Lactoferrin-lipid A-lipopolysaccharide interaction: Inhibition by anti-human lactoferrin monoclonal antibody AGM 10.14

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bact ericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal ef fect. In this study, we evaluated, by means of an enzyme-linked immunosorbe nt assay, the binding of biotinylated LF to the S (smooth) and R (rough) (R a, Rb, Re, Rd1, Rd2, and Re) forms of LPS and different lipid A preparation s. In addition, the effects of two monoclonal antibodies (AGM 10.14, an imm unoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specif ically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The bind ing of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccha ride core structures may interfere with the LF-lipid A interaction. In addi tion, we found that soluble lipid A also inhibited LF binding to immobilize d LPS, demonstrating that, in the whole LPS structure, the lipid A region c ontains the major determinant recognized by LF, AGM 10.14 inhibited LF bind ing to lipid A and LPS in a dose-dependent fashion, indicating that this mo noclonal antibody recognizes an epitope involved in the binding of LF to li pid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS, There fore, AGM 10.14 may represent a useful tool for neutralizing selectively th e binding of LF to lipid A. In addition, the use of such a monoclonal antib ody could allow better elucidation of the consequences of the LF-lipid A in teraction.