ABSTRACT: The objective of this study is to develop high affinity monoclonal antibody (MAb) probes recognizing all major serotypes of Listeria monocytogenes cells. From 500 candidate hybridoma clones, 2 new monoclonal antibody-producing hybridomas were selected and evaluated. MAbs 22D10 and 24F6 reacted strongly with live cells of most serotypes of L. monocytogenes except 4c and 4e and with some L. innocua strains; MAb 22D10 reacted strongly with both live and heat-killed cells (100 °C for 20 min) of Listeria. Both MAbs 22D10 and 24F6 did not show any cross-reactions with the other non-Listeria G(+) bacteria tested in ELISA. The mixture of EM-7G1 and 22D10 or 24F6 reacted with all 13 major serotypes of live L. monocytogenes except serotype 4c, while none of these 3 MAbs when tested alone did so. MAb 22D10 mixed with 7G1 reacted with all heat-killed L. monocytogenes serotypes except 4c and 4e. In Western blots, MAbs 22D10 and 24F6 reacted with 1 major protein band of 66 kDa in extracts from L. monocytogenes, but with 2 major protein bands of 66 kDa and 76 kDa in extracts from L. innocua. These results suggest that MAbs 22D10 and 24F6 have high affinity for 11 of 13 serotypes of L. monocytogenes, both live and heat-killed cells. MAbs 22D10 and 24F6—in combination with species-specific MAb EM-7G1—should be useful candidates for use in an ELISA sandwich assays for detecting L. monocytogenes in RTE meat and poultry products.