Moss D M, Arrowood M J
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 30341, USA.
J Parasitol. 2001 Apr;87(2):406-12. doi: 10.1645/0022-3395(2001)087[0406:QOCPOI]2.0.CO;2.
Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.
尽管已证明单色流式细胞术在定量微小隐孢子虫卵囊方面比荧光显微镜更灵敏，但该方法尚未得到优化。针对卵囊细胞壁的单克隆抗体OW50与超顺磁性颗粒、异硫氰酸荧光素和藻红蛋白偶联。然后用荧光染料标记的OW50对卵囊进行双重染色，并将其置于含有已知数量高荧光聚苯乙烯珠的试管中，从而可通过流式细胞术对卵囊进行定量，而无需依赖采集的样本体积。使用卵囊和珠子的逻辑门控进行双色流式细胞术得到的数据显示，纯化卵囊悬液的稀释度与检测到的卵囊平均数之间呈线性关系（r2 = 1.00）。在理论浓度为12个卵囊/毫升的稀释液中，平均计数到15个纯化卵囊/毫升。将已知数量的纯化卵囊接种到正常小鼠粪便标本中，用OW50标记的免疫磁性颗粒捕获，在低pH值下用5%重铬酸钾洗脱，并用荧光染料标记的OW50进行双重染色。通过流式细胞术，平均回收率为43.1%（±8.3%），检测到的卵囊数量少至133个。捕获并洗脱的卵囊对新生BALB/c小鼠具有感染性。这种双色流式细胞术方法与通过免疫磁性颗粒捕获和洗脱卵囊相结合，不仅为从不同来源定量和纯化微小隐孢子虫卵囊提供了强大工具，也为体外和体内卵囊的特性鉴定提供了有力手段。
