红内期间日疟原虫浓集纯化及免疫反应性研究

Study on purification and immunoreactivity of erythrocytic stage Plasmodium vivax

目的:建立白细胞滤器(Plasmodipur)联合Percoll密度梯度离心浓集纯化红内期间日疟原虫(P.v)的方法。分析和比较皂素与冻融两种处理方法制备的P.v抗原与P.v感染者混合血清免疫反应性的差别。方法:P.v感染者血样,用Plasmodipur滤器分离去除白细胞,经60%Percoll浓集其中的感染红细胞(iRBC)。采用皂素法和冻融法从iRBC中释放疟原虫,经超声粉碎,所获P.v可溶性抗原和同样处理的正常红细胞(nRBC)成分经SDS-PAGE电泳分析其组分差别,并应用P.v感染者、正常对照混合血清与相应的抗原进行免疫印迹分析,确定具有免疫原性的抗原组分。结果:115例P.v感染者血样,用Plasmodipur滤器分离去除白细胞,每10个油镜视野中WBC残留≤5个99例(86.1%),经60%Percoll浓集的35例样本中,共有30例(85.7%)能提高iRBC比例至60%以上。SDS-PAGE电泳分析,皂素处理与冻融处理的P.v抗原分别显示出6条和2条P.v特异性蛋白条带。免疫印迹分析表明,P.v感染者混合血清能特异性地识别22、24.5、29、35、36 kDa皂素处理的P.v抗原,26、49、59、63、115、120 kDa冻融处理的P.v抗原。结论:血样中疟原虫皂素与冻融两种处理方法制备的P.v抗原与P.v感染者混合血清免疫反应性存在明显差异,其中皂素处理的22 kDa抗原组分具有较强的免疫原性,其作为P.v特异性诊断抗原的价值有待进一步研究。

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% Pereoll 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% Pereoll demonstrated of high etficiency 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.