Production and characterization of a cross-reactive monoclonal antibody to lipopolysaccharide

Lipopolysaccharide (LPS) is located on the cell wall of gram-negative bacteria and plays an important role in the pathogenesis of sepsis, which continues to be a leading cause of death in the intensive care units. There are many strains and serotypes of gram-negative bacteria and each individual has a unique kind of LPS. In addition, LPS belongs to thymus-independent (TI) antigen, making it difficult to produce high-affinity, cross-reactive monoclonal antibodies (MAb) against LPS. Here we report a novel method to produce cross-reactive murine MAbs against LPS by mixed immunization with whole cell bacteria, commercial LPS, and synthetic peptide, which simulates the structure of LPS. Using this approach, an MAb designated SMU-3A8 was generated, which can react with four commercial LPSs and seven gram-negative bacteria with high affinity suited for ELISA, dot-ELISA, Western blotting, immunofluorescence, and flow cytometry. Our results provide a new strategy for the generation of high-affinity, cross-reactive MAbs against LPS and other TI antigens.