X omat 1000a

The ECL-SB was carried out as described previously [12 (link)] with several modifications to streamline the procedure. Briefly, serial dilutions of rPfCSP (ranging from 1–0.008 pg) and PfOocyst lysate (ranging from 0.25–0.004 PfOocysts/20 μl) in TBS plus 0.5% SDS were loaded in duplicate in the wells in the ECL-SB apparatus. The protein samples were allowed to bind to nitrocellulose paper for 15 minutes. The membrane was then subjected to 3× 10 minute incubations in iBlock blocking buffer (Applied Biosystems, T2015, Foster City, CA) and probed with AP-conjugated anti-PfCSP mAb 2A10 or C3103. After subsequent 3× 10 minute washes in iBlock blocking buffer, the membrane was rinsed twice for two minutes with 25 ml of 1X assay buffer and then incubated with 6 ml of ECL-substrate solution at RT for five minutes and bands were visualized by either exposure to an AR film and developed (Kodak X-OMAT 1000A) or digitally captured on the C-Digit blot scanner (LI-COR, Lincoln, NE). The ECL-reagents used in this assay were purchased as a kit (Life Technologies,Western-StarTM Immunodetection System, T1046, Grand Island, NY) from Applied Biosystems, (Bedford, MA).

This assay was performed by using the procedure described earlier by our laboratory [17] (link). rPf CSP and midgut lysates were used for antigen detection. mAb 2A10 (at 0.31 µg/ml) was used as primary antibody and an ECL-goat anti-mouse-IgM+IgG conjugated to AP (1∶5000 dilution); as source of secondary antibody. Finally, the membrane was incubated with the ECL substrate solution (Life Technologies,Western-Star^TM Immunodetection System, T1046, Grand Island, NY) and exposed to an autoradiograph (AR) film (KODAK X-OMAT AR, IB1651579; Radnor, PA) and developed using a Kodak developer (X-OMAT 1000A).