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.