METHODS FOR SANITARY SURVEILLANCE OF LIVESTOCK PRODUCTION. VII. ENZYMOIMMUNOASSAY OF STREPTOMYCIN

Abstract

Summary The polyclonal rabbit antibodies against streptomycin conjugated with bovine serum albumin in reaction with diglycidyl ether of 1,4-butanediol presents high specificity to streptomycin and its similar structural analog — dihydrostreptomycin. In the con- ditions of indirect competitive enzymoimmunoassay with immobilized antigens, heterologous to immunogene on protein carrier and methods of synthesis, the limit of streptomycin detection is 0.1 ng/ml. The authors consider the use of developed immunoenzyme test- systems for control of antibiotic contamination in milk and eggs with the limit of detection of 10 mkg/kg and in meat with the limit of 20 mkg/kg. Streptomycin (SM) is aminoglycoside antibiotic widely used for many years in our country and abroad for the treatment of acute infectious diseases of animals, despite its dangerous side effects such as allergic reactions, disorders of neuromuscular conduction, and ototoxicity (1 2). In modern practice, the control of SM residues in animal products is often based on enzyme-linked immunosorbent assay (ELISA). This method was implemented in several countries in the form of test systems of various types including biosensor technology (3-12). Russian researchers also have proposed a test system based on direct ELISA and the evaluation test using immunochromatography for the control of SM in milk and dairy products (13, 14). The purpose of this work was obtaining specific immunoreagents based on streptomycin, optimization of conditions of the indirect competitive solid-phase immunoassay, and assessing the applicability of this test system for the control of SM residues in milk, eggs and meat. Technique. The experiment was performed using the following chemicals: streptomycin sulfate, diglycidyl ether of 1,4- butanediol, adipic acid dihydrazide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), human transferrin (HT) (Sigma, USA ), sodium borohydride (Serva, Germany), dihydro-streptomycin (G.F. Gauze Research Institute of New Antibiotics, Moscow), dimethylformamide (Fluka, Germany), bovine serum albumin (BSA), gelatin (Gel) and antispecies enzyme conjugate prepared according to the description (15) from horseradish peroxidase (EC 1.11.1.7) and donkey antiserum to rabbit immunoglobulin, sodium periodate of domestic production. ELISA was conducted on high-binding polystyrene plates (Costar, USA) using a photometer Dynatech MR 5000 (Dynatech, Germany). The reaction with diglycidyl ether of 1,4-butanediol for the synthesis of conjugates of SM with BSA and Gel - BSA- SM(100)e, Gel-SM(10)e, Gel-SM(30)e, and Gel-SM(100)e: 6,8 mg SM sulfate (10 umol) in 1 ml 1% NaHCO3 was added with 14 ul (10 umol) diglycidyl ether of 1,4-butanediol (10% solution in dimethylformamide) and stirred at room temperature for 24 hours. Then, 4 mg BSA (0,06 umol) in 0,5 ml of 0,05 M carbonate-bicarbonate buffer (CBB, pH 9,5) was added with 600 ul of the abovementioned mixture (100-fold molar excess of hapten), and three portions of 4 mg Gel (0,025 umol) in 0,5 ml CBB - with, respectively, 25; 75, and 250 ul (10-, 30-, and 100-fold molar excesses), then stirred for 3 hours and dialyzed . The reaction with adipic acid dihydrazide for the synthesis of SM conjugates with BSA and Gel- BSA-SM(100)h, Gel- SM(10)h, Gel-SM(30)h, and Gel-SM(100)h: firstly, 4 mg BSA (0,06 umol) and 12 mg Gel (0,075 umol) (resp., in 0,5 and 1,5 ml water) were added with EDCI (resp., 15 and 30 mg), stirred for 30 minutes, and then the mixture with Gel was divided to 3 equal portions. Then, an aqueous solution of 6,8 mg SM sulfate (10 umol) was added with 1,8 mg of adipic acid dihydrazide and stirred at room temperature overnight. After that, the portion with BSA was added with 600 ul of the mixture containing the reaction product of SM and adipic acid dihydrazide (100-fold molar excess), while the portions with Gel - with, respectively, 25; 75, and 250 ul (10-, 30-, and 100-fold molar excesses), then stirred for 2 hours at room temperature and dialyzed.