title: Immunological recognition of fungi
Creators: name: Marshall, Margaret
Publisher: name: University of Kent
Year: 2022

description: Polyclonal antisera were raised against the fungi Neurospora crassa and Paxillusinvolutus, respectively. These antisera were used in the development of an ELISA for subsequent use in monoclonal antibody (MAb) screens. Fungal cell walls were chosen as the most suitable immunogen for in situ ecological studies. Hyphal walls were isolated and bound to a poly-/-lysine coated ELISA plate. Both antisera showed cross-reactions with other species of fungi. These reactions were not diminished by pre-absorption of the antisera with cell walls from species of fungi which react with the antiserum. The ELISA was then used in the production of specific MAbs raised against fungal cell wall antigens. Hybridoma supernatants were screened following fusions of spleen cells from immunised mice and Sp2.0 myeloma cells. A successful immunisation protocol was established using fungal cell walls, and a titre of tailbleed plasma used to determine whether a mouse would be used for fusion. Four MAb cell lines were established (S4D1, S3B3, S1E5 and Pax-1).Of these MAbs, three were derived from mice immunised with cell walls of N. crassa and the fourth from a mouse immunised with the cell walls of P. involutus. In immunofluorescence studies the three MAbs raised against N. crassa recognised epitopes which show different patterns of distribution at the cell surface of N. crassa. MAb S4D1 recognises an epitope which is present on the surface of both conidia and hyphae; MAb S3B3 an epitope seen only at the end of conidia or in the septal region of hyphae and conidial chains; and S1E5 an epitope present on the surface of hyphae, but not mature conidia. MAb Pax-1, raised against P. involutus, reacts with cell wall fragments of P.involutus and reacts with N. crassa conidia in a similar way to S3B3 in immunofluorescence studies, i.e. with septa and the ends of conidia. The MAbs S3B3 and Pax-1 are, however, differentiated by their reaction with P. involutus, Pax-1 reacts with P. involutus, but S3B3 does not.S4D1 reacted with an epitope found in a-1,3 glucan fractions from different fungi. The surface distribution of this epitope varies across a taxonomic spectrum. It is found on the surface of both conidia and hyphae of N. crassa and Aspergillus nidulans, but only on the basidiospore surface of Amanita muscaria. Conversely it appears on the hyphae but not the conidia of Penicillium chry’sogenum. Immunogold studies revealed that the epitope was present throughout the wall of N. crassa. In quantification studies, S4D1 showed a linear relationship between the reaction of the MAb with its epitope, recorded as absorbance in an ELISA, and cell wall biomass (freeze-dried weight).S3B3 and Pax-1 also reacted with other fungi. Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycotina. The nature of the epitopes of these two MAbs was not determined, nor was that of S1E5. Immunogold studies revealed that epitopes of these three MAbs were not present at the surface in all the morphological forms of N. crassa, but that they were present in inner layers of these walls. For example, the epitope of Pax-1 is found in a discrete layer next to the plasma membrane of N. crassa cells but not at the surface, except in the septal region. The epitope of Pax-1 is not chitin. The MAbs produced did not show species-specificity. The specific reaction of the MAbs with their epitopes enabled the localisation of the epitopes at the cell surface of fungi and their distribution throughout the wall of N. crassa to be studied. The distribution of the epitopes within the walls of hyphae and conidia has been schematically represented.