CRYOPRESERVATION OF PRIMARY SPLENOCYTES FROM ANTIBODY PRODUCED MICE AGAINST Bitis arientans SNAKE VENOM
Abstract
Bitis Arientans are associated with the highest number of morbidity and mortality in Nigeria.The most effective treatments against snakebite is the administration of antivenom.Antibodies that is specific for a single epitope of an antigen are obtain by isolating antibody-secreting lymphocytes. These lymphocytes are found within the Splenocytes cells which can be used for a wide variety of immunology-based applications. However, development of hybridoma cell is rare and time consuming. Therefore, valuable time and cell materials can be saved through cryopreservation. This research aimed at Cryopreservation of primary splenocytes from Antibody produced mice against Bitis arientans snake venom. The LD50 of Bitis Arientans venom toxicity was determined in mice according to World Health Organization guidelines. Antibody production was achieved using six mice after immunization for six weeks and antibody titer were determined by indirect ELISA method. Myeloma cells line X63 Ag8.653 were cultured and Mouse Splenocytes with the highest immune response were removed aseptically by mechanical method and cells viability was determined then the isolated splenocytes were cryopreserved in cryovial tube stored at -80C.The LD50 was found to be 1.8mg/kg and Elisa analysis showed mice 2 and 6 to elicited highest immune response with IgG Concentration 3.1µg/ml and 4.6µg/ml. isolated splenocytes were counted to be 6.5x10 and 1.2x10 cells for mice 2 and 6 and myeloma cells to be 1.95x105 cells.In this finding antibody against Bitis Arientans venom were produced and mouse splenocytes were isolated and cryopreserved. Thus, cryopreserved splenocytes and myeloma can be used for the generation of monoclonal antibody
References
Ascoli, C.A. and Aggeler, B. (2018) Overlooked Benefits of Using Polyclonal Antibodies, Biotechniques 65 (3) 127–136.
Barral-netto, M., Schriefer, A., Ainhas, V. And Almeida, AR. (1990). Enzyme-linked immunosorbent Assay for the Detection of Bothrops jararaca venom. Toxicon, 28, 1053-61.
Cavender B.J., Holbrook, N.M. and Zwieniecki, M.A. (2005). Impacts of Freezing on Long Distance Transport in Woody Plants", Vascular Transport in Plants, Physiological Ecology, Burlington: Academic Press, pp. 401–424
Chippaux, J.P. (2017). Snakebite Envenomation Turns Again Into A Neglected Tropical Disease! Journal of Venom Animal. Toxin incl. Tropical Disease, 23(5): 1–2.
Colo, G., Chacur, M., Gutierrez, J.M., Teixeira, C.F. And Cury, Y. (2002). Evaluation of Antivenoms in the Neutralization of Hyperalgesia and Edema Induced by Bothrops jararaca and Bothrops Asper Snake Venoms. Braz. Journal of Medical Biology. 35, 1221–1228.
Daltry, J. C., Ponnudurai, G., Shin, C. K., Tan, N. H., Thorpe, R. S. And Wuster, W. (1996a). Electrophoretic Profiles and Biological activities: Intraspecific Variation in the Venom of the Malayan Pitviper (Calloselasma rhodostoma). Toxicon, 34, 67-79
Fawzi O. E., Abdul M.A., Adam A.E., Fadella, A. and El Meshri S.E. (2020). Isolation of primary mice splenocytes for in vitro research. Alexandria journal of veterinary sciences. Vol. 64 (2): 9-12. Jan 2020
Gutiérrez, J.M. Calvete, J.J. Habib, A.G. Harrison, R.A. Williams, D.J. and Warrell, D.A. (2017). Snakebite envenoming. Transactions of the Royal Society of Tropical Medicine and Hygiene 3, 70-79.
Habib, A.G. (2013). Public health aspects of snakebite care in West Africa: perspective from Nigreia. Journal Venomous Animal and Toxin including tropical disease. 19:27
Habib, A.G. Brown, N.I. (2018). The Snakebite Problem and Antivenom Crisis from a Health Inventory and Spatial Analysis. Lancet Glob Heal 6, 342–50
Hutz. R.J., DeMayo, F.J. and Dukelow, W.R. (1985).The use of vital dyes to assess embryonic viability in the hamster, Mesocricetus Auratu. Stain Technol. 60(3):163-167
Ibrahim, S., Ibrahim, H., Muge, S., Safak, I.C., Deniz, D. and Aynur, B. (2011) cryopreservation of spleen and lymph nodes as a source of mononuclear cells to be used for the development of monoclonal antibody producing hybridoma cells. CryoLetters 32 (3), 266-274
Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L & Hovatta O (2005) Human Reproduction 20, 1676–1687.
Mallow, D., Ludwig, D. and Nilson, G. (2003). True Vipers: Natural History and Toxinology of Old World Vipers. Krieger Publishing Company, Malabar, Florida. 359
Nghorn, R., Persson, F., Åblad, B., Goddard, A., Schoeman, J.P., Willesen, J.L., Tarnow, I. And Kjelgaard-Hansen, M. (2014). Myocardial Injury in Dogs with Snake Envenomation and Its Relation to Systemic Inflammation. Journal Vetenary. Emergency Critical Care, 24, 174–181.
Pegg DE (2006) Cell Tissue Banking 7, 349–358
Spawls S, Howell K, Drewes R, Ashe J. (2004). A Field Guide to the Reptiles of East Africa. A & C Black Publishers Limited. London. 543.
Tabll, A., Abbas, A.T., El-KTafrawy, S. and Wahid, A. Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus. World J Hepatol. 2015. 7(22): 2369–2383
Tini, M., Jewell, U.R., Camenisch, G., Chilov. D. and Gassmann, M. (2002). "Generation and Application of Chicken Egg-yolk antibodies. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology. 131 (3): 569–74
Wang, J. J., Qiu, L., Fernandez, R., Yeap, X. Y., Lin, C. X. and Zhang, Z. J. (2019). A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity. Journal of Science.8 (4) 34-45.
World Health Organization, (2010). Regional Office for Africa. Guidelines for the prevention and Clinical Management of Snakebite in Africa. World Health Organization. Regional Office for Africa.
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