Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.Methods:Mice were immunized with a schizont extract to stimulate the immune system...Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.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 production of antibodies against different domains of Plasmodium falciparum(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.The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains.The positive clones were expanded to large(75 cm^2)flask and cultured under the same conditions,checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.Results:A total number of 7 fusions including 26 plates(2496 wells)were performed,of which 1336 hybrids were produced and the overall efficiency(1336/24%×100)was about 53%.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 Ft hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test.Western blotting results also 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.Conclusions: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.展开更多
文摘Objective:To assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.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 production of antibodies against different domains of Plasmodium falciparum(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.The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains.The positive clones were expanded to large(75 cm^2)flask and cultured under the same conditions,checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.Results:A total number of 7 fusions including 26 plates(2496 wells)were performed,of which 1336 hybrids were produced and the overall efficiency(1336/24%×100)was about 53%.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 Ft hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test.Western blotting results also 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.Conclusions: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.
