Study on purification and immunoreactivity of erythrocytic stage Plasmodium vivax

Objective: To establish a method of concentration and purification of erythrocytic stage Plasmodium vivax(P.v) by using combined WBC filtration and Percoll density centrifugation,and compare and analyze the immunoreactivity difference between infected RBC(iRBC) and normal RBC(nRBC) antigens prepared by saponin lysis and freeze-thawing by using pooled sera from P.v patients and normal controls.Methods: Leukocytes were removed by Plasmodipur filter from P.v patients' blood,iRBC was concentrated by 60% Percoll density gradient.Parasites were released by two methods:saponin lysis or freeze-thawing from iRBC.After sonication,P.v and nRBC soluble antigens which were prepared by the same method were compared by SDS-PAGE.Specific P.v antigen bands were defined by immunoblotting,pooled sera from P.v patients and normal controls were used as primary antibody to recognize P.v and nRBC antigen strip.Results: Ninety-nine of 115(86.1%) P.v infected blood samples filtrated by Plasmodipur filter to remove leukocytes and retained RBC reached an acceptable level(≤ 5/10 oil immersion field).Thirty-five WBC free samples were concentrated by 60% Percoll,and the iRBC percentage of 30 cases were raised over 60%.By SDS-PAGE analysis,there were 6 and 2 specific bands found in saponin lysis and freeze-thawing treatment P.v soluble antigens respectively.Immunoblotting analysis were showed that pooled P.v patients' sera can specifically recognized 22,24.5,29,35,36 kDa bands from saponin lysis treatment and 26,49,59,63,115,120 kDa bands from freeze-thawing treatment P.v soluble antigens.Combined using of Plasmodipur filter and 60% Percoll demonstrated of high efficiency in removing WBC and concentrating iRBC from P.v infected blood.Conclusions: There was an significant immunoreactivity difference between P.v soluble antigens prepared by saponin lysis and freeze-thawing treatment through using pooled P.v patients' sera.Twenty-two kDa P.v antigen prepared by saponin lysis treatment showed high immunoreactivity,it needs further study to evaluate its diagnostic usage.