Monoclonal antibody production with hybridoma technique against the antigen of Leishmania infantum, causative agent of visceral leishmaniasis

Leishmaniasis is a parasitic disease which is transmitted to mammalian hosts by the bites of sand flies (phlebotomus) with infected intracellular Leishmania protozoon during bloodsucking. Different Leishmania species cause to the various important forms of the disease such as; visceral (VL), cutaneous (CL) and mucocutaneous (MCL) leishmaniasis depending on the parasite types and the host factors. In every year 1-1,5 million people suffer from Cutaneous Leishmaniasis (CL) and 500.000 people from visceral leishmaniasis (VL) all around the world. Furthermore impact area of the disease is expected to extend over the world as a result of developing resistance to antileishmanial drugs in parasites and to insecticides in vectors, also by virtue of global warming. Therewithal leishmaniasis is one of the serious public health problems that occurs prevalently in Aegean, Mediterranean, Middle Anatolian and Southeast Anatolian regions of Turkey. Untreated VL form of the disease ends up with deaths. A variety of techniques (microscopic, culture, molecular and serological) are used in the diagnosis of the disease. Among these techniques, monoclonal antibody based serological techniques provide a great advantage with high sensitivity and specificity for diagnosis that is special to the parasite antigens. However, diagnostic kits used in the diagnosis of leishmaniasis are generally imported products and make a great expense to the economy of the country. The studies about the issue to overcome these kinds of problems are inadequate in our country. Up to now, only one medical specialization thesis has been completed about this issue and in that study monoclonal antibody has been produced against Leishmania tropica which is a factor of Cutaneous Leishmaniasis. However as it is known in literature serological techniques are not sensitive techniques for diagnosis of CL. On the other hand, in our country there is no study about development of monoclonal antibody against VL which is known as fatal if doesn?t diagnose in time and to be treated. Furthermore, it is known that there are some problems in hybridoma techniques for providing in vitro whole processes. According to this, the aim of this thesis study as a first time in our country is to produce monoclonal antibody against L. infantum antigen which was used in the diagnosis of VL and to optimize the present method by optimizing the hybridoma technology process into in vitro stage. Within the scope of the thesis, in Cell Culture and Tissue Engineering Laboratory, cell culture, ELISA, western blot, hybridoma and purification techniques were used. In the experiments; L. infantum whole parasite antigens were injected to 6-8 week old BalB/c mice with adjuvant. Then the fusion studies were started with the mice which gave response of high antibody. In the fusion experiments the spleen cells which are the source of B lymphocyte were fused with the mouse myeloma cells (P3-X63-Ag8.653) in the existence of polyethylene glycol (MV 4000). After the fusion, in order to support hybridoma cells besides mouse peritoneal macrophages, J774 mouse monocyte-macrophage cell line metabolite was also used. According to the experimental results, it was detected that hybridoma cells were formed in examined 233 of 456 wells. It was seen that the 46 of 233 wells were positive according to ELISA test. The culture of three wells which showed the highest absorbance in cross-reaction tests, was largely produced. The monoclonal antibodies, the products of 1C3 hybridoma cell line which was obtained as a result of limiting dilutions. It was detected that the antibodies which were examined with western blot method, are specific to the L. infantum antigen which weights 15kda. At the end-stage of the study, supernatant which are obtained from culture were purified with dialysis and protein G affinity chromatography methods, so the monoclonal antibody which is specific to 15 kDa antigen of L. infantum was obtained.As a result of this study for the first time in Turkey monoclonal antibody producing hybridoma cell line have been obtained against L. infantum and cryopreserved in cryobank. Additionally, in order to render whole in vitro processes of hybridoma technology, hybridomas were firstly supported with J774 macrophages cell line metabolites and in vitro current processes of hybridoma technique was optimized. Furthermore we showed that J774 metabolites provide the appropriate supporting environment. Obtained results within this thesis can lead to development of diagnostic kits to VL that is one of the important public health problems of Turkey. Furthermore the obtained monoclonal antibodies in this study is important in terms of showing that they can be also used in various diagnosis systems (such as; Western blot (WB), Enzyme Linked Immune Sorbent Assay (ELISA), Immune Fluorescein Assay (IFAT)and Immuno chromatographic test ICT)) by conjugating with enzymatic, chemiluminescence and magnetic agents.