Example 5
Pneumocystis KEX1 Antisera Cross-Reactivity with Other Fungal KEX Recombinant Proteins
To determine the nature of immunologic cross-reactivity among fungal KEX recombinant proteins, antisera from Pneumocystis KEX1 immunized monkeys were tested for reactivity by Western blotting using recombinant KEX proteins following purification and TEV cleavage of the fusion partner. Proteins were resolved by 15% SDS/PAGE and transferred to 0.2 μm nitrocellulose membranes. Membranes were then blocked overnight at 4° C. in 5% BSA/5% non-fat dry milk in PBS-T (0.05% Tween-20) and then incubated with high titer antiserum from monkey number 17911 (dilution 1:500) or low titer antiserum from monkey number 8015 (dilution 1:500) for 2 hours at room temperature to test for construct cross-reactivity. Blots were then incubated with secondary antibody goat anti-monkey IgG (H+L)-HRP (1:10,000) in blocking buffer for 1 hour at room temperature and visualized with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher). As a negative control, parallel blots were probed with secondary antibody only. The presence of maltose binding protein (MBP)-tagged proteins was confirmed by blotting with mouse anti-MBP antibody (1:10,000) (New England BioLabs, Ipswich, Mass.) and secondary antibody goat anti-mouse IgG immunoglobulin (H+L)-HRP (1:10,000) (ThermoFisher),