Jensen H E, Halbaek B, Lind P, Krogh H V, Frandsen P L
Laboratory of Veterinary Pathology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
J Vet Diagn Invest. 1996 Jan;8(1):68-75. doi: 10.1177/104063879600800111.
Murine monoclonal antibodies (MAbs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured hybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immunosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous reactivity with immunohistochemical techniques using formalin-fixed, paraffin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs raised against A. fumigatus WSSA and WF were selected for a further evaluation of cross-reactivity (diagnostic specificity) in immunohistochemical and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fungal species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted strongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiospermum were also strongly labeled by MAb-WF-AF-3 and -4. The 2 specifically reacting MAbs (MAb-WF-AF-1 and -2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106-kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatus. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesions, to culture results. In the vast majority of lesions (n = 133), the MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted in a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that were not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics of the 2 MAbs (MAb-WF-AF-1 and -2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify their application for the in situ diagnosis of systemic aspergillosis of cattle.
通过将免疫的BALB/c小鼠的脾细胞与小鼠骨髓瘤X63-Ag 8.653细胞融合，制备了针对烟曲霉水溶性体细胞抗原（WSSA）和细胞壁组分（WF）的鼠单克隆抗体（MAb）。体外培养杂交瘤的上清液首先在酶联免疫吸附测定（ELISA）中筛选与烟曲霉的WSSA和WF以及其他真菌的WSSA的反应性。随后，使用来自实验感染小鼠的福尔马林固定、石蜡包埋组织，通过免疫组织化学技术筛选仅与烟曲霉抗原反应的上清液的同源和异源反应性。由于与同源真菌具有高免疫组织化学反应性，选择了4种针对烟曲霉WSSA和WF产生的MAb，用于在免疫组织化学和免疫印迹测定中进一步评估交叉反应性（诊断特异性）。在免疫组织化学测定中，所有针对WSSA产生的MAb与许多其他真菌物种发生强烈交叉反应。所有4种针对WF产生的MAb（MAb-WF-AF-1-4）与曲霉属的菌丝强烈反应；MAb-WF-AF-3和-4也强烈标记了尖端赛多孢的菌丝。2种特异性反应的MAb（MAb-WF-AF-1和-2）为IgM生物型且具有沉淀性，在免疫印迹实验中均与WF的106-kD抗原结合，而它们不与烟曲霉的WSSA结合。2种曲霉病特异性MAb之一（MAb-WF-AF-1）用于筛选145例牛的霉菌病变。将MAb-WF-AF-1对牛病变的诊断结果与基于与异源吸收多克隆抗体反应性的结果进行比较，对于一些病变，还与培养结果进行比较。在绝大多数病变（n = 133）中，MAb-WF-AF-1和多克隆抗曲霉抗体以相似模式反应，即在41例曲霉病病变中呈阳性，在92例接合菌病病变中呈阴性。12例未被多克隆抗体染色的病变中有3例的菌丝与特异性MAb-WF-AF-1反应；即诊断为曲霉病。在ELISA以及免疫印迹和免疫组织化学测定中，针对烟曲霉WF产生的2种MAb（MAb-WF-AF-1和-2）的特性证明了它们可用于牛全身性曲霉病的原位诊断。
