Figure 2. Screening of murine hybridoma clones for selection of candidate therapeutic anti-Shh antibodies directed at the C-terminal of the Sonic Hedgehog protein.

(A) Supernatants from antibody- producing hybridoma clone cells were incubated with synthetic peptide mimics of Shh as measured by an ELISA-based assay. Clone 1C11 (red arrow) showed the strongest binding to the same Shh peptide mimics used to generate an immune response. (B) Hybridoma clonal supernatants were incubated with the following cell lysates to check for Shh protein detection and analyzed by Western blotting: 1) 293T (endogenous Shh) and 2) 293T (exogenously transfected with pCMV-Shh) (C) Flow cytometric evaluation of cell-surface Shh recognition performed with clonal supernatants incubated with non-permeabilized 293T cells containing the vector control or (D) pCMV-Shh. Clone 1C11 detected the highest percentage of cell-surface Shh compared with the other clones. The secondary antibody alone incubated with cells was run as a negative control.