Bosompem K M, Assoku R K, Nantulya V M
Noguchi Memorial Institute for Medical Research (NMIMR), Accra, Ghana.
J Immunol Methods. 1995 Nov 16;187(1):23-31. doi: 10.1016/0022-1759(95)00163-5.
Gut samples prepared from laboratory-reared tsetse flies and applied in dots onto nitrocellulose (NC) membrane were found to stain the membrane with differing coloration and intensity. The stains were, predominantly, either reddish to brown or blackish-brown to black and occasionally greenish to almost colourless, depending on the stage of digestion of the bloodmeal in the fly. NC membrane strips applied with tsetse gut samples from T. brucei infected and uninfected control flies were tested with the standard antigen detection dot enzyme-linked immunoassay (dot-ELISA), using a T. brucei specific monoclonal antibody (MoAb) and horseradish peroxidase goat anti-mouse conjugate. The stains in both infected and uninfected sample dots persisted through the assay. Furthermore, the staining intensity of some assayed uninfected sample dots were enhanced as a result of non-specific reactivity, making it difficult to distinguish between the infected and uninfected flies. This necessitated the development of a simple technique by which the non-specific stains and reactions could be removed. Sample 'dotted' NC membrane strips were destained by incubation with 5% hydrogen peroxide (H2O2) diluted in 5% skimmed milk in Tris buffer, pH 8.0. After washing, the destained strips were tested in the dot-ELISA. This method gave satisfactory reproducible results, since the most intense stains could be removed, and it had no effect on trypanosome antigens detected by a panel of four T. brucei species-specific, three T. vivax species-specific, four T. congolense species-specific and four Nannomonas subgenus-specific MoAbs. Using the destaining process in a modified dot-ELISA, 86 out of 95 (90.5%) of Glossina morsitans centralis flies experimentally infected with T. brucei, were identified. The destaining method was also used successfully to decolorize NC membrane bound tsetse faecal material.
从实验室饲养的采采蝇制备的肠道样本，点涂在硝酸纤维素（NC）膜上后，发现膜上会出现不同颜色和强度的染色。根据采采蝇中血餐的消化阶段，染色主要为红棕色至棕色或黑棕色至黑色，偶尔为绿色至几乎无色。使用布氏锥虫特异性单克隆抗体（MoAb）和辣根过氧化物酶山羊抗小鼠偶联物，通过标准抗原检测斑点酶联免疫吸附测定（斑点ELISA），对涂有感染布氏锥虫的采采蝇和未感染对照采采蝇肠道样本的NC膜条进行检测。在整个检测过程中，感染和未感染样本点的染色都持续存在。此外，一些检测的未感染样本点由于非特异性反应，染色强度增强，使得难以区分感染和未感染的采采蝇。这就需要开发一种简单的技术来去除非特异性染色和反应。将点涂有样本的NC膜条与在pH 8.0的Tris缓冲液中用5%脱脂牛奶稀释的5%过氧化氢（H2O2）孵育进行脱色。洗涤后，对脱色后的膜条进行斑点ELISA检测。该方法给出了令人满意的可重复结果，因为最强的染色可以被去除，并且对由一组四种布氏锥虫物种特异性、三种活泼锥虫物种特异性、四种刚果锥虫物种特异性和四种小无鞭毛体亚属特异性MoAb检测的锥虫抗原没有影响。在改良的斑点ELISA中使用脱色过程，在实验感染布氏锥虫的95只中，有86只（90.5%）的莫氏采采蝇被鉴定出来。脱色方法也成功用于使NC膜结合的采采蝇粪便物质脱色。
