Yeast cell surface display system for determination of humoral response to active immunization with a monoclonal antibody against EpCAM.
Even though an immunogenic formulation of the murine monoclonal anti-EpCAM (epithelian cell adhesion molecule) antibody Mab 17-1A, has been shown to evoke a strong humoral immune response in both, monkey studies and early clinical trials, conventional anti-EpCAM ELISA could not identify anti-EpCAM immune response in relation to treatment with Mab17-1A. In contrast, usage of cellulose membranes prepared by SPOT technology presenting overlapping EpCAM peptides allowed the unequivocal determination of EpCAM related antibodies present in monkeys sera after immunization with IGN101. Based on such contradictory results, it was of high interest to compare obtained data to a different method for better assessment of their possible interpretation. Therefore, in the present studies, some EpCAM peptides, determined as reactive by binding of IgG isolated from sera of treated monkeys on membranes prepared by SPOT technology, were represented on yeast surface using the pYD1 yeast display vector system. Binding of biotinylated IgG from sera was detected with streptavidin-FITC and quantity of binding was determined by FACS measurement. Though using this completely different method, experiments with pre-immune and immune sera of four monkeys exemplarily are comparable to the results obtained by analysis with synthetic peptide arrays.
Dürauer
Astrid
A
Department of Biotechnology, Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
Berger
Eva
E
Zandian
Marlene
M
Mersich
Christa
C
Schuster
Manfred
M
Loibner
Hans
H
Jungbauer
Alois
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D058846:Antibodies, Monoclonal, Murine-Derived; D000917:Antibody Formation / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000276:immunology*; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D007114:Immunization / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology; D008253:Macaca mulatta / Q000276:immunology; D040081:Protein Array Analysis; D012441:Saccharomyces cerevisiae / Q000276:immunology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000276:immunology*; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007074:Immunoglobulin G; D029701:Saccharomyces cerevisiae Proteins; C400149:Edrecolomab