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https://doi.org/10.29013/ESR-19-9.10-16-20
Eshboev Farkhod Bakir o'g'li, junior researcher of the Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan E-mail: farkhod.eshboev@gmail.com Yusupova Elvira Gaynatovna, Ph D., Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan Piyakina Galina Aliksandrovna, Ph D., Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan Sasmakov Sobirdjan Anarmatovich, Ph D., Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan
E-mail: sasmakov@web.de Azimova Shakhnoz Sadikovna, Professor, Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan
OBTAINING OF MORPHINE-BSA CONJUGATE FOR USING IN ELISA AS AN ANTIGEN FOR SORBSION
Abstract. Morphine-hemisuccinate / BSA conjugate was synthesized and specification of this conjugate was confirmed with ELISA method. Seventy blood samples of drug addicts were analyzed by ELISA method using the synthesized morphine-BSA conjugate as sorption antigens. According to the results of the ELISA, anti-opioid antibodies were determined in the 88.5% samples of tested opioids addicts.
Keywords: morphine, BSA, conjugate, ELISA, Drug addicts, HPLC.
Introduction diagnosis of drug addiction are immunochemical
Drug addiction is one of the important health and HPLC-MS methods for the determination of problems in the world. This pathology results in the narcotic substances. These include: immunochro-death of about 500000 individuals annually around matographic, immunofluorescence, radioimmunoas-the globe. Opioids continue to cause the most harm, say methods of analysis [3; 4]. The listed diagnostic accounting for 76 per cent of deaths where drug us- methods of drug addiction are based on the determi-ing disorders were implicated [1; 2]. The current nation ofthe metabolic products ofthe used narcotic situation requires the creation of new methods of substances in the human biological fluid. However, analysis of identification of drug addictions. It is at the 1st stage of drug addiction, when people rarely known that the most widespread methods for the take drugs (1-2 times in a month), these methods
cannot determine metabolites of drugs in biological fluids of drug users, because the narcotic substances saved very short period (24-48 h.) in the body. This is unacceptable for the early detection of drug users with a period of more than 2 days. Therefore, enzyme-linked immunosorbent assays (ELISA) based on the determination of specific antibodies to drugs were used in the early diagnosis of drug addiction, because, antibodies circulate and retain in the bloodstream for a long time (2-3 months). Hapten-protein conjugates are considered the main components of the ELISA test kits of the determination of anti-hap-ten antibodies. There were works used from bovine serum albumin, ovalbumin, lysozyme, human serum albumin, tetanus toxoid protein and fibrinogen as a carrier protein to obtain protein-hapten conjugates for developing vaccines to drug addicts [5; 6].
The aim of this work, obtaining of morphine-bovine serum albumin (BSA) conjugate for using ELISA test in order to detecting the antibodies to opiates in human blood serum.
Materials and methods
Materials: The following reagents were used for synthesis of morphine-BSA conjugates: morphine HCl, succinic anhydride (Sigma-Aldrich, USA), pyridine, ethyl alcohol, benzene, N-Ethyl-N- ( 3-dimethylaminopropyl) -carb o dimide hydrochloride (AppliChem, Germany), BSA fraction V (Himedia, India.), dimethylformamide.
The following materials were used for performing ELISA test: 96 well ELISA plate (Nunc maxiSorp, ThermoFisher Scientific), Anti-Human IgM, IgG (^-chain cpecific)-Peroxidase antibody produced in goat (Sigma, USA), 3,3',5,5'-Tetramethylbenzidine (TMB), H2O2, tween 20 and sulfuric acid.
Methods: Synthesis of morphine hemisuccinate. Obtaining of morphine-hemisuccinate was performed as follows: 0.2 gr morphine and 0.4 gr suc-cinic anhydride was added to reaction solutions (7 ml benzene and 3 ml pyridine). Reaction mixture was warmed up to 80 °C during 4 hours, after heating the reaction mixture was cooled slowly at
room temperature then benzene and pyridine were evaporated by vacuum pump. The reaction product was washed 5 times with 99% ethanol for recrystal-lization of morphine-hemisuccinate. The obtained morphine-hemisuccinate was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods.
Identification of morphine-hemisuccinate by TLC. The obtained morphine hemisuccinate was identified by TLC on silica gel (5/40 with chloroform, methanol and ammonia (40 : 10 : 0.5ml) as a solvent system and tested by the regent of Dragindorf. Morphine was used as a standard substance.
HPLC analysis of morphine-hemisuccinate. The synthesized morphine hemisuccinate was identified by reverse phase high performance liquid chromatography (HPLC). The HPLC system consisted of Agilent 1100 series equipment and Zorbax-Eclipse XDB-C18 reversed-phase column (3.0 x 150 mm, particle size 3.5 ^m). The UV detector wavelength was set at 226 nm. HPLC was performed under the following conditions: Mobile phase: acetonitrile-wa-ter, 1 min: 5:95 (v/v); 10 min: 95:5 (v/v); 15 min: 5:95 (v/v). Fluid flow rate - 0.5 ml /min. Injection volume: 20 ^l. Concentration of samples: 1 mg/ml.
