Lymphocytes from 9 Malaria immune donors were isolated  using Ficoll-hypaque gradient centrifugation. The peripheral blood mononuclear cells were then used for the immortalization of B cells by using Epstein Barr Virus. Supernatants from successfully immortalized B cells were used to detect total IgG and IgM. Schizont extract from FCR3S1.2 strain was obtained by using the Magnetic activated cell sorting method. 

Before the process of immortalization of B cells by EBV, the plasma samples from both the immune donors and non immune donor were tested against Plasmodium falciparum schizont extract to detect any anti-P. falciparum antibodies using ELISA test. Results from this analysis confirmed exposure of immune donors to P.falciparum parasite and increased chances of obtaining circulating anti-malaria B cells.

Two methods were used to isolate malaria specific B cells:

Method 1 involved isolating infected RBC (iRBC) surface reactive B cells- Schizont extract from FCR3S1.2 strain was incubated with immortalized B cells then purified by Magnetic activated cell sorting method (MACS).

Method 2 involved isolating total malaria antigen specific B cells- Labeled parasite antigen-DSB-X biotin with immortalized B cells were incubated then Dynabeads Streptavidin was used for isolation.

Combinations of cytokines IL2,IL6,IL10,IL15 and IL21 were used to stimulate the parent cell lines and Malaria specific B cells.

ELISA test was done to detect Plasmodium falciparum specific antibodies.