Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons

Klaile, Esther; Klassert, Tilman E; Scheffrahn, Inka; Müller, Mario M; Heinrich, Annina; Heyl, Kerstin A; Dienemann, Hendrik; Grünewald, Christiane; Bals, Robert; Singer, Bernhard B; Slevogt, Hortense

CEACAM1-, CEACAM5-, and CEACAM6-specific Sandwich-ELISA Frozen BALF samples were thawed and centrifuged at 2000 × g and 4°C for 10 min. Then CEACAM1, CEACAM5, and CEACAM6 were assessed in the supernatants. 96 well micro titer plates (MaxiSorb TM plates, Nunc) were coated for 2 h at room temperature with 3 μg/ml rabbit anti-CEA-antibody (Dako) diluted in PBS. After washing the plate twice with 0.05% Tween (Carl Roth GmbH) in PBS all unbound sites were blocked for 2 h at room temperature with PBS containing 3% bovine serum albumin (Carl Roth GmbH). To quantify the different CEACAMs in the samples appropriate standard curves were prepared by making serial dilutions of recombinant human CEACAM1-Fc, recombinant human CEACAM6-Fc and purified CEA (from 0.25 ng/ml to 100 ng/ml). The standard and the undiluted samples were incubated over night at 4°C and washed three times. Then 20 μg/ml C5-1X (anti-CEACAM1), 5C8C4 (CEACAM5), or 1H7-4B (CEACAM6) were added. Plates were washed three times with PBS and supplemented with HRPcoupled goat anti mouse antibody (Dianova) for 2 h followed by three washing steps. Then 100 μl TMB-X-tra substrate (Biotrend Chemikalien GmbH) were added and incubated for up to 30 min. The reaction was stopped by 20 μl of 2 N H 2 SO 4 (Carl Roth) and optical densities were read at 450 nm in a microplate reader (Tecan). All antibodies, samples and standard curves were diluted in PBS containing 1.5 % BSA. The linear ranges for CEACAMs 1, 5, and 6 were 0.25 ng/ml -100 ng/ml.