derivatives of barbituric acid with an immunotenic carrier having a higher sensitivity and allowing a reduction in the percentage of cross-reactions in an immunochemical analysis of barbamil.

This goal is achieved by a process for the preparation of bovine serum albumin (BSA) conjugates with 5-isosmyl-5- (5-carboxypentyl) -barbituric acid pre-modified with isobutyl chloroformate.

The process is carried out as follows: to a solution of 5-isoamyl-5- (5-carboxypentyl) barbituric acid in dioxane. triethylamine was added to bind the hydrochloric acid released during the reaction, and isobutyl chloroformate was added while cooling. The resulting reaction mixture was stirred for 3 minutes at 0-10PC and added to a solution of BSA in 0.01 M carbonate buffer (pH 8-9). in a molar ratio of 50: 1-100: 1, maintained at 0-10 ° C for 24 hours. The resulting product is purified by dialysis in running water for 2 days and then lyophilized,

The amount of barbituric acid residues covalently linked to the BSA molecule is determined spectrophotometrically by the difference in the content of free amino groups in the starting protein and the resulting conjugated antigen, using ka00

with y

go

Yu

Chestses reacting to amino groups of trinitrobenzenesulfonic acid.

. The following examples illustrate the synthesis of conjugated derivatives of bug litchic acid and their use for the analysis of barbzmil.

 r and me R -1. Preparation of bovine serum albumin conjugate with 5-iso-8-myl-5- (5-czrboxypent11l) barbituric acid (at a ratio of 1:50).

To a solution of 31 mg (100 µmol) of 5-iso-amyl-5- (51-sarboxypentyl) barbituric acid in 1 ml of dioxane was added 50 µl of tritzylmins, cooled to 0-10 ° C and 20 µl of isobutyl chloroform lat. The reaction is possible at a temperature of 0-10 ° C for 2-3 minutes. The mixture was then added to a solution of 140 MI (2 µmol) BSA and 5 ml of 0.01 M carbonate buffer (pH B) containing 5 ml of dioxans. The reaction is carried out for 24 hours at 0-10 ° C. The synthesized conjugate is isolated by dialysis in running water for 2 days and then lyophilized. In the obtained product, 14-16 molecules of 5-mzoamyl-5- (5-carboxypentyl) barbituric acid are covalently bound to one BSA molecule,

Example 2. Preparation of a bovine serum albumin conjugate with 5-iso-amyl-5- (5-carbo sipeityl) barbituric acid (at a ratio of 1: 100).

The content of 5-isoamyl-5- (5-chicken6o sipentk

barbituric acid in konyogate, pier.

14-16

 18-20

The resulting koyyugiroian antigen induces highly specific antibodies to bzrbamil in animals. Cross reactions with other structurally similar compounds do not exceed 8%.

Example 4. Determination of baroaml using reagents on ss:; this 5-isoamyl-5- (5-czrboxylene) bzrbiturope acid,

In plastic containers placed in a measuring carousel with cuvettes, 50 µl of model urine samples containing various concentrations of barbamil are introduced. 40-fold diluted rabbit anti-carbamyl antiserum, solution j3 of amnofluoresceintioksrbamyl-ethylamide 5-isoamyl-5- (5-carboxylene-tent () barbituric acid at a concentration of 130 nmol / l and phosphate buffer (pH.7.6) containing 1 g of bovine gotamma-globulin and 1 g of sodium ezide are poured into three separate bottles installed in one special box: Box with reagents i.kara

According to the described method, from 31 mg (100 m mol) 5-amyl and -5- (5 - carboxy and pentyl) barbituric acid and 70 mg (T micromol) ) BSA at pH 9 and a temperature of 0-10 ° C receive a conjugated antigen in which 18-20 molecules of 5-isoamyl-5- (5-carboxypentyl) barbituric acid are covalently bound to one BSA molecule. er 3. Antigenic properties

received cat ogatov.

Rabbits are immunized subcutaneously by administration of a conjugated antigen in which 14-16 barbituric acid residues are covalently linked to the BSA molecule. Immunization is carried out with a 50% emulsion of an aqueous solution of konyogate and Freund's complete a / vowant at a dose of 2 mg / kg for 3 weeks with an interval of 7 days. Reimg tuning is carried out after a month,

for 3 days, daily iodine conjugate at a dose of 5 mg / kg intravenously. After a week, blood was collected and serum was isolated. Immunization, rabbit in conjugated antigen, which is associated with the BSA molecule1

18-20 RESIDUAL barbituric acid is carried out similarly. The antigenicity of all the compounds obtained is determined by the amount of antibody induced by the conjugate using direct polarization fluoroimmunoassay. The resulting antiserum tyre values are presented below.

Antiserum titer

about

1: 1200-1: 1800 1: 2400-1: 3200

The farmer is installed in a device (TDX analyzer from Abbott), which automatically performs the analysis according to the specified program. The concentration of barbamil in the urine sample is automatically determined by the magnitude of the fluorescence polarization on a calibration graph built in advance and stored in the memory of the device; barbamil concentration - polarization of fluorescence. Sensitivity

determination of 2.2 x 1 IV barbamil in urine.

Thus, the proposed production method allows to increase the detection sensitivity of barbamil by 2.5 times, and to reduce the% of cross-reactions from 100

up to 8%.

Claims (1)

. The claims A method of producing conjugated derivatives of barbituric acid for immunochemical analysis of bzrbamil zaig, 18321226 5-isoamyl-5- (5-kzrboxypenic acid with a protein compound on an alkali) barbituric acid is used with the action of a barbituric derivative; bovine serum is used as a pH in the medium of an organic soluble compound tel. that as albumin and the process is carried out at a pH of 8-9 and barbituric acid derivative is-5 at a temperature of 0-10 ° C.