SU792869A1 - Process for producing conjugated antigenes barbituric acid-immunogenic carrier - Google Patents

Description

The new chloroformate is reacted with an immunogenic carrier in a molar ratio of 30-100: 1. Serum albumin, and, unfortunately bovine serum albumin, is used as the immunogenic carrier. The reaction is carried out in a water-dioxane solution at -10 ° C and a pH of 8-9 for 24 hours, while the pH of the solution is maintained by the addition of a 10% solution of sodium hydroxide. The resulting conjugated antigen is separated from low molecular weight impurities by dialysis against distilled water and freeze-dried. The amount of barbituric acid residues covalently bound to the protein carrier molecule is determined spectrophotometrically by the difference in the content of free amino groups in the original protein and the conjugated antigen obtained, using trinitrobenzenesulfonic acid as a reagent for the amino groups. Depending on the molar ratio, the chloroformate and protein introduced into the reaction produce conjugating antigens containing various amounts of barbituric acid residues covalently linked to the protein molecule. So, if the molar ratio of barbituric acid chloroformate to protein is 30-100: 1, then conjugated barbiturate acid-immunogenic antigens are obtained, containing 3-17 barbituric acid residues covalently bound to 1 mole of protein, respectively (see Table 1). In a known method, a conjugated antigen is obtained, containing 2-3 barbituric acid residues bound to a carrier molecule using a molar ratio of barbituric acid derivative to carrier 500-1000: 1. The conversion of the reactive derivative of barbituric acid in the proposed method is 5-16%, while in the known method 0.1-0.3%. Example 1. a) Method for preparing 5- (1-methylbutyl) -5- (2-hydroxypropyl) -barbituric acid. In 20 ml of concentrated sulfuric acid, dissolve and 2 g (0.09 mol) of 5- (1 methylbutyl) -5 - allyl barbituric acid. The solution is kept at room temperature for 20 minutes, poured into ice water. The resulting precipitate is separated, dried and recrystallized from aqueous ethyl alcohol. 1.3 g of 5- (1-methylbutyl) -5- (2-hydroxypropyl) - barbituric acid are obtained. Yield 60%. T. pl. 205 ° C. - Literary data: t. Pl. 205-206 ° C. B) A method for preparing conjugated antigen with 5- (1-methylbutyl) -5-propylbarbituric acid with bovine serum albumin. To a solution of 4 g (0.015 mol) of 5- (1-methylbutyl) -5- (2-hydroxypropyl) -barbituric acid in 60 ml of tetrahydrofuran was added a solution of 3 g (0.030 mol) of phosgene in 10 ml of tetrahydrofuran and 12 ml of pyridine. The reaction mixture is stirred overnight at room temperature. The precipitated pyridine hydrochloride is filtered off, the filtrate is evaporated. The residue is extracted several times with ether. The ethereal solution is washed with water, dried over calcium chloride, evaporated, and 4.6 g of 5- (1-methylbutyl) -5- (2-hydroxypropyl) -barbituric acid chloroform are obtained. Yield 85%. The resulting chloroformate was reacted with bovine serum albumin without further purification. For this, 10 g of protein (0.000145 mol) is dissolved in 150 ml of water, the pH of the solution is adjusted to 8-9 with 10% sodium hydroxide solution. With stirring, a solution (0.0145 mol) of chloroformate in 100 ml of dioxane is added dropwise to the obtained solution at-10 ° C while maintaining the pH at 8-9 by adding 10% sodium hydroxide solution. The reaction mass is stirred for 4 hours and left overnight at O-4 ° C. The resulting conjugated antigen is purified from low molecular weight impurities by dialysis against distilled water for 7 days. The solution is lyophilized. Get 10 g of coyugirovina aktigena. It is determined spectrometrically that 17 moles of the corresponding barbituric acid are covalently bonded with 1 mole of protein in it. Example 2. According to the method described above, 1.6- (2 g) and barbituric acid and. 10 g (0.000145 mol) of protein are obtained from co-conjugated antigen 5- (1.6 g (0.005 mol) of 5- (1-methylbutyl) -5- (2-hydroxypropyl) - barbituric acid. 1-methylbutyl) -5-propyl - barbituric acid with bovine serum albumin containing 2-3 moles of barbituric acid associated with 1 mole of protein. Example 3. According to the above described procedure, conjugated antigen 5- (1-methylbutyl) is obtained from 4 g (0.012 mol) of 5- (1-methylbutyl) 5- (2-chloroformate-propyl) - barbituric acid and 10 | G (0.000145 mol) of protein -5-propyl barbituric acid with bovine serum albumin containing 10 barbituric acid residues covalently bound to 1 mole of protein. ,one

These examples are given in table. one.

Table 1

Example 4. Antigenic properties of semi | Chenna conjugates.

Rats and rabbits are immunized by intramuscular injection of conjugated antigen-barbituric acid-protein,

The barbitur content of which with 1 mol of protein is covalently bound to 3 mol of barbituric acid, in a dose of 25 mg / kg 2 times a week for 2 months. Two weeks after the last immunization with prepar atom, blood is taken from the animals and blood serum is isolated. The immunization of rabbits and rats with conjugated antigens, in which 10 and 17 moles of barbituric acid are covalently linked to a carrier protein molecule, is carried out in a similar way. The antigenicity of all the compounds obtained is determined by the amount of antibodies induced by the conjugates, by passive hemagglutination reaction on tanned erythrocytes by a known method. The obtained antibody titer values are given in Table. 2

table 2

Tntry antibodies

Claims (1)

DETAILED DESCRIPTION OF THE INVENTION A method for producing conjugated antigen of a barbituric acid-immunogenic carrier, characterized in that 5-alkyl-5 - chloroformate alkyl barbaric acid is reacted with an immunogenic carrier at a molecular ratio of 5-alkyl-5-chloroformate barbituric acid: 30-100: 1 at pH 8-9 and temperature O-10 ° C. Source of information taken into account during the examination: 1. US Patent No. 3766162, cl. 260-112, publ. 1973.