F. Tobeyani, Dr. S. Milani, Dr. F. Yari,
Volume 19, Issue 2 (7-2022)
Abstract
Background and Objectives
in-vitro immunization is one of the common methods of immunization as a preliminary step in the production of Monoclonal Antibodies. In this method, peripheral blood mononuclear cells (PBMC) are isolated and incubated in culture medium with necessary factors for immunization. Antigen-specific B lymphocytes proliferate and differentiate into cells that secrete specific antibodies. In the current study, we aimed to identify various factors and contributing elements on B cell In-vitro immunization process for the generation of antibody against Kell blood group antigen.
Materials and Methods
In this experimental study, Kell antigen was purified from Kell+ whole blood. Peripheral blood mononuclear cells were isolated using a density gradient approach from Kell- whole blood bag. After exposure to L-Leucyl-L-leucine methyl ester (LLME), they were incubated with other required factors for immunization, including antigen (500-1000 ng/ml), adjuvant (5-20 mg/ml), and cytokines (9.5 pg/ml). After 7-11 days, cells supernatant was evaluated using a sensitive method, ELISA, for antibody production.
Results
It was observed that the most desirable culture condition for immunization and further production of specific anti-Kell antibody from mononuclear cells was 0.25 mM concentration of LLME employing 500-1000 ng of Kell antigens and 10 microliter MLC. In the mentioned condition, the optical density was read at 450 nm as 2.052 ± 0.03, which was the representative of production of specific anti-Kell antibody.
Conclusions
Based on the study, immunization in culture medium against Kell antigen can be done. By employing different factors, the production of anti-Kell producing cells in culture condition can be optimized.
