, Volume 68, Issue 3, pp 469–480 | Cite as

Development and characterization of a monoclonal antibody against the putative T cells of Labeo rohita

  • Sanjay C. Rebello
  • Gaurav Rathore
  • Peyush PuniaEmail author
  • Neeraj Sood
Original Research


In this study, we have described the development and characterization of monoclonal antibodies (MAbs) directed against thymocytes of rohu, Labeo rohita. MAbs were obtained by immunizing BALB/c mice with freshly isolated and nylon wool column enriched mononuclear cells of thymus. Positive clones against thymocytes were screened by cellular ELISA. The hybridoma showing strong reactivity with nylon wool enriched mononuclear cells, and non-reactivity with a rohu thymus macrophage cell line and rohu serum was selected and subjected to single cell cloning by limiting dilution. The MAbs secreted by a positive clone were designated as E6 MAb. Western blotting of reduced protein from enriched thymocytes showed that E6 reacted with a 166.2 kDa polypeptide and belongs to the IgG1 subclass. Flow cytometric analysis of gated lymphocytes, revealed that the percentage of E6 positive (E6+) cells in thymus (n = 5, 720.4 ± 79.70 g) was 89.7 %. Similarly, the percentage of E6+ cells in kidney, spleen and blood (n = 5) was 6.71, 1.71 and 1.88 %, respectively. In indirect immunoperoxidase test, E6+ cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio and were densely packed in the central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. E6 MAb also reacted with a small population of lymphocytes in blood smear. This MAb appears to be a suitable marker for T lymphocytes and can be a valuable tool in studying immune response and ontogeny of L. rohita immune system.


Labeo rohita Monolonal antibody T-lymphocytes Flow cytometry Thymus cELISA Immunoperoxidase test Western blotting 


