Trang Dai Thi Xuan, Huy Nguyen Tien, Kariu Tohru, Tajima Kunihiko, Kamei Kaeko
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Malar J. 2004 Mar 17;3:7. doi: 10.1186/1475-2875-3-7.
Isolation of a concentrated, living preparation of malarial parasite-infected red blood cells (PRBCs) that have low contamination of white blood cells (WBCs) facilitates research on the molecular, biochemical and immunological aspects of malarial parasites. This is currently carried out by a two-step method, including the concentration of PRBCs using density gradient centrifugation through Percoll or Nycodenz, followed by the removal of host WBCs using a cellulose powder column or a commercially available filtration unit. These two-step methods can help isolate sufficient PRBCs, but they are laborious. In this study, a simplified one-step procedure that takes advantage of the difference between diamagnetic low-spin oxyhaemoglobin and paramagnetic haemozoin (haem polymer) was described. The paramagnetic polymer is deposited in the food vacuoles of the parasite, allowing the use of magnetic separation to efficiently and rapidly concentrate PRBCs while removing contaminating host WBCs.
The magnetic removal of WBCs using a commercial LD column (MACS) was evaluated as a new method for concentrating and purifying PRBCs. To compare this method with the two density gradient centrifugation methods using Percoll or Nycodenz, we analysed the quantities of enriched PRBCs and contaminating host WBCs as well as the viability of malarial parasites in the final preparations.
The quantity of PRBCs and the viability of malarial parasites in the isolated PRBCs were similar between magnetic and centrifugation methods. However, 90-99% of the contaminating WBCs were removed from the starting material using a magnetic column, whereas WBC content did not change using the Percoll or Nycodenz methods.
The use of a commercially available magnetic LD column is effective, safe and easy for the one-step purification of PRBCs. This simple method does not affect the viability of malarial parasites.
分离出白细胞（WBC）污染低的浓缩活疟原虫感染红细胞（PRBC）制剂，有助于对疟原虫的分子、生化和免疫学方面进行研究。目前这是通过两步法进行的，包括使用通过Percoll或Nycodenz的密度梯度离心法浓缩PRBC，随后使用纤维素粉末柱或市售过滤装置去除宿主WBC。这两步法有助于分离出足够的PRBC，但操作繁琐。在本研究中，描述了一种利用抗磁性低自旋氧合血红蛋白和顺磁性疟色素（血红素聚合物）之间差异的简化一步法程序。顺磁性聚合物沉积在寄生虫的食物泡中，从而可以利用磁分离高效快速地浓缩PRBC，同时去除污染的宿主WBC。
评估使用商用LD柱（MACS）磁去除WBC作为浓缩和纯化PRBC的新方法。为了将该方法与使用Percoll或Nycodenz的两种密度梯度离心法进行比较，我们分析了最终制剂中富集的PRBC数量、污染的宿主WBC数量以及疟原虫的活力。
磁分离法和离心法分离出的PRBC中PRBC数量和疟原虫活力相似。然而，使用磁柱从起始材料中去除了90 - 99%的污染WBC，而使用Percoll或Nycodenz方法时WBC含量没有变化。
使用市售磁LD柱对PRBC进行一步纯化有效、安全且简便。这种简单方法不影响疟原虫的活力。
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