(LASV-GP-017) LASV Glycoprotein antigen, Strain Josiah, Ectodomain, Pre-fusion GP monomer, lineage IV

Glycoproteins secreted into the supernatant of Drosophila S2 lines are purified via streptactin affinity. Following elution proteins are dialyzed into PBS, filtered, aliquoted and flash frozen in LN2. Non-reducing and reducing SDS-PAGE gels are run to analyze purity from strep-affinity purification and, for GPCysR4 proteins, efficient furin-mediated processing (A.).
New constructs are analyzed by SEC-MALS to determine molecular weight, oligomeric state and glycan content (B.).
For each new lot of GP, an ELISA screen is performed with a variety of antibodies to ensure the same antibody reactivity profile is consistent (C.). Antibodies used in ELISA screens include: 2.4F and 5.6H – GP1, non-neut; 12.1F and 19.7E – GP1, neut; binds upper portion of GP1 subunit. NB: 19.7E is not cross reactive to Lineage III; 3.6H and 13.9H – GP2, post fusion specific, non-neut; 18.5C – GPC-B, neut; quaternary – binds at the base of GP and simultaneously contacts two GP monomers; 22.5D – linear GP2, pan arena; 25.10C and 36.1F – GPC-A – neut, quaternary epitope; binds mid-upper portion of GP, contacts both GP1 and GP2 subunits from the same monomer, does not bind across trimers. NB: 36.1F is Lineage IV specific in neutralization studies.