Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: Implication for diagnosis of Kala-azar

Document Type : Original Article


1 School of Biology, College of Science, University of Tehran, Tehran, Iran

2 Faculty of Biology, College of Science, University of Mohaghegh Ardabili, Ardabil, Iran

3 Department of Immunology, Pasteur Institute of Iran, Tehran, Iran


Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.
Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c mice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.
Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones.
Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.


Main Subjects

1.  Kennedy PJ, Oliveira C, Granja PL, Sarmento B.Monoclonal antibodies: Technologies for early discovery and engineering. Crit Rev Biotechnol 2017; 8:1-15.
2. Ready PD. Epidemiology of visceral leishmaniasis. Clin Epidemiol 2014; 6:147–154.
3. Bhattacharya SK, Dash AP. Treatment of visceral leishmaniasis: options and choice. Lancet Infect Dis 2016; 16:142–143.
4. Pezeshkpoor F, Rezaei AR, Shirdel A, Khajedaluee M, Alizadeh M, Yazdanpanah MJ. Association between HTLV-I infection with chronic lupoid Leishmaniasis. Iran J Basic Med Sci 2013; 16:281-283.
5. Galgamuwa LS, Sumanasena B, Yatawara L, Wickramasinghe S, Iddawela D. Clinico-Epidemiological patterns of cutaneous Leishmaniasis patients attending the anuradhapura teaching hospital, Sri Lanka. Korean J Parasitol. 2017; 55: 1-7.   
6. Heidari A, Mohebali M, Kabir K, arati H, Soultani Y, Keshavarz H, Akhoundi B, Hajjaran H, Reisi H. Visceral Leishmaniasis in rural areas of Alborz Province of Iran and implication to health policy. Korean J Parasitol. 2015; 53: 379-383.   
7. Rajabi O, Sazgarnia A, Abbasi F, Layegh P. The activity of ozonated olive oil against leishmania major promastigotes. Iran J Basic Med Sci 2015; 18:915‐919.
8. Sayyahfar S, Ansari S, Mohebali M, Behnam B. Visceral Leishmaniasis without Fever in an 11-month-old infant: A rare clinical feature of Kala-azar. Korean J Parasitol. 2014; 52: 189-191.   
9. Nadim A. Present status of kala-azar in Iran. Am J. Trop. Med. Hyg 1978; 27: 25-28.
10. Bettini S, Gradoni L. Canine leishmaniasis in the Mediterranean area and its implications for human leishmaniasis. Insect. Sci. Appl 1986; 7: 241-245.
11. Mohebali M, Edrissian GH, Shirzadi MR, Akhoundi B, Hajjaran H , Zarei Z,Molaei S, Sharifi I, Mamishi S, Mahmoudvand H, Torabi V, Moshfe A, Malmasi A, Motazedian MH, Fakhar M.An observational study on the current distribution of visceral leishmaniasis in different geographical zones of Iran and implication to health policy. Travel Med Infect Dis 2011; 9: 67-74.
12. Akhavan AA, Yaghoobi-Ershadi MR, Khamesipour A, Mirhendi H,Alimohammadian MH , Rassi Y, Arandian MH, Jafari R, Abdoli H, Shareghi N, Ghanei M, Jalali-zand N. Dynamics of Leishmania infection rates in Rhombomys opimus (Rodentia: Gerbillinae) population of an endemic focus of zoonotic cutaneous leishmaniasis in Iran. Bull Soc Pathol Exot 2010; 103: 84-89.
13. Farajnia S, Darbani B, Babaei H, Alimohammadian MH, Mahboudi F, Gavgani AM. Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran. Parasitology J 2008; 19:1-7.
14. Alimohammadian MH, Kivanjah M, Pak F, Gaznavi A, Kharazmi A. Evaluation of the efficacy of Iran leishmanin and comparison with leishmanins form Wellcome (UK) and Roma (Italy) in cured cutaneous leismaniasis patients. Trans R Soc Trop Med Hyg 1993; 87: 550- 551.
15. Alimohammadian MH, Almasi H, Khabiri AR, Hatam GR, Karimi A, Mahboudi F, Tehrani SM, Amirkhani A. Identification of species and characterization of an outbreak of cutanous leishmaniasis in a new focus of Iran. Iran Biomed J 1999; 3: 31-39.
16. Tashakori M, Ajdari S, Kariminia A, Mahboudi F and Alimohammadian MH. Chracterization of Leishmania species and L. major strains in different endemic areas of cutaneous leishmaniasis in Iran. Iran Biomed J 2003; 7: 43-50.
17. Tavaresa J, Ouaissic BA, Lind PKT. Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes. Int J Parasitol 2005; 35: 637–646.
18. Sereno D, Holzmuller P, Lemesre JL. Efficacy of sec line drugs on antimonyl-resistant amastigotes of Leishmania infantum , Acta Tropica 2000; 74 : 25–31.
19. Alain D, Manju BJ, Pimenta PFP, Dennis M D. Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics , Int J Parasitol 2004; 34: 205–217.
20. Rochettea A, Raymond F, Corbeil J, Ouellette M, Papadopoulou B. Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum. Mol Biochem Parasitol 165; 2009; 165: 32–47.
21. Herwldt BL. Leishmaniasis. Lancet 1999; 354: 1191-1199.   
22. Gupta N, Goyal N, Rastogi AK. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 2001; 173: 150-153.
23. Pan AA. Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium. Exp Parasitol 1984; 58: 72–80.
24.  Bates PA. Axenic culture of Leishmania amastigotes. Parasitol Today1993; 9:143–146.
25. Bates PA. Complete developmental cycle of Leishmania mexicanain axenic culture. Parasitology J 1994; 108: 1–9.
26. Nasereddin A, Schweynochb C, Schonianb G, Jaffe JL. Characterization of Leishmania (Leishmania) tropica axenic amastigotes. Acta Tropica 2010; 113: 72–79.
27. Sereno D, Cavaleyra M, Zemzoumi K, Maquaire S , Ouaissi A, Lemesre JL. Axenically grown amastigotes of Leishmania infantum used as an In vitro model to investigate the pentavalent antimony mode of action. Antimicrob Agents Chemother 1998;42: 3097–3102.
28. Kurtzhals   JAL, Hey  AS,  Jardim  A, Kemp M, Schaefer KU,  Odera EO, Christensen CBV, Githure  JI,  Olafson RW, Theander TG, Kharazmi A. Dichotomi  of  the  human  T cell  response  to Leishmania antigens. II. Absent  or   Th2-like   response  to  gp63  and  Th1-like response  to  lipophosphoglycan-associated   protein  in  cells  from  cured visceral  leishmaniasis  patients.  Clin  Exp  Immunol 1994;96: 416-421.
29. Zoghlami Z, Chouihi E, Barhoumi W, Dachraoui K, Massoudi N, Ben Helel K, Habboul Z, Hadhri MH, Limam S, Mhadhbi M, Gharbi M, Zhioua E. Interaction between canine and human visceral leishmaniases in a holoendemic focus of Central Tunisia. Acta Trop 2014; 139: 32–38.
30. Savoia D. Recent updates and perspectives on leishmaniasis.  J Inf Dev Ctries 2015; 96: 588–596.
31. Debrabant A1, Joshi MB, Pimenta PF, Dwyer DM. Generation of Leishmania donovani axenic amastigotes: Their growth and biological characteristics. Int J Parasitol 2004; 34: 205–217.
32. Saar Y, Ransford A, Waldman E, Mazareb S, Amin-Spector S, Plumblee J, Turco SJ, Zilberstein D. Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani. Mol Biochem Parasitol 1998; 95:9–20.
33. Gupta N, Goyal N, Rastogi AK. In vitro cultivation and characterization of axenic amastigotes of Leishmania. Trends Parasitol 2001; 17: 150-153.
34. Tomita M, Tsumoto K: Hybridoma technologies for antibody production. Immunotherapy 2011; 3: 371–380.
35. Moritaa M, Sugiharaa  H, Tokunakaa K, Tomuraa A, Saigaa K, Satoa T, Imamurab Y, Hayashic T. Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain. Biochem Biophys Rep 2017; 9: 128–132.
36. Kholer G, Milestine C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature1975; 125: 495-497.
37. De Ibarra AA, Howard JG, Snary D.Monoclonal antibodies to Leishmania tropica major: specificities and antigen location. Parasitology J 1982; 85: 523-531.
38. Greenblatt CL, Slutzky GM, De Ibarra AA, Snary D. Monoclonal antibodies for  serotyping Leishmania strains. J Clin Microbiol 1983; 18: 191-193.
39. Chaves CS, Soares DC, Da Siva RP, Saraiva EM. Characterization of the species-and stage specificity of two monoclonal antibodies against Leishmania amazonensis. Exp. Parasitol 2003; 103: 152-159.
40. York DS, Heinz S, Beatrice M, David GR, Margret Q, Peter O. Monoclonal antibodies to Leishmania mexicana promastigote antigen. J Cell Sci 1991; 99: 181-186.
41. Mandira M, Anindita B, Swadesh D. Monoclonal antibody affinity purification of a 78 KDa membrane protein of Leishmania donovani of Indian origin and its role in host –parasite interaction. Indian Academy Science 2002; 27: 665-672.
42. Goding JW.Theory Monoclonal antibodies. In: Monoclonal antibodies: Principles and practice‏. 2nd ed. Academic Press; 1986: 5-58.
43. Macken C. Design and analysis of serial limiting dilution assays with small sample sizes. J Immunol Meth 1999; 222: 13-29.
44. Galfre G, Howe SC, Milstein C, Butcher GW. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 1977; 266: 550-552.
45. Debrabanta A, Joshib MB, Pimentac PFP, Dwyer DM. Generation of Leishmania donovani axenic amastigotes: Their growth and biological characteristics. Int J Parasitol 2004; 34: 205–217.
46. Tomita M, Tsumoto K. Hybridoma technologies for antibody production. Immunotherapy 2011; 3: 371–380.
47. Tomita M, Tsumoto K. Recent advances in antigen-based generation of monoclonal antibodies. Curr Immunol Rev 2010; 6: 56–61.
48. Pasqualini R, Arap W: Hybridoma-free generation of monoclonal antibodies. Proc Natl Acad Sci USA 2004; 101: 257–259.
49. Schmitz K, Geisslinger G, Tegeder I. Monoclonal antibodies in preclinical EAE models of multiple sclerosis: A systematic review. Int J Mol Sci 2017; 18:1-18.
50. Kivi G, Teesalu K, Parik J, Kontkar E, Ustav M J, Noodla L, et al. HybriFree: A robust and rapid method for the development of monoclonal antibodies from different host species. BMC Biotechnol 2016; 16: 2.