Hybridoma generation by in vitro immunization of murine splenocytes with cytosolic proteins of Chinese hamster ovary (CHO) mitotic cells.
Despite the potential uses of polyclonal antisera, monoclonal antibodies (MAbs) are preferred for target-specific applications. In vitro immunizations toward the development of hybridomas have become more advantageous in situations of limited antigen availability, small molecular size, and the duration required for the production of MAbs. Cells in the mitotic stage are distinct in their protein profiles compared to cells in the other stages of the cell cycle, and studying these proteins can give various insights into the mechanisms of cell cycles and the interventional scenario, such as mitotic inhibition. Murine splenocytes were immunized in vitro with protein extracts of Chinese Hamster Ovary mitotic cells, and healthy, secretory hybridomas were generated. A 10 day incubation post-immunization in serum-free conditions, 10:1 ratio of fusion partners, and limiting dilutions in the presence of serum, conditioning medium, and syngenic feeder cells resulted in the stable hybridoma clones secreting IgG antibodies. While cell ELISA assay indicated B cells in a population of murine splenocytes and the final antigen-specific secretory cells, double diffusion and ELISA resulted in the fusion and specific efficiencies of the protocols adopted. An antigenic concentration of 2.775 microg produced the maximum fusion efficiency while 3.7 microg of the antigen produced the best specific efficiency.
Ravi
Maddaly
M
Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, India. maddalyravi@hotmail.com
Sundar
Sukanya Shyama
SS
Kumar
Kaavya Krishna
KK
Parvathi
Deepa
D
Paul
Solomon F D
SF
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006825:Hybridomas / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis / Q000276:immunology*; D013154:Spleen / Q000166:cytology / Q000276:immunology* / Q000378:metabolism