Binding of conformational-dependent and -independent MAbs to mutated CEACAM1-3-Fc constructs.

MAb binding to (A) the initial CEACAM1-3-Fc mutants and (B) to mutants surrounding the V39 residue and involved in R64 to D82 intrafold salt bridge. Each mutated CEACAM1-3-Fc protein, the unmodified CEACAM1-4-Fc protein, or BSA at concentrations of 1 μg/100 μL were plated in triplicate onto 96-well Immulon 3 plates that had been precoated with goat antihuman Fc and their reactivity with the MAbs indicated on each graph assessed by ELISAs as described in “Materials and methods.” The binding of each MAb to the unmodified CEACAM1-4-Fc (A) or CEACAM1-3-Fc (B) constructs was normalized to 100% and the relative binding of these MAbs to each mutated constructed or to the BSA (Nil) negative control was calculated as a percentage of binding to the unmodified CEACAM1-4-Fc or CEACAM1-3-Fc, respectively. The graphs are from a representative experiment and show means ± SD of 3 to 6 replicate measurements. The reactivity of each construct was analyzed on at least 2 independent occasions and generated similar results.