Generation of hybridoma technology based antibodies against immunogen molecules isolated from leishmania tropica parasites in order to use in diagnosis

Leishmaniasis is a zoonotic disease which is one of the serious public health problem in our country and the world. Data released in the framework of the World Health Organization, 2 million new cases occur each year and in worldwide 350 million people are living under the threat of this disease is endemic in 90 countries. Cutaneous leishmaniasis is caused by Leishmania tropica, while Leishmania major causes Mucocutaneous, Leishmania infantum causes the Visceral Leishmaniasis. Antimonial drugs used in the treatment and vectors used in the development of resistance to insecticidal in failure to develop an effective vaccine until now and also that the diagnosis of these problems can not be solved until now it is among the causes of the disease show wide distribution. The diagnosis of leishmaniasis are used microscopy, culture and micro culture-based systems, serological and molecular methods. Sensitivity ratings given against parasite species and different forms of the disease is different in the current diagnostic methods. Giemsa stained preparations form the basis of the microscopic method. The most important disadvantage of the microscopic method which is cheap but partially applicable, depending on the number of samples contained parasites change the degree of sensitivity. In another method is culture methods, the observation of parasite development in a closed system is also possible, although it is required to put the 15-30 days long in the diagnosis of this disease. Despite these disadvantages of the culture method, the micro-culture method developed in the following years and it was achieved more sensitive response in a shorter period of time (1-7 days). This method is not only used in the diagnosis of Leishmaniasis. This method has been a role model also used in the samples obtained from the donor blood, vaccine development, mice to study without being sacrificed, stem cell isolation and the diagnosis of Helicobacter. There are also molecular methods such as isoenzyme analyses, polymerase chain reactions, realtime polymerase chain reactions to diagnose Leismaniasis. The biggest disadvantages of these methods are that they are susceptible to paracites and also very expensive. In the diagnosis of disease, serologic methods that targets the antigens on the surface of the parasites have become very important. The most used serologic methods that provide an early diagnosis are immunofluorescence colouring based IFAT, ELISA and rk39 kits. The underlying source in these serologic methods is the detection of antibodies in the serum. Commercially produced r39 kits have a high susceptibility on patients with Visseral Leismaniasis however their susceptibility level on patients with Cutaneous Leishmaniasis is 20%. According to this, it could be said that the low levels of susceptibility towards Cutaneuous Leismanasis might be because the parasite has a particular kind of antigen in its lesion area. We have not come across a method like RK39 kits that is used to diagnose patients with Cutaneous Leishmaniasis in our country and in the world. Antibodies that are produced based upon hybridoma technologies are rooted in why these kinds of test kits are prepared. Diagnostics systems that have very high affinity and specificity towards many kinds of diseases have been developed with this technology. The studies that have used hybridoma technology against Leishmaniasis in the world has only examined the cross reactions of the produced antibodies but they did not develop an effective diagnostics system. Previously in the scope of a master's thesis, monoclonal antibody was produced to work against the factor L.infantum of Leishmaniasis in our laboratory. The reasons as to why the studies in this field are insufficient is because the difficulties in getting immune answers. One of the common subjects of hybridoma studies is the use of Freund's adjuvant in the immunisation stage. This adjuvant is established in some studies as effective but also toxic. This toxic nature brings the need to have new adjuvants. According to this, the purpose of this thesis study is to produce antibodies with hybridoma technology to use in the diagnostics of cutaneous leishmaniasis against the immunogen molecules of L.tropica parasites and to examine the effectiveness of this antibody production on various adjuvants. To achieve this goal, hybridoma technology created as a result of fusion based antibodies were produced by fusion of myelomas cancer cells with B-lymphocytes isolated from bone marrow of mices that are immunized with surface molecules obtained from Leishmania tropica. In the steps of this technology, the immune mice responses against all-surface antigens of Leishmania tropica with Freund's adjuvant complex that is frequently used in literature or unlike literature using new polymeric based Polioksidony I (PO) adjuvant that is FDA approved and non-toxic, were compared. The Mice with sufficient response is sacrified by cervical dislocation method and B-lymphocytes isolated to used in the fusion studies with myeloma bone marrow cancer cells. Antibody produced by the hybrid cell produced by hybridoma technology were tested by ELISA system. Also secreted that antibodies produced by hybridoma cells not only against Leishmania parasites, has also been examined against the adjuvant which used in the immunization step. These antibodies produced by hybridoma cells is also tested against the L. major, L. infantum and the parasite's surface and the immunogenic molecule lipophosphoglycan (LPG). However, the amount of antibody produced by hybridoma cells was examined before and after cryopreservation depending on freezing protocols in the literature. Within the framework of the obtained results, FREUND after'S mice immunized with adjuvant 6. immunization in mice immunized with PO 8. It is found that immunization resulted in increased approximately 5.5 times compared to the control antibody level. Also the same serum samples were also tested against the adjuvant. Accordingly to this 8. Immunization result in Freund's adjuvant as compared to the control mice used in the antibody response generated against Freund's it was determined that approximately three-fold increase. But the mice immunized against Polyoxidonium adjuvant, there was no significant changes in responses compared the control after 8. Immunization. The next step resulting from the fusion of hybridomas cultured in selective medium and produced polyclonal antibody responses were determined by ELISA method. According to the these antibody responds, polyclonal antibodies against the L.tropica was observed in hybridoma cultures in the 10th and 20th days. Furthermore the effectiveness of antibody were examined not only against to the antigen coating but also the adjuvant coatings. Freund's group hybridoma cell responses (antigen-specific) were more decreased 20th day compared to 10th day. PO group of hybridoma cells produced specific antibodies to the antigen was detected 20th days higher than 10th day. Freund's group of the antibody produced by the hybrid cells is not only specific to the antigen, the adjuvant also has been found to create a response to it. The PO coated ELISA plate results, produced by the hybridoma cell antibody response, 10th day versus 20th days was seen more reduced. Produced antibodies by Hybridomas have been identified to generate the response against whole leishmania lysate and Leishmanias' most immunogenic surface molecules; LPG. Hybridoma cells' antibody producing ability before and after cryopreservation were comparatively analyzed and this antibody response not just for L.tropica has also been found to show effects in the different susceptibility to L.infantum and L.major. In the light of this study, suggested the use of these antibodies in the diagnosis of cutaneous leishmaniasis. In addition, the experimental value of having these antibodies also added into the medium of parasite antigens were produced, the properties of infectivity, as they allow to investigate the evolutionary process promastigote-amastigote differentiation.