Antigenic heterogeneity of lipid A of Haemophilus influenzae

The chemical structure and biologic function of the lipid A portion of lipopolysaccharide are not identical among gram-negative bacteria. This study indicates that antigenically heterogeneous lipid A exists among strains of Haemophilus influenzae. An immunoglobulin G3 murine monoclonal antibody, 3D2, produced against a nontypable H. influenzae strain 3524 has specificity for a site on the lipid A portion of the H. influenzae lipopolysaccharide. With the Western blot and immunodot assay, 3D2 recognized this lipid A determinant on 14 of 24 (58%) of strains of nontypable H. influenzae and in 51 of 95 (54%) strains of H. influenzae type b. This lipid A epitope has a high degree of specificity for H. influenzae, since it is not present on the lipid A of 39 gram-negative strains from 14 non-Haemophilus species. In addition, studies of 36 strains of six Haemophilus species other than H. influenzae and 8 strains of 4 species of Actinobacillus did not contain the 3D2 epitope. Enzyme-linked immunosorbent assay analysis with a kinetic assay and enzyme-linked immunosorbent assay inhibition confirmed the antