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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 g/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. [ABSTRACT FROM AUTHOR]

Background and Objectives Alloimmunization is the most serious problem in thalassemia patients and Anti-K is the most prevalent antibody in these patients. So accurate identification of this antigen can significantly decrease the rate of alloimmunization. Serological phenotyping is usually not reliable in multitransfused patients. Molecular genotyping can overcome limitations of hemagglutination assays, resolve discrepant serologic typing and guide RBC selection for them. In this regard, we intended to determine the K and k among alloimmunized thalassemia patients using molecular methods and compare the results between different methods. Materials and Methods In this descriptive study, a total of 200 blood samples were collected randomly from alloimmunized thalassemia patients of Tehran Adult Thalassemia Clinic. The phenotype of all samples was determined for K and k. PCR-SSP was performed for all samples. The discrepant results between the phenotype and genotype were re-evaluated by PCR-RFLP and were confirmed by DNA sequencing. Results Sixty-three (28%) out of 200 patients developed Anti-K. Molecular typing of samples for K and k antigens revealed 96% (192 patients) KEL*02/KEL*02 and 4% (8 patients) KEL*01/KEL*02 among our samples. Discrepancy between the serology and genotyping results were detected in 8 cases that in all cases correction of genotype results was confirmed by DNA sequencing. Conclusions This study demonstrates that antibodies against K antigen continue to develop in thalassemia patients at high rate. Our findings suggest that RBC molecular genotyping is superior to serological phenotyping and is a good alternative and more reliable method especially in multi transfusion patients such as thalassemia patients. [ABSTRACT FROM AUTHOR]