Heterologous expression, purification and generation of specific antibodies for Annexin A8

Annexin A8 (ANXA8) is a member of the Annexin gene family, with high levels of its mRNA and protein observed in various tumor tissues, making it a potential tumor biomarker. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) to specifically detect the presence and accurately determine the concentration of ANXA8. To this end, we expressed a series of proteins using both Pichia pastoris and Escherichia coli expression systems. Particularly, human ANXA8 expressed in Pichia pastoris was used as the antigen and for antibody screening, while human ANXA2 and ANXA5 expressed in Pichia pastoris and murine ANXA8 expressed in E. coli BL21 (DE3) were used as counter screens to eliminate cross-reactive antibodies and to select antibodies that show specificity to ANXA8 from both human and mouse. Through research efforts with production and purification of recombinant proteins, immunization, specificity screening and selection of epitope pairing, two specific monoclonal antibodies, E9 and B7, apparently targeting different epitopes of ANXA8, were obtained. The specificity of the antibody pair was checked against recombinant human ANXA2 and ANXA5 with ANXA8 as the positive control. Western Blot and Sandwich ELISA demonstrate superb sensitivity and specificity with a detection limit of about 0.065 ng/mL for the latter. In summary, this study provides an experimental tool for the quantitative determination of ANXA8 concentration and a potential tumor biomarker to monitor disease progression.