Jensen H E, Aalbaek B, Lind P, Krogh H V
Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Vet Pathol. 1996 Mar;33(2):176-83. doi: 10.1177/030098589603300207.
Murine monoclonal antibodies (Mabs) against water-soluble somatic antigens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopus oryzae) were produced in vitro by fusion of splenocytes from immunized BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants reacting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) revealed a high homologous reactivity on tissues and the cross-reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On tissues and on immunoblots all four Mabs reacted identically and specifically with members of the family Mucoraceae, i.e., Absidia corymbifera, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous antigens with molecular masses from I4 to 110 kDa. With WSSA from A. corymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bovine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal antibodies. In most lesions (n = 140 [approximately 97%]) the Mab and the polyclonal antibodies reacted in a similar pattern, i.e., positively for zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, and negatively in 10 undiagnosed lesions. Hyphae within two of four lesions in lymph nodes, which were not stained by the polyclonal antibodies, reacted with the specific Mab. However, in another three lesions of lymph nodes stained by the polyclonal antibodies no reactivity was seen with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA-RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justify their application for the in situ diagnosis of systemic bovine zygomycosis.
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，在体外制备了针对米根霉（Rhizopus oryzae）水溶性体细胞抗原（WSSA）和细胞壁组分（WF）的鼠单克隆抗体（Mab）。随后，在酶联免疫吸附测定中仅与同源抗原反应的上清液，通过免疫组织化学技术筛选其与同源真菌的反应性。所有针对米根霉WF产生的四种Mab在组织上均无反应。然而，针对米根霉WSSA产生的四种Mab（Mab-WSSA-RA-1至Mab-WSSA-RA-4）在组织上显示出高同源反应性，随后在含有毛霉科其他成员和其他无关真菌的组织上评估了它们的交叉反应性。在组织和免疫印迹上，所有四种Mab对毛霉科成员，即伞枝犁头霉（Absidia corymbifera）、米根霉和微小根毛霉（Rhizomucor pusillus）的反应相同且具有特异性。这些Mab均被鉴定为IgM抗体，无沉淀反应，并且与分子量从14至110 kDa的同源抗原反应。对于来自伞枝犁头霉和微小根毛霉的WSSA，这四种Mab分别与14至52 kDa和20至28 kDa的抗原结合。将其中一种特异性Mab（Mab-WSSA-RA-1）对145例牛病变的诊断结果与异源吸收的多克隆抗体的诊断结果进行比较。在大多数病变（n = 140 [约97%]）中，Mab和多克隆抗体的反应模式相似，即89例接合菌病病变呈阳性，41例曲霉病病变呈阴性，10例未确诊病变呈阴性。淋巴结中四个病变中的两个病变内的菌丝，多克隆抗体未对其染色，但与特异性Mab发生反应。然而，在另外三个被多克隆抗体染色的淋巴结病变中，未观察到Mab-WSSA-RA-1的反应性。针对米根霉WSSA产生的Mab（Mab-WSSA-RA-1至Mab-WSSA-RA-4）的免疫反应性证明了它们在牛全身性接合菌病原位诊断中的应用价值。
