OBTAINING MONOCLONAL ANTIBODIES TO EPITOPES OF IVERMECTIN

С.Сейфуллин атындағы Қазақ агротехникалық университетінің Ғылым жаршысы / Вестник науки Казахского агротехнического университета им. С. Сейфуллина. 2013. - 4 (79). C.8-14 OBTAINING MONOCLONAL ANTIBODIES TO EPITOPES OF IVERMECTIN Bulashev A.K., Akibekov O.S. Abstract Ivermectin belongs to the low molecular weight compounds that are not capable of independently eliciting an immune response due to the small size of its molecule. In order to give the hapten s molecule ability to cause antibody formation it must be conjugated with a macromolecular carrier. Balb/c mice were immunized with cоllodial gold and Ivermectin (CGI) conjugate through two schemes with a duration of immunization 16 days and 80 days. Both methods turned to be applicable for inducing antibody formation against Ivermectin. Two stable strains of Hybridomas producing monoclonal antibodies to epitopes of Ivermectin are obtained which can be used in the development of immunoassays for the rapid determination of the anthelmintic in animal products. Keywords: ivermectin, colloidal gold, immunization, hybridomas, monoclonal antibodies Introduction Ivermectin is the active substance of preparations (Ivomek, Baymek, Aversekt, Novomek, Ivertin, Iversect, Ivermek, Narvak, Rustomektin and others) which are widely used for the destruction of nematodes, scabies mites, gadfly larvae and other parasites in farm animals. The preparations of Ivermectin have a low toxicity for an organism and decent environmental properties. However, uncontrolled using them in animal husbandry represents a threat to human health because this group of preparations may be excreted with milk, accumulate in the organs and tissues of animals. In the case of non-compliance with the holding time of livestock after treatment with Ivermectin can enter the human body with products of animal origin and cause various pathologies [1]. At the present time to control of residual amounts of Ivermectin in foods High Performance Liquid Chromatography (HPLC) with fluorescence detector and its various modifications are mainly used [2]. However, drawbacks of the method, such as high cost of equipment, routine sample preparation, duration of analysis limit its widespread use. Recently, as a substitute for the HPLC the options of one of the highly sensitive immunological tests ELISA based on the antibodies specific to Ivermectin are used. It has some significant advantages over HPLC such as specificity, speed and simplicity of setting the test, and relatively low cost of the assay procedure. However, ELISA can be used only in

laboratories equipped with special devices. In this regard, the development of simple, but specific tests designed for rapid detection of Ivermectin s residues in animal products is one of the urgent tasks of Veterinary science. Nowadays Immunochromatographic assay remains the single immunological method which allow to determine for a few minutes the presence or absence of specific substances in biological samples (whole blood, urine, serum or plasma, saliva, feces, etc.) However, the sensitivity and specificity of this test is largely determined by the nature of the antibodies. In this regard, monoclonal antibodies (mabs) that can be used as "reliable tool" in the development of rapid tests are the great theoretical and practical interest. However, the production of mabs against Ivermectin is not easy job. By its chemical composition Ivermectin refers to the class of the macrocyclic lactones and, as a hapten, is not capable of stimulating the immune system to synthesize antibodies. In order to impart immunogenicity to the hapten s molecule, i.e. ability to evoke antibody production it must be conjugated with a macromolecular carriers. For this purpose different substances were tested among which colloidal gold turned to be the most suitable [3,4,5]. It does not cause allergies, non-toxic and has immunomodulatory properties. In addition, the gold itself does not act as an antigen which can not be said about other carriers, keeps the composition of antigen molecules attached to it, and hence capable to present them to cells responsible for immunity in an unmodified form [6,7 ]. The purpose of researches was to get mabs determinants of Ivermectin, conjugated with colloidal gold. against antigenic Materials and methods Balb/c mice, X63-Ag 8.6.5.3 myeloma cells line and the following reagents were used in the research: chemically pure preparation of Ivermectin (Sigma); IgG-Free, protease-free bovine serum albumin (Jackson Immuno Research); colloidal gold (~ 0.01% HAuCl4, ~ 1A520 unit / ml, a monodisperse particle, size 20nm, Sigma); hydrochloride 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide - EDC (Sigma-Aidrich); 2 - mercaptoethanol (Ferak); complete and incomplete Freund's adjuvant (Sigma); RPMI 1 640 (Sigma); hypoxanthine-guanine phosphoribosyl transferase - GGFRT; hepes (Sigma); solution of PEG 4000 with 10% dimethyl sulfoxide DMSO (Fluca); sodium pyruvate (Sigma); L -glutamine (Sigma); coomassie G -250 ( Диэм); ethyl alcohol 96 º; fetal calf serum (Hy Clon); pristane - 2,6,10,14-tetrametilpentadekan (Fluca); nitrocellulose membrane (Millipore), 1% agar (Difco) and set of reagents for ELISA. Conjugation of Ivermectin and polyclonal rabbit antibodies with colloidal gold was performed as described by Dykman L. A. et.al (2008) [8]. Immunization of Balb/c mice with colloidal gold and Ivermectin (C GI) conjugate was performed according to the following two schemes. By the first scheme (the short scheme) mice were injected intraperitoneally with 0.1 ml of CGI conjugate (concentration 1mg/ml) in a

