Assessment of non-glycosylated domain of CD۱۳۳ marker for detection of cancer stem cells

Introduction:Since antibodies must be able to detect the native cell surface markers, the selection of the appropriate fragment of the cell surface marker as an antigen is very important for the production of antibodies. CD۱۳۳, as a suitable biomarker candidate in the cancer stemcells (CSCs), is a glycosylated protein. The antibodies used for analyzing it recognize glycosylated epitopes of CD۱۳۳ instead of the native protein. Since the glycosylated motifs have a dynamic nature over the lifetime of a protein, they limit the detection of CD۱۳۳.Method:In this study, we expressed the third domain (D-EC۳) (serine۶۴۱-leucine۷۱۰) in E. coli BL۲۱ (DE۳), then the purified recombinant antigen was used for mouse immunization. Finally, the dignity of an epitope of pure recombinant antigen has been approved by the interactions of antibody and antigen with the use of mouse immunized serums via ELISA and CD۱۳۳-cells florescent staining in the flow cytometry experimentation.Results:The results showed that the selected non-glycosylated fragment can compete well with the commercial antibody against the glycosylated epitopes to identify the native cell surface markers.Conclusion:Since both ELISA and Flow cytometry tests verify our designed D-EC۳ structure, this structure is a suitable antigenic domain to generate the anti-CD۱۳۳ mono-clonal and poly-clonal antibodies.