Synthesis of morphine-hemisuccinate/BSA conjugate. Morphine/protein conjugates are considered important components of ELISA kits of drug addicts. Synthesis of morphine-hemisuccinate/BSA conjugate was performed on the following condition: a solution of 25 mg of BSA in 2.5 ml of distilled water was mixed with 1 ml of dimethylformamide containing 7.5 mg of morphine-hemisuccinate and 5 mg of water-soluble carbodimide in 1.5 ml of distilled water. The reaction mixture was incubated for 5 hours at 4 °C. The resulting conjugate was dialyzed in 0.02 M carbonate buffer pH 9.5.
ELISA analysis of drug addictions. In this study examined the blood of 70 opioid addicts and 10 blood samples of healthy people. These blood samples were delivered from republican center of nar-cology of Uzbekistan. The analysis was performed
as follows: the morphine-BSA conjugate was absorbed at 30 ^g to holes of ELISA well plates within 48 hours at 4 °C, then free pores of plate was blocked over night with 2% BSA solution and the ELISA plate was washed twice with a washing buffer (1-PBS pH 7.4; 0.05% Tween-20). The blood serum of people was diluted (1:100) with washing buffer, added to holes of ELISA plate into 100 ^l and incubated at 37 °C for 1 hour. After that, it was washed 5 times with a washing buffer and second conjugates (Anti-Human IgM, IgG (^-chain cpecific)-Peroxidase antibody produced in goat) were added to 100 ^l to ELISA holes and incubated 37 °C for 30 minutes. ELISA plate was washed again 5 times with a washing buffer then 3,3',5,5'-tetra-methylbenzidine (TMB) in 0.05 M phosphate-citrate buffer and H2O2 into 100 ^l were added as
the peroxidase substrate. The reaction was stopped
approximately 10 minutes later by the addition of
2 M sulphuric acid to reaction mixture by 50 ^l and result of ELISA was spectrophotometrically read at 450 nm and expressed as optical density.
Results and discussion
Morphine cannot bound to surface of ELISA well plate, therefore, it was connected with carrier protein (BSA) for adsorbing to well plate. First, morphine-hemisuccinate was obtained to bound with amino group of amino acids. The authors have developed numerous methods for obtaining morphine-protein conjugates, such as the carrier proteins were bounded to second carbon atom of morphine and nitrogen atom of morphine [7]. In the present study, morphine-hemisuccinate was synthesized by the method of succinic anhydride (figure 1).
A B
Figure 1. A. morphine B. morphine-hemisuccinate
Figure 2. Result of TLC analysis. 1. Obtained morphine-hemisuccinate. 2. Morphine
It was found that the melting temperature of the obtained morphine hemisuccinate was 237-239 °C [8]. TLC analysis of morphine hemisuccinate was per-
formed and morphine was used as standard substance. According to the TLC results, morphine hemisuc-cinate remained at the starting point, but morphine moved and its Rf point was been 0.4 (figure 2).
According to the picture, the product of reaction did not contain free morphine.
HPLC is considered an effective analytical method and it is often used for drug analysis and standardization of substances. In this work, morphine-hemis-uccinate was also standardized using HPLC method. According to HPLC results, morphine-hemisucci-nate formed a peak at 5.99 min in the selected HPLC conditions. It indicates that the reaction was finished and product was individual (figure 3).
Figure 3. HPLC analysis of obtained morphine-hemisuccinate
Conjugate was synthesized by creating a cova-lent bond between the carboxyl group of morphine-
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Authors used different proteins as a carrier protein for the synthesis of morphine-protein conjugates. In this study, BSA was chosen as a carrier protein for a number of reasons such as price, and easy dissolving in ordinary solvents.
Obtained morphine-BSA conjugate was used in the ELISA analysis of blood samples of drug addictions and healthy people. According to results of ELISA, optic density of all blood samples of healthy people did not exceed than 0.3. As the result, 0.3 optic densities were chosen as a cutoff point of ELISA analysis. The optical density of 62 samples was higher than 0.3 (between 0.4 and 1.2 optic densities), but the optical intensity of the other 8 drugs
Q min
hemisuccinate and the free amino groups of amino acids of BSA (figure 4).
was below than 0.3. It is appropriate 88.5% specification of ELISA analysis and it is considered that the obtained morphine conjugate appropriates to use on the ELISA analysis of opioid addictions.
In conclusion, the synthesized morphine-hemisuccinate and morphine-BSA conjugate were standardized using the methods described above and confirmed by ELISA analysis. The specificity of the ELISA analysis performed was sufficiently high (88.5%). The obtained morphine-BSA conjugate appropriate to use as the main component (sorption antigen) of ELISA test kits to firstly identification (common monitoring) of opioid addictions.
A B
Figure 4. Reaction of conjugation. A. morphine-6-hemisuccinate. B. obtained morphine/BSA conjugate
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