volume of 0.1 ml complete Freund's adjuvant in the first day of immunization. On the 7th day of immunization immunogen was injected at the indicated dose in a solution of incomplete Freund's adjuvant and then immunization was continued for 11, 12, 13th days using only CGI conjugate in a buffered saline. The second immunization scheme (the long scheme) envisages fivefold immunization. The interval between the first, second and third immunization was two weeks. Fourth immunization was performed in three, and fifth - in four weeks after the previous administration of immunogen. CGI conjugate was injected intraperitoneally at a concentration of 0.2 mg/ml in a mixture with Freund's complete adjuvant for the first immunization and at the subsequent immunization it was administrated with Freund's incomplete adjuvant. Booster dose in a volume of 0.2 mg/ml per head without Freund's adjuvant was injected to mice in both groups 3 days before the isolation of immune lymphocytes. Indirect ELISA was used to determine antibodies titers in serum of immunized Balb/c mice against Ivermectin. Hybridization of X63-Ag 8.6.5.3 myeloma cells and immune mice s splenocytes was conducted by G.Kohler and C.Milstein [9]. «Dot» variant of sandwich ELISA was used for determining that produce antibodies against Ivermectin. For this purpose, samples of the hybridoma culture fluids were applied to a nitrocellulose membrane. After blocking free sites of nitrocellulose paper with bovine serum albumin it was transferred to a solution of Ivermectin. The presence of antibody+antigen complex was deteсted by using polyclonal rabbit antibodies, conjugated with colloidal gold. Cloning hybridoma cells was performed by limiting dilution [10]. Hybrid cells are cultured in the peritoneal cavity of syngeneic mice to produce preparative amounts of mabs. Affinity of mabs was established by the method of J.Beaty et al. [11]. Determination of isotypes of mabs performed by ELISA using the set of antisera to the different classes of mouse immunoglobulin. Results The serological test results of mice immunized with CGI conjugate are shown in table 1. As seen from table 1, the conjugate comprising Ivermectin showed its immunogenicity to Balb/c mice. Both schemes showed their sufficient efficiency in stimulating humoral response of immune system to Ivermectin. The groups of mice did not differ significantly among themselves by serum antibodies titers. Table 1 Studying blood sera of Balb/c mice by ELISA

Immunization schemes Short scheme Long scheme Numbers of mice Duration of immuni-za tion Numbers of isolated splenocytes, mln.cells Antibody titers 1 2 3 4 5 1 10х10 6 1:12 800 2 16 days 3х10 6 1:12 800 3 5 х10 6 1: 6 400 4 80 days 7 х10 6 1:6 400 5 10х10 6 1:12800 6 6х10 6 1:6 400 * Note - The samples of blood serum from each mouse were investigated in five replicates. The table shows the prevailing titers. From the spleen of each mouse splenocytes were obtained in an amount of 3,0 mln.cells up to 10,0 mln.cells. The data of table 1 indicate that for hybridization 41.0 million spleen cells were collected. The first hybridization carried out using immune lymphocytes of mouse 5 taken immediately before the cell fusion, and for the second fusion frozen lymphocytes of mouse 1 were selected. At the first of hybridization the number of splenocytes and myeloma cells were 1 10 6 and 5 10 6, i.e. cell ratio was equal to 1:5. After the fusion the cells were seeded in 384 wells of culture plates. Formation of hybrid cells was observed in 63 wells i.e. percentage of hybridomas output was low and amounted to 16.4%. Furthermore, none of the of hybrid cells showed activity against Ivermectin, ie they did not synthesize antibodies specific to this anthelmintic. Considering low yield of hybridomas in the preceding fusion, for the second hybridization myeloma cells and lymphocytes were taken in a ration of 1:8,5. Splenocytes of the mouse 1 cryopreserved in liquid nitrogen were taken for the second hybridization. The viability of defrosted spleen lymphocytes was rather high (90%). Increased percentage of lymphocytes in the mixture of cell-partners positively affected on the hybrid output. Thus, the growth of hybrid cells was observed in 155 out of 384 seeded wells, or 40.4% of cases. The results of studying hybrid for antibody activity by ELISA showed the presence of mabs in the culture fluid of 4 hybridoma strains designated as 1H1, 1H9, 2A3 and 4B1. These positive were transferred to 24-well plates for further culturing. After accumulating a sufficient cell mass all hybridomas were cloned by limiting dilution to obtain genetically homogeneous lines. Antibody activity of sub was determined by ELISA in 10-14 days after cloning. In this case the culture fluid samples were taken only from those wells where there were

microscopically distinguishable single cell colonies. Cloning results shown in table 2. Num-b ers of Table 2 - Activity of sub at the first cloning of hybridomas Designation of Numbers of formed sub total active sub 1 1Н1 30 11 (36,6%) 2 1Н9 23 9 (39,1%) 3 2А3 18 5 (27,7%) 4 4B1 13 7 (53,8%) Table 2 shows that at the first cloning the activity of sub, ie the ability to produce antibodies was rather low. Thus, in the supernatant of sub from hybridoma 4B1 specific antibodies were identified in 53.8% of cases, and the share of active sub from other three hybridomas (1H1, 1H9 and 2A3) was much lower: 36.6%, 39.1% and 27.7%, respectively. Taking into account the genetic heterogeneity of hybridoma after getting a sufficient amount of cells recloning of sub was immediately carried out (Table 3). Num-b ers of Table 3 - Activity of hybridomas sub after the second cloning Designation of Numbers of formed sub total Proportion of active 1 1Н1 17 0 (0%) 2 1Н9 11 6 (54,5%) 3 2А3 20 0 (0%) 4 4B1 9 5 (55,6%) Table 3 shows that the cells of two hybridomas (1H1 and 2A3) had lost the ability to synthesize mabs during cloning procedure. Hybrid sub 1H9 and 4B1 maintained their antibody activity. Thus, if the share of active sub of the first hybridoma was higher (54.5%) than that of in the first cloning (39,1%), then this indicator of the second one remained almost unchanged (53.8% and 55.6%). The resulting sub were cultured for one week, and then were subjected to the secondary recloning. The growth of isolated sub of hybridomas 1H9 and 4B1 was noted in 19 and 22 wells respectively and in all cases antibodies to Ivermectin had been registered. Thus, as a result of repeated recloning two of hybridomas were obtained producing mabs specific for antigenic determinants of Ivermectin.

For the accumulation of mabs in vitro hybridomas cells were transferred from the wells of 96-well to 24-well plates. With the accumulation of cells and the transition to the stage of logarithmic growth hybridomas were transferred and cultured in polystyrene mattresses. Antibody titers remained stable and were within 1:8-1:16. Protein concentration determined by Bradford s method was in the range of 30-60 ug/ml. The cells of mabs producers were injected into the peritoneal cavity of four syngeneic mice (two heads per hybridoma), pretreated with pristane at a dose of 0.5 ml per head in the amount of 0.5 million cells per 1 ml. After two or three weeks, with the tumor formation ascitic fluid from the peritoneal cavity was aspirated. Table 4 shows some properties of the mabs which are synthesized by two of hybridomas. Table 4 - Characteristics of hybridoma 1Н9 и 4B1 Designation of Affinity Isotypes of mabs Produc-tivity of mabs in vitro, mg / ml Produc-tivity of mabs in vivo, mg / ml * mabs titers in the culture mediu m * mabs titers in ascitic fluid 1Н9 2,0х10-9 М IgG1 0,06 4,0 1:16 1:1 600 4B1 2,6х10-9 М IgG1 0,03 4,0 1:8 1:800 * Note: Samples of the culture and ascitic fluids were tested by ELISA in five replicates. The table shows predominant titles. The results of studing individual properties of mabs showed that antibodies synthesized by the 1H9 and 4B1, belong to IgG1 and had a binding constant (affinity) with Ivermectin which is equal to 2.0x10-9 М and 2.6x10-9 М, respectively. As seen from the table 4, the hybridoma in a cell culture and ascitic fluid characterized by satisfactory productivity. Cultivating hybridomas in the peritoneal cavity of mice allowed to receive ascites fluid with the antibodies titers against Ivermectin within 1:800-1:1600. The samples of both mabs were subjected to SDS-PAGE electrophoresis on the apparatus for vertical electrophoresis to determine the molecular mass of immunoglobulins. The results showed that the heavy and light chains of mabs constitute the bands corresponding to 66 and 25 kd. Discussion and conclusion Ivermectin belongs to the low molecular weight compounds that are not capable of independently eliciting an immune response due to the small size of the molecule. In order to give the hapten s molecule ability to cause antibody

formation it must be conjugated with a macromolecular carrier. Immunization is the most "creative" stage in the process of obtaining antibodies, because its effectiveness determines ultimate success of the research. Balb/c mice were immunized with CGI conjugate through two schemes with a duration of immunization 16 days and 80 days. Both methods turned to be applicable for inducing antibody formation against Ivermectin "presented" to the immune system of Balb/c mice by colloidal gold. In serum samples of Balb/c mice antibodies against Ivermectin were detected in ELISA until dilutions of 1:6400-1:12800. The lymphocytes isolated from spleens of immune mice were used for fusion with myeloma X63-Ag 8.6.5.3 to produce hybrid - producers of mabs. Four hybrid synthesizing mabs to Ivermectin were obtained. Hybridomas were subjected to cloning and recloning in order to obtain lines which free from cells that lost their ability to synthesize of mabs. At the first cloning the activity of sub, i.e. the ability of cells to produce of antibodies was rather low. The share of sub that kept an antibody activity after the first cloning ranged from 27.7% up to 53.8%. These results indicate the presence of "descendants" among hybridoma cells that have lost their ability to produce antibodies as a result of mutation. Two hybridomas (1H1 and 2A3) which had a high percentage of passive sub following the results of first cloning lost their activity against Ivermect during the recloning. Sub of hybridomas 1H9 and 4B1 retained their ability to synthesize mabs after recloning. Moreover, the share of active sub of the first hybridoma was significantly higher (54.5%) than that of in primary cloning (39.1%). Hybridomas selected for further studies were cultured in vivo and in vitro for accumulation of preparative amounts of mabs specific to Ivermectin s epitopes followed by immunochemical study of their properties such as specificity, antibody activity, class and subclass of immunoglobulins, molecular weight of light and heavy chains of immunoglobulins and the binding constant of antibodies with the antigen (affinity). Monoclonal antibodies which are synthesized by cells of 1H9 and 4B1 belong to the class IgG1 and have affinity for Ivermectin equal to 2.0x10-9 М and 2.6x10-9 М with the titers of ascitic fluid 1:800 and 1:1 600, respectively. The concentration of mabs in the culture fluid was in the range of 0,03-0,06 mg/ml, but in ascitic fluid it reached 4.0 mg/ml. Thus, two stable strains of hybridomas were obtained producing mabs that can be used in the development of immunoassays for the rapid determination of Ivermectin in animal products.

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Резюме Ивермектин относится к низкомолекулярным веществам, которые самостоятельно не способны вызывать иммунный ответ организма. Для придания молекуле гаптена способность стимулировать антителообразование его необходимо конъюгировать с высокомолекулярными носителями. Мыши линии Balb/c были иммунизированы конъюгатом, приготовленным из ивермектина и коллоидного золота, по двум схемам с продолжительностью иммунизации 16 и 80 дней, соответственно. Оба метода оказались пригодными для индуцирования образование антител против ивермектина. С помощью метода гибридомной техники были созданы два стабильных штамма гибридных культивируемых клеток, продуцирующие моноклональные антитела к эпитопам ивермектина. Полученные антитела могут быть использованы в разработке иммунологических тестов для экспресс-определения остаточного количества антгельминтика в животноводческих продуктах. Түйін Ивермектин өздігінен организмнің иммундық жауабын тудыра алмайтын молекулярлық салмағы жеңіл заттар қатарына жатады. Гаптен молекуласына иммуногендік қасиетті беру мақсатында оны жоғарымолекулярлық тасымалдағыштармен конъюгациялау керек. Balb/c линиясының тышқандары ивермектин мен коллоидты алтынның қоспасынан әзірленген конъюгатты 16 және 80 күн аралығында егу арқылы иммунизацияланды. Сыналған тәсілдердің екеуі де организмнің ивермектинге қарсы антидене түзуін қоздыра алды. Гибридома техникасының көмегімен ивермектиннің эпитоптарына телімді моноклоналдық антиденелерді тұрақты түрде түзейтін гибридті жасушалардың екі штамы алынды. Мұндай антиденелер мал өнімдерінен даярланған тағамдарда антгельминтиктің қалдық мөлшерін тез арада анықтай алатын иммунологиялық тәсілдерді әзірлеуде қолданыс табуы мүмкін.