DENV Envelope Protein Detection

DENV-containing culture supernatants were obtained from C6/36 cells and divided into two groups. The reducing group was added with loading buffer containing 100 mM beta-mercaptoethanol while the unheated and non-reducing group was added with loading buffer without beta-mercaptoethanol. The samples were then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (10600023; Amersham) by electroblotting. Non-specific binding was blocked using 5% skimmed milk, after which the membranes were treated with DENV-specific HMAb 1G5 (1 μg/ml) and then horseradish peroxidase-conjugated antibody against human IgG (1:5000, v/v). The membranes were then treated with an enhanced chemiluminescence substrate (RPN2106, Amersham). DV69.6 (1 μg/ml) (Beltramello et al., 2010 (link)), which is a cross-reactive antibody against DENV, recognizes the prM protein and is an indicator of the molecular weight of prM. 4G2 (1 μg/ml) (Henchal et al., 1985 (link)), which is a cross-reactive antibody against flavivirus, recognizes a conserved epitope that is present on the fusion loop of the E protein; it is an indicator of the molecular weight of the E protein.