Human transferrin receptor (hTfR1), a type II homodimeric transmembrane protein, is extensively expressed at low levels in most normal human tissues. However, the expression level of hTfR1 in some tumor tissues and the blood-brain barrier (BBB) is comparatively higher. Therefore, we developed an hTfR1 monoclonal antibody 2C9, which may be utilized to deliver drugs across the BBB through receptor-mediated endocytosis in the future. 2C9 was obtained by hybridoma cells fused by the SP2/0-Ag14 cell line and splenic B cells from hTfR1-immunized BALB/c mice. In this study, we used indirect ELISA, Western blot, and immunofluorescence to explore the characterization and application of MAb 2C9. The results showed that the affinity constant (Kaff) of the MAb 2C9 produced from ascites was 2.85 × 10-8 M and its isotype is IgG2a (Kappa chain). Above all, this report provides a more comprehensive protocol to produce monoclonal antibodies against the extracellular domain of hTfR1.
Keywords: blood-brain barrier; human transferrin receptor; hybridoma; monoclonal antibody.