An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.
An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.
复制标题
DOI：
10.1186/1475-2875-10-95
复制
发表时间：
2011-04-18
期刊：
影响因子：
3
通讯作者：
Trenholme KR
中科院分区：
文献类型：
--
作者：
Butterworth AS;Robertson AJ;Ho MF;Gatton ML;McCarthy JS;Trenholme KR
疟疾研究中的许多技术都需要获得单个寄生虫克隆。对寄生虫的生长进行基于显微镜的评估，通过限制稀释进行克隆是一个艰巨而劳动密集型的过程。在限制稀释法中检测寄生虫生长的另一种方法是使用商品化的富含组氨酸蛋白II(HRP2)检测试剂盒。用HRP2-EL ISA检测寄生虫的生长，并与厚膜显微镜进行比较。HRP2蛋白标准被用来确定HRP2酶联免疫吸附试验的检测阈值，而HRP2释放模型被用来推断阳性结果所需的寄生虫生长量。在检测寄生虫生长方面，HRP2酶联免疫吸附试验比镜检更敏感。该方法的最低检测水平为0.11 ng/ml。对HRP2释放的模拟结果表明，需要2,116个寄生虫才能完成一个完整的红细胞周期，才能产生足够的HRP2以供ELISA法检测。在标准的培养条件下，寄生虫的数量很可能在培养的8至14天之间达到。该方法为限制稀释法检测寄生虫的生长提供了一种准确、简便的方法，减少了传统方法所需的时间和资源。此外，该方法使用废弃的培养基来代替感染寄生虫的红细胞，使培养能够继续进行。
Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.
登录
查看更多内容
影响因子：
3
作者：
Maher SP;Balu B;Shoue DA;Weissenbach ME;Adams JH
通讯作者：
Adams JH
影响因子：
4.9
作者：
Anderson, TJC;Nair, S;Nosten, F
通讯作者：
Nosten, F
影响因子：
56.9
作者：
ROSARIO, V
通讯作者：
ROSARIO, V
影响因子：
2.1
作者：
Goodyer, ID;Taraschi, TF
通讯作者：
Taraschi, TF
DOI：
10.1016/j.trstmh.2004.11.014
发表时间：
2005-07-01
影响因子：
2.2
作者：
Desakorn, V;Dondorp, AM;White, NJ
通讯作者：
White, NJ