Clone chk 48

Mouse monoclonal anti-chikungunya virus E2 envelope glycoprotein antibodies, clone CHK-48 (produced in vitro) (NR-44002) were obtained from BEI Resources, NIAID, NIH. These antibodies were shown to be cross-reactive with MAYV antigens of both BE AN343102 and BE AR505411 strains. Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibodies, Alexa Fluor 488 (A-11029) were purchased from Invitrogen. Both antibodies were used in focus-forming assays at a dilution of 1:1000.

Samples were prepared by mixing 10μg of total protein in Laemmli buffer with βME, unless otherwise noted. These samples were boiled at 100°C for 5 min and proteins were separated on Bolt 10% Bis-Tris Plus gel as before. Next, the proteins were transferred from the gels onto PVDF membranes using the Trans Blot Turbo apparatus [Bio-Rad, Hercules, CA, USA]. The membranes were blocked for 5–10 min in iBind solution [Novex]. Polyclonal mouse anti-E1 and anti-E2 sera were recovered from mice vaccinated with E1 or E2 proteins in the lab and used to probe for these proteins. Mouse monoclonal antibody against E2 [Clone CHK-48, BEI Resources, Manassas, VA, USA] was also used to probe for E2. Goat anti-mouse conjugated with horseradish peroxidase [Southern Biotech, Birmingham, AL] was used as the secondary antibody. The membrane, antibody, and iBind solutions were loaded into the iBind Western System [Life Technologies, Carlsbad, CA] from which point all steps in the membrane blotting process proceed automatically by sequential lateral flow. Blotting using the iBind system was complete after 2.5 h. Following washing of the membrane twice more with PBS with 0.05% Tween-20 [PBS-T], the membrane was exposed with Clarity Western ECL Substrate [Bio-Rad]. Images were captured using my ECL Imager [Thermo Fisher Scientific].