Duque-Beltrán Sofía, Nicholls-Orejuela Rubén Santiago, Arévalo-Jamaica Adriana, Guerrero-Lozano Rafael, Montenegro Sonia, James Mark A
Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, D.C., Colombia.
Mem Inst Oswaldo Cruz. 2002 Dec;97(8):1165-8. doi: 10.1590/s0074-02762002000800018. Epub 2003 Jan 20.
The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 g/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.
本研究开发并标准化了一种酶联免疫吸附测定（ELISA）法，使用兔多克隆抗体检测粪便中的贾第虫抗原。通过蔗糖和 Percoll 梯度从人类粪便样本中纯化贾第虫包囊。用蒙古沙鼠（Meriones unguiculatus）感染以获得滋养体。用 14 株哥伦比亚贾第虫分离株的包囊或滋养体抗原接种兔子，以产生针对相应阶段的抗体。通过辛酸和硫酸铵分步沉淀法纯化抗贾第虫 IgG。将这些多克隆抗体的一部分与碱性磷酸酶（偶联物）连接。对 196 份来自不同患者的人类粪便样本进行寄生虫学诊断检测：69 份贾第虫包囊呈阳性，56 份未发现贾第虫寄生虫，71 份显示有除贾第虫以外的寄生虫。用于抗原捕获的多克隆抗体的最佳浓度为 40 μg/ml，最佳偶联物稀释度为 1:100。吸光度临界值为 0.24。检测贾第虫抗原的 ELISA 试验参数为：灵敏度 100%（95%CI：93.4 - 100%）；特异性 95%（95%CI：88.6 - 97.6%）；阳性预测值 91%（95%CI：81.4 - 95.9%）；阴性预测值 100%（95%CI：96.1 - 100%）。这种 ELISA 将改善哥伦比亚贾第虫感染的诊断，并有助于治疗后对患者的随访。