  1. Arunachalam B, Talwar GP, Raghupathy R (1990) A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens. J Immunol Methods 135:181–189CrossRefGoogle Scholar
  2. Bartos JM, Sommer CV (1981) In vivo-cell mediated immune response to M. tuberculosis and M. salmoniphilum in rainbow trout, Salmo gairdneri. Dev Comp Immunol 5:75–83CrossRefGoogle Scholar
  3. Beelen R, Pavanelli GC, Boyd B, Garavello JC, Guimaraes-Beelen PM, Ainsworth AJ (2004) Production and characterization of a putative T-cell marker in hybrid surubim catfish Pseudoplatystoma corruscans (Agassiz) × Pseudoplatystoma fasciatum (Linnaeus). Acta Sci Biol Sci 26:201–207CrossRefGoogle Scholar
  4. Botham JW, Grace MF, Manning MJ (1980) Ontogeny of first set and second set alloimmune reactivity in fishes. In: Manning MJ (ed) Phylogeny of immunological memory. Elsevier, North Holland/Amsterdam, pp 83–92Google Scholar
  5. Chaudhary DK, Sood N, Pradhan PK, Agarwal NK, Rathore G (2012) Production and characterization of a monoclonal antibody against putative T lymphocytes of Catla catla. In Vitro Cell Dev Biol Anim 48:483–492CrossRefGoogle Scholar
  6. Chowdhury PS, Pastan I (1999) Analyses of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunization. J Immunol Methods 231:83–91CrossRefGoogle Scholar
  7. FGIS (2007) Fisheries global information system. FAO, RomeGoogle Scholar
  8. Frindlay C, Tatner MF (1994) A comparative study of T and B lymphocytes in rainbow trout (Oncorhynchus mykiss) following their separation by nylon wool adherence and lectin agglutination techniques. Comp Haematol Int 4:55–60CrossRefGoogle Scholar
  9. Giraud A, Ataman-Önal Y, Battail N, Piga N, Brand D, Mandrand B, Verrier B (1999) Generation of monoclonal antibodies to native human immunodeficiency virus type 1 envelope glycoprotein by immunization of mice with naked RNA. J Virol Methods 79:75–84CrossRefGoogle Scholar
  10. Graham S, Secombes CJ (1990) Cellular requirements for lymphokine secretion by rainbow trout, Salmo gairdneri, leucocytes. Dev Comp Immunol 5:75–83Google Scholar
  11. Hathcock KS (2001) T cell enrichment by nonadherence to nylon. Curr Protoc Immunol Chapter 3, Unit 3.2Google Scholar
  12. Hetland DL, Jorgensen SM, Skjodt K, Dale OB, Falk K, Xu C, Mikalsen AB, Grimholt U, Gjoen T, Press CM (2010) In situ localisation of major histocompatibility complex class I and class II and CD8 positive cells in infectious salmon anaemia virus (ISAV)-infected Atlantic salmon. Fish Shellfish Immunol 28:30–39CrossRefGoogle Scholar
  13. Julius MH, Simpson E, Herzenberg LA (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur J Immunol 3:645–649CrossRefGoogle Scholar
  14. Laing KJ, Hansen JD (2011) Fish T cells: recent advances through genomics. Dev Comp Immunol 35:1282–1295CrossRefGoogle Scholar
  15. Laing KJ, Dutton S, Hansen JD (2007) Molecular and biochemical analysis of rainbow trout LCK suggests a conserved mechanism for T-cell signaling in gnathostomes. Mol Immunol 44:2737–2748CrossRefGoogle Scholar
  16. Lopez DM, Sigel MM, Lee JC (1994) Phylogenetic studies on T cells 1. Lymphocytes of shark with differential response to PHA and ConA. Cell Immunol 10:287–292CrossRefGoogle Scholar
  17. Manning MJ (1994) Fishes. In: Turner RJ (ed) Immunology: a comparative approach. Wiley, Chichester, pp 68–100Google Scholar
  18. Miller NW, Deuter A, Clem LW (1986) Phylogeny of lymphocyte heterogeneity the cellular requirements for the mixed leucocyte reaction with channel catfish. Immunology 59:123–128Google Scholar
  19. Moonsom S, Khunkeawla P, Kasinrerk W (2001) Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein. Immunol Lett 76:25–31CrossRefGoogle Scholar
  20. Nakanishi T, Fischer U, Dijkstra JM, Hasegawa S, Somamoto T, Okamoto N, Ototake M (2002) Cytotoxic T cell function in fish. Dev Comp Immunol 26:131–139CrossRefGoogle Scholar
  21. Nishimura H, Akamatsu N, Ikemoto M, Kawai K, Miyazawa H, Fujimoto S, Kusuda R (1995) Monoclonal antibody against yellowtail thymic lymphocytes recognizing a lymphocytic subpopulation. Fish Sci 61:181–185Google Scholar
  22. Nishimura H, Ikemoto M, Kawai K, Kusuda R (1997) Cross-reactivity of anti-yellowtail thymic lymphocyte monoclonal antibody (YeT-2) with lymphocytes from other fish species. Arch Histol Cytol 60:113–119CrossRefGoogle Scholar
  23. Partula S (1999) Surface markers of fish T cells. Fish Shellfish Immunol 9:241–257CrossRefGoogle Scholar
  24. Passer BJ, Chen CH, Miller N, Cooper MD (1996) Identification of a T lineage antigen in the catfish. Dev Comp Immunol 20:441–450CrossRefGoogle Scholar
  25. Polak JM, Noorden SV (2003) Introduction to immunocytochemistry. BIOS Scientific Publishers Ltd., OxfordGoogle Scholar
  26. Rathore G, Kumar G, Sood N, Kapoor D, Lakra WS (2008) Development of monoclonal antibodies to rohu [Labeo rohita] immunoglobulins for use in immunoassay. Fish Shellfish Immunol 25:761–774CrossRefGoogle Scholar
  27. Rebello SC, Rathore G, Punia P, Sood N, Elangovan V (2014) Development and characterization of a continuous macrophage cell line, LRTM, derived from thymus of Labeo rohita (Hamilton 1822). In Vitro Cell Dev Biol Anim 50:22–38CrossRefGoogle Scholar
  28. Rombout JHWM, van de Wal JW, Companjen A, Taverne N, Taverne-Thiele JJ (1997) Characterization of a T cell lineage marker in carp (Cyprinus carpio L.). Dev Comp Immunol 21:35–46CrossRefGoogle Scholar
  29. Rombout JH, Joosten PH, Engelsma MY, Vos AP, Taverne N, Taverne-Thiele JJ (1998) Indications for a distinct putative T cell population in mucosal tissue of carp (Cyprinus carpio L.). Dev Comp Immunol 22:63–77CrossRefGoogle Scholar
  30. Rowley AF, Hunt TC, Page M, Mainwaring G (1988) Fish. In: Rowley AF, Ratcliffe NA (eds) Vertebrate blood cells. University Press, Cambridge, pp 18–127Google Scholar
  31. Scapigliati G, Mazzini M, Mastrolia L, Romano N, Abelli L (1995) Production and characterisation of a monoclonal antibody against the thymocytes of the sea bass Dicentrarchus labrax (L.) (Teleostea, Percicthydae). Fish Shellfish Immunol 5:393–405CrossRefGoogle Scholar
  32. Scapigliati G, Scalia D, Marras A, Meloni S, Mazzini M (1999) Immunoglobulin levels in the teleost sea bass Dicentrarchus labrax (L.) in relation to age, season, and water oxygenation. Aquaculture 174:207–212CrossRefGoogle Scholar
  33. Secombes CJ, Van Groningen JJM, Van Muiswinkel WB, Egberts E (1983) Ontogeny of the immune system in carp (Cyprinus carpio L.). The appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocytes monoclonal antibodies. Dev Comp Immunol 7:455–464CrossRefGoogle Scholar
  34. Secombes CJ, Hardie LJ, Daniels G (1996) Cytokines in fish: an update. Fish Shellfish Immunol 6:291–304CrossRefGoogle Scholar
  35. Shibasaki Y, Toda H, Kobayashi I, Moritomo T, Nakanishi T (2010) Kinetics of CD4 + and CD8alpha + T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii. Dev Comp Immunol 34:1075–1081CrossRefGoogle Scholar
  36. Talwar PK, Jhingran AG (1991) Inland fishes of India and adjacent countries, vol 1. Oxford & IBH Publishing Co. Pvt. Ltd, New DelhiGoogle Scholar
  37. Timmusk S, Jansson E, Pilstrom L (2003) The generation of monoclonal antibodiesby genetic immunisation: antibodies against trout TCRalpha and IgL isotypes. Fish Shellfish Immunol 14:187–206CrossRefGoogle Scholar
  38. Toda H, Shibasaki Y, Koike T, Ohtani M, Takizawa F, Ototake M, Moritomo T, Nakanishi T (2009) Alloantigen-specific killing is mediated by CD8-positive T cells in fish. Dev Comp Immunol 33:646–652CrossRefGoogle Scholar
  39. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRefGoogle Scholar
  40. Yamaguchi K, Kodama H, Miyoshi M, Nishi J, Mukamoto M, Baba T (1996) Inhibition of cytotoxic activity of carp lymphocytes (Cyprinus carpio) by anti-thymocyte monoclonal antibodies. Vet Immunol Immunopathol 51:211–221CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Sanjay C. Rebello
    • 1
  • Gaurav Rathore
    • 2
  • Peyush Punia
    • 1
    Email author
  • Neeraj Sood
    • 1
  1. 1.National Bureau of Fish Genetic ResourcesLucknowIndia
  2. 2.Central Institute of Fisheries EducationMumbaiIndia

Personalised recommendations