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Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidemica: a novel method using antigen-coated magnetic beads for specific B cell isolation

¹ Department of Virology, National Bacteriological Laboratory
² Department of Virology, Karolinska Institute, c/o National Bacteriological Laboratory, S-105 21 Stockholm
³ Department of Surgery, University Hospital, S-901 85 Umeå
⁴ Department of Cell and Molecular Biology, Laboratory of Medical Cell Genetics, Hybridoma Group, Karolinska Institute, S-104 01 Stockholm
and⁵ National Defense Research Establishment, FOA-4, S-901 82 Umeå, Sweden

Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein—Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four antiglycoprotein 2-specific clones. Identical isotypes (IgGl ) and isoelectric points (9·2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partner SPAM-8. Several of the established human x (human x mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.

Received 21 January 1993; accepted 24 February 1993.

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