Specificity Testing: BOSC cells were transiently transfected with expression vectors containing either the cDNA of CEACAM1, CEACAM3, CEACAM5-CEACAM8 or a recombinant transmembrane-anchored PSG1|fusion protein. Recognition of CEACAM4 was tested on CHO cells stably transfected with a CEACAM4 expression vector. Expression of the constructs was confirmed with monoclonal antibodies known to recognize the corresponding proteins (CEACAM1, 3, 4, 5 and 6 green curves). An irrelevant monoclonal antibody served as a negative control (black curves). For specificity testing, protein G purified MBS606731 was tested on all CEACAM transfectants. A positive signal was obtained with CEACAM1, CEACAM3, CEACAM4, CEACAM5 and CEACAM6 expressing cells (red curves).
Domain organization of the human CEACAM family. The CEACAM family consists of two subgroups, the CEACAM and the PSG subgroup. CEACAM family members are membrane-bound by either via a transmembrane domain or a GPI anchor (green arrow) whereas the PSGs are secreted glycoproteins. N-linked glycosylation sites are shown as drum sticks. The IgV domains are shown. in red, the IgC domains in blue.
Immunofluorescence of stable CHO-K1/CEACAM1-transfectant with MBS606731. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% saponine and then stained with 50ug/ml MBS606731 diluted in PBS with 1% BSA (A). Untransfected CHO-K1 parental cells served as negative control (B). Detection of antibody was performed with a FITC-labeled anti-mouse-IgG secondary antibody.
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