Arrowood M J, Hurd M R, Mead J R
Parasitic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30341-3724, USA.
J Parasitol. 1995 Jun;81(3):404-9.
A flow cytometric method for the quantification of Cryptosporidium parvum oocysts in stool specimens was developed to replace conventional microscopic immunofluorescent assays. Fecal pellets were collected from control (uninfected) severe combined immune-deficient mice, suspended in 2.5% potassium dichromate at a ratio of 400 microliter per pellet, and homogenized by vortexing. Purified oocytes were added to the samples (10(5), 10(4), 10(3), and 10(2)/ml). Aliquots (200 microliters) of the vortexed samples were centrifuged over microscale discontinuous sucrose gradients. The oocyst-containing fractions were collected, washed, and incubated with an oocyst-specific monoclonal antibody (labeled with fluorescein isothiocyanate) for 30 min at 37 degrees C. Sample volumes were adjusted to 600 microliters with phosphate-buffered saline and assayed by using logical gating of forward/side scatter and fluorescence signal on a flow cytometer. Seeded samples showed a linear correlation with the number of oocysts recovered from the gradients. Analyses of stool samples from chronically infected mice demonstrated that the flow cytometry method was approximately 10 times more sensitive than conventional immunofluorescent assays.
开发了一种用于定量粪便标本中小隐孢子虫卵囊的流式细胞术方法，以取代传统的显微免疫荧光测定法。从对照（未感染）重度联合免疫缺陷小鼠收集粪便颗粒，以每颗粒400微升的比例悬浮于2.5%重铬酸钾中，并通过涡旋进行匀浆。将纯化的卵囊添加到样品中（10⁵、10⁴、10³和10²/ml）。将涡旋后的样品等分试样（200微升）在微型不连续蔗糖梯度上进行离心。收集含卵囊的级分，洗涤，并与卵囊特异性单克隆抗体（用异硫氰酸荧光素标记）在37℃孵育30分钟。用磷酸盐缓冲盐水将样品体积调整至600微升，并在流式细胞仪上通过前向/侧向散射和荧光信号的逻辑门控进行测定。接种的样品与从梯度中回收的卵囊数量呈线性相关。对慢性感染小鼠粪便样本的分析表明，流式细胞术方法的灵敏度比传统免疫荧光测定法高约10倍。
