Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2

Abstract

Background: Several methods such as SDS-PAGE analysis are frequently used for monitoring the recombinant proteins during the expression and purification. As monoclonal antibodies are extremely specific, they can be used in association with Western blotting for confirming the expression of MSP-2 proteins of different domains and also to assess their quality and purity. Methods: Mice were immunized with a schizont extract, to stimulate the immune system to make antibodies against different antigens of the late stage parasite, including the production of antibodies against different domains of P. falciparum MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. Results: Overall from all 7 fusions performed 243 clones were grown in 96 wells plates as they were detected positive against crude schizont extract and only 10 clones were finally detected positive against different domains of the MSP-2 recombinant protein after sub-cloning in tissues culture flasks and before freezing. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results showed that, different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera. Conclusion: Bringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.