Tritten Lucienne, Matile Hugues, Brun Reto, Wittlin Sergio
Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland. lucienne.tritten@unibas
Malar J. 2009 Oct 12;8:226. doi: 10.1186/1475-2875-8-226.
The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The method is highly reproducible, however, the handling of radioactive material is costly, hazardous and requires the availability of appropriate technology and trained staff. Several other ways to evaluate in vitro anti-malarial activity do exist, all with their own assets and limitations.
The newly developed double-antibody sandwich ELISA described here is based on the properties of a non-overlapping pair of monoclonal antibodies directed against Plasmodium falciparum aldolase. This glycolytic enzyme possesses some unique nucleotide sequences compared to the human isoenzymes and has been highly conserved through evolution. Out of twenty possibilities, the most sensitive antibody pair was selected and used to quantitatively detect parasite aldolase in infected blood lysates.
A total of 34 compounds with anti-malarial activity were tested side-by-side by ELISA and the [3H]hypoxanthine incorporation assay. The novel ELISA provided IC 50s closely paralleling those from the radioactivity-based assay (R = 0.99, p < 0.001). At the investigated assay conditions (72 h incubation time, parasitaemia = 0.3%), the assay was found to be reproducible and easy to perform.
The newly developed ELISA presents several advantages over the comparative method, the [3H]hypoxanthine incorporation assay. The assay is highly reproducible, less hazardous (involves no radioactivity) and requires little and cheap technical equipment. Relatively unskilled personnel can conduct this user-friendly assay. All this makes it attractive to be employed in resource-poor laboratories.
评估化合物抗疟活性的标准体外试验是[3H]次黄嘌呤掺入试验。这是一种基于放射性的方法，用于测量疟原虫在红细胞中的DNA复制。该方法具有高度可重复性，然而，放射性物质的处理成本高、危险性大，并且需要合适的技术和经过培训的人员。确实存在其他几种评估体外抗疟活性的方法，它们都有各自的优点和局限性。
本文所述新开发的双抗体夹心酶联免疫吸附测定（ELISA）基于一对针对恶性疟原虫醛缩酶的不重叠单克隆抗体的特性。与人类同工酶相比，这种糖酵解酶具有一些独特的核苷酸序列，并且在进化过程中高度保守。从二十种可能性中，选择了最敏感的抗体对，并用于定量检测感染血液裂解物中的寄生虫醛缩酶。
通过ELISA和[3H]次黄嘌呤掺入试验对总共34种具有抗疟活性的化合物进行了并行测试。新型ELISA提供的半数抑制浓度（IC50）与基于放射性的试验结果密切平行（R = 0.99，p < 0.001）。在研究的试验条件下（孵育时间72小时，疟原虫血症 = 0.3%），该试验具有可重复性且易于操作。
新开发的ELISA与比较方法[3H]次黄嘌呤掺入试验相比具有几个优点。该试验具有高度可重复性，危险性较小（不涉及放射性），并且所需的技术设备少且成本低。相对不熟练的人员也可以进行这种用户友好型试验。所有这些使得它在资源匮乏的实验室中具有吸引力。
