Accoceberry I, Thellier M, Datry A, Desportes-Livage I, Biligui S, Danis M, Santarelli X
Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitaire de Bordeaux, Hôpital Saint André, 33075 Bordeaux Cedex, France.
J Clin Microbiol. 2001 May;39(5):1947-51. doi: 10.1128/JCM.39.5.1947-1951.2001.
An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.
本文描述了一种从人粪便中纯化微小隐孢子虫孢子的原始、可靠且可重复的方法。我们最近报道了一种针对微小隐孢子虫孢子外壁产生的种特异性单克隆抗体（MAb）6E52D9免疫球蛋白G2a（IgG2a）。该单克隆抗体被用作配体来开发免疫亲和基质。小鼠IgG2a单克隆抗体直接与Streamline rProtein A吸附剂结合，该吸附剂用于免疫球蛋白的扩张床吸附，以实现抗体的最佳空间定向和抗原的最大结合效率。然后使用二盐酸二甲基庚二酸酯将该复合物共价交联。在用含有大量微小隐孢子虫孢子的过滤粪便样本孵育免疫亲和基质后，在用6 M盐酸胍洗脱之前，吸附剂床的膨胀消除了所有粪便污染物。通过间接免疫荧光抗体试验（IFAT）测定洗脱级分中孢子的存在情况。在所有四个实验的洗脱级分中均发现了微小隐孢子虫孢子，并且如IFAT所示，其仍具有高度抗原性。光学显微镜检查的涂片含有非常纯净的孢子，没有粪便残渣或背景细菌和真菌污染物。然而，孢子回收率相对较低：每次运行平均纯化10(7)个孢子。这种直接从人粪便样本中高度纯化分离微小隐孢子虫孢子的技术为研究这种寄生虫开辟了新途径。
