Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies
Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies
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DOI：
10.1128/jcm.44.5.1665-1673.2006
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发表时间：
2006-05-01
影响因子：
9.4
通讯作者：
Beeson, James G.
中科院分区：
文献类型：
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作者：
Persson, Kristina E. M.;Lee, Chee T.;Beeson, James G.
抑制恶性疟原虫在红细胞中复制的抗体被认为在对疟疾的获得性免疫中以及作为由候选血液阶段疫苗产生的免疫的介质两者中都是重要的。然而，一些限制因素限制了这些功能性抗体在人群研究和疫苗试验中的研究。我们报告的发展和优化高通量生长抑制试验，提高灵敏度，使用最小体积的测试血清。暴露供体血清的主要抑制活性是抗体介导的，但在未处理的血清中发现非特异性抑制因子。培养体积可以有效地减少到25 μ l，以限制试验中使用的试验血清或抑制剂的量。在两个循环的寄生虫复制进行抑制试验，得到了更大的灵敏度比单循环试验，它简单的两个循环抑制试验的开发，产生高度可重复的结果。通过流式细胞术测定寄生虫生长最适合于使用小培养体积的高通量测定，并且比寄生虫乳酸脱氢酶测定更敏感，并且比显微镜更不易出现错误和变化。我们评估并优化了从血清中去除抗疟药物和非特异性抑制因子的方法，这些方法适合用于通常从临床研究中获得的小体积样本。微透析法和硫酸铵沉淀法纯化免疫球蛋白是有效和实用的。这些方法应有助于评价疫苗试验和免疫临床研究，也适用于测试药物和其他化合物的抗疟活性。
Antibodies that inhibit replication of Plasmodium falciparum in erythrocytes are thought to be important both in acquired immunity to malaria and as mediators of immunity generated by candidate blood-stage vaccines. However, several constraints have limited the study of these functional antibodies in population studies and vaccine trials. We report the development and optimization of high-throughput growth inhibition assays with improved sensitivity, that use minimal volumes of test serum. The major inhibitory activity of serum from exposed donors was antibody mediated, but nonspecific inhibitory factors were found in untreated serum. Culture volumes could be effectively reduced to 25 mu l to limit amounts of test serum or inhibitors used in assays. Performing inhibition assays over two cycles of parasite replication gave greater sensitivity than single-cycle assays, and it simple two-cycle inhibition assay was developed that yielded highly reproducible results. Determination of parasite growth by How cytometry was most suitable for high-throughput assay's using small culture volumes and was more sensitive than parasite lactate dehydrogenase assays and less prone to error and variation than microscopy. We evaluated and optimized methods to remove antimalarials and nonspecific inhibitory factors front serum that are suitable for use with small volumes of samples that are typically obtained from clinical studies. Both microdialysis and immunoglobulin purification by ammonium sulfate precipitation were effective and practical. These methods should facilitate evaluation of vaccine trials and clinical studies of immunity and are also suitable for testing drugs and other compounds for antimalarial activity.