A llama (Lama glama) was immunized five times (days 1, 21, 35, 49, and 63) subcutaneously with approximately 100 µg of recombinant CEACAM6 [N- and A-domains, residues 35–232; (14 (link))] per injection, kindly provided by Helix BioPharma (Aurora, ON, Canada), with Complete Freund’s Adjuvant on day 1 and Incomplete Freund’s Adjuvant on the remaining days. On days 1, 22, 36, 49, 64, and 71 blood (50 mL) was collected, from which sera and peripheral blood lymphocytes were isolated (15 (link)). This study was carried out in accordance with Animal Use Protocols approved by the National Research Council Canada Animal Care Committee.
Microtiter plates were coated with 10 µg/mL of CEACAM6 overnight at 4°C in 15 mM Na2CO3, pH 9.6, followed by blocking with 2% fat-free dry milk (BioRad Laboratories, Mississauga, ON, Canada) in PBS. Serially diluted serum was then added to the wells. Detection of llama IgGs was performed with goat anti-llama IgG (Bethyl Laboratories, Montgomery, TX, USA) and a horseradish peroxidase anti-goat IgG conjugate (Cedarlane Laboratories, Burlington, ON, Canada). Finally, peroxidase substrate (KPL, Gaithersburg, MD) was added followed by 1 M H3PO4 after 15 min. The absorbance at 450 nm was then measured.
Microtiter plates were coated with 10 µg/mL of CEACAM6 overnight at 4°C in 15 mM Na2CO3, pH 9.6, followed by blocking with 2% fat-free dry milk (BioRad Laboratories, Mississauga, ON, Canada) in PBS. Serially diluted serum was then added to the wells. Detection of llama IgGs was performed with goat anti-llama IgG (Bethyl Laboratories, Montgomery, TX, USA) and a horseradish peroxidase anti-goat IgG conjugate (Cedarlane Laboratories, Burlington, ON, Canada). Finally, peroxidase substrate (KPL, Gaithersburg, MD) was added followed by 1 M H3PO4 after 15 min. The absorbance at 450 nm was then measured.