Selection of anti-galectin 9 hybridomas and epitope mapping of the corresponding monoclonal antibodies. A) Seven hybridomas were selected for their performance in ELISA against recombinant c-terminus galectin-9 (2E12, 1E12, 2E6, 1F4, 1G3, 1D8, 2D12). Their crude supernatants gave about the same reactivity as the undiluted mouse serum sample taken prior to spleen collection (mouse polyclonal). Other hybridomas such as 1H12 and 2F4 were set aside due to their lower reactivity with c-terminus galectin-9 in ELISA. B) Mapping of the 1G3 epitope (TPAIPPMMYPHPA) contained in the three natural isoforms of galectin-9. This epitope is close to the n-terminus extremity of the recombinant c-terminus galectin-9 used for mouse immunization. C) Selective inhibition of 1G3 binding to the recombinant S-isoform of human galectin-9 by the P4 oligopeptide (TPAIPPMMYPHPA). Plates were coated with human galectin-9 S. Subsequent binding of the mabs 1G3 and 9S2-3 were done after pre-incubation with the P4 peptide or a control peptide. The binding of 1G3 to the galectin-9 is selectively inhibited by the P4 peptide at concentrations above 0.5 μg/ml. In constrast the binding of 9S2-3 is not affected.