بهينه سازي شرايط رشد هيبريدهاي مولد آنتي بادي مونوكلنال عليه آنتي ژن ليشمانيا اينفنتوم

Monoclonal Antibody Against Antigens of Leishmania infantum: Optimize the Growth Condition of Monoclonal Antibody-producing Hybrids

زﻣﯿﻨﻪ و ﻫﺪف ﻟﯿﺸﻤﺎﻧﯿﺎ اﯾﻨﻔﺎﻧﺘﻮم ، ﻋﺎﻣﻞ اﺗﯿﻮﻟﻮژﯾﮏ ﻟﯿﺸﻤﺎﻧﯿﻮز اﺣﺸﺎﯾﯽ در اﯾﺮان اﺳﺖ واﮐﺴﻦ ﻫﺎي ﻣﺆﺛﺮ و روش ﻫﺎي ﺗﺸﺨﯿﺼﯽ ﺑﺮاي ﮐﻨﺘﺮل ﻟﯿﺸﻤﺎﻧﯿﻮز ﻻزم اﺳﺖ. آﻧﺘﯽﺑﺎديﻫﺎي ﻣﻮﻧﻮﮐﻠﻮﻧﺎل اﺑﺰاري ﺑﺎارزش ﺑﺮاي ﺗﺸﺨﯿﺺ، درﻣﺎن و ﺗﻌﯿﯿﻦ وﯾﮋﮔﯽ آﻧﺘﯽژﻧﯽ اﻧﮕﻞﻫﺎ ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﮔﺮﻓﺘﻪاﻧﺪ. ﻫﺪف از اﯾﻦ ﻣﻄﺎﻟﻌﻪ، ﺑﻬﯿﻨﻪ ﺳﺎزي ﺷﺮاﯾﻂ رﺷﺪ ﻫﯿﺒﺮﯾﺪﻫﺎي ﻣﻮﻟﺪ آﻧﺘﯽﺑﺎدي ﻣﻮﻧﻮﮐﻠﻨﺎل ﻋﻠﯿﻪ آﻧﺘﯽ ژن ﭘﺮوﻣﺎﺳﺘﯿﮕﻮت ﻟﯿﺸﻤﺎﻧﯿﺎ اﯾﻨﻔﻨﺘﻮم اﺳﺖ. ﻣﻮاد و روشﻫﺎ: ﻣﻮش ﻫﺎ ﺗﻮﺳﻂ آﻧﺘﯽژن ﻟﯿﺸﻤﺎﻧﯿﺎ اﯾﻨﻔﻨﺘﻮم واﮐﺴﯿﻨﻪ ﺷﺪﻧﺪ و ﺗﯿﺘﺮ آﻧﺘﯽﺑﺎديﻫﺎي آﻧﺎن ﺗﻮﺳﻂ روش اﻻﯾﺰا ﺗﻌﯿﯿﻦ ﺷﺪﻧﺪ. ﺳﻠﻮل ﻫﺎي ﻃﺤﺎل ﻣﻮش ﻫﺎﯾﯽ ﮐﻪ ﺑﻪ ﺧﻮﺑﯽ ﺗﻮﺳﻂ آﻧﺘﯽ ژن ﻟﯿﺸﻤﺎﻧﯿﺎ اﯾﻨﻔﻨﺘﻮم اﯾﻤﻦ ﺷﺪه ﺑﻮدﻧﺪ ﺑﺎ ﺳﻠﻮل ﻫﺎي SP2/0در ﺣﻀﻮر ﭘﻠﯽ اﺗﯿﻠﻦ ﮔﻠﯿﮑﻮل ادﻏﺎم ﺷﺪﻧﺪ. ﺗﺄﺛﯿﺮ ﺳﻮﭘﺮﻧﺎﺗﺎﻧﺖ ﺳﻠﻮلﻫﺎي SP2/0و ﺳﻠﻮلﻫﺎي ﻣﺎﮐﺮوﻓﺎژ ﺻﻔﺎﻗﯽ ﻣﻮش ﻫﺎ ﺑﺮ ﺗﮑﺜﯿﺮ ﺳﻠﻮل ﻫﺎي ﻫﯿﺒﺮﯾﺪوﻣﺎ ﺑﺮرﺳﯽ ﺷﺪﻧﺪ. ﯾﺎﻓﺘﻪﻫﺎ: در ﻣﯿﺎن 12ﻓﯿﻮژن درﻣﺠﻤﻮع 26 ﻣﻮﻧﻮﮐﻠﻮن ﻣﺜﺒﺖ ﺑﻮدﻧﺪ ﮐﻪ 12 ﻣﻮﻧﻮﮐﻠﻮن ﺟﺬب ﻧﻮري )OD( ﻗﺎﺑﻞﻗﺒﻮﻟﯽ داﺷﺘﻨﺪ. 4 ﮐﻠﻮن ﺑﻪ ﻋﻨﻮان6G2 FV4 ، 8D2 FVI3 ، 8 D2 FVI6 و 6G2 FV3ﺗﻌﯿﯿﻦ ﺷﺪﻧﺪ. از اﯾﻦ ﻫﯿﺒﺮﯾﺪﻫﺎ، ﻣﻮﻧﻮﮐﻠﻮﻧﺎل آﻧﺘﯽﺑﺎديﻫﺎ ﻋﻠﯿﻪ ﻟﯿﺸﻤﺎﻧﯿﺎ اﯾﻨﻔﺎﻧﺘﻮم ﺑﻪدﺳﺖ آﻣﺪ. ﻫﻤﭽﻨﯿﻦ اﯾﻦ ﻣﻄﺎﻟﻌﻪ ﻧﺸﺎن داد ﮐﻪ ﻣﺎﯾﻊ روﯾﯽ )ﺳﻠﻮل ﻫﺎي SP2/0و ﺳﻠﻮل ﻫﺎي ﻣﺎﮐﺮوﻓﺎژ ﺻﻔﺎﻗﯽ( ﻧﻘﺶ ﮐﻠﯿﺪي در ﺗﮑﺜﯿﺮ ﻫﯿﺒﺮﯾﺪوﻣﺎ و ﺗﻮﻟﯿﺪ آﻧﺘﯽﺑﺎديﻫﺎي ﻣﻮﻧﻮﮐﻠﻮﻧﺎل دارد ﮐﻪ ﺳﺮﺷﺎر از ﻓﺎﮐﺘﻮرﻫﺎي رﺷﺪ اﺳﺖ. ﻧﺘﯿﺠﻪ ﮔﯿﺮي: ﺑﻪ ﻧﻈﺮ ﻣﯽرﺳﺪ در آﯾﻨﺪه ﻧﺰدﯾﮏ ﻣﺎﯾﻊ روﯾﯽ ﻣﺬﮐﻮر ﻣﯽ ﺗﻮاﻧﺪ ﺑﻪﻋﻨﻮان ﯾﮏ ﻓﺎﮐﺘﻮر رﺷﺪ ﺑﺮاي ﺳﻠﻮل ﻫﺎي ﺳﺮﻃﺎﻧﯽ و ﻏﯿﺮﺳﺮﻃﺎﻧﯽ در ﻣﺮاﮐﺰ ﺗﺤﻘﯿﻘﺎﺗﯽ در ﺳﻄﺢ وﺳﯿﻊ ﺗﺮي ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﮔﯿﺮد.

Leishmania (L.) infantum is the etiologic cause of visceral leishmaniasis in Iran. Efficient vaccines and diagnosis methods are required to control leishmaniasis. The aim of this study is produce and optimize monoclonal antibodies against promastigotes forms of L. infantum antigen. Materials and Methods: The mice were vaccinated with the L. infantum antigen and their antibody titers were determined by the ELISA method. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol.The effect of supernatant of SP2/0 and mice peritoneum macrophage cells culture (SSMCC) on hybridoma cell proliferation was studied. Results: Among the 12 fusion, a total of 26 monoclonal were positive.12 of which had acceptable optical absorbance in OD 450 nm. Finally, 4 clones, designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. From these hybrids, anti-promastigotes L. infantum monoclonal antibodies were obtained. SSMCC was shown to play a key role in hybridoma proliferation and of mAb production. It seemed that SSMCC is rich of growth factors. Conclusion: It seems in the near future, this SCCSM can be used as a growth factor for cancerous and non-cancerous cells in research centers at a wider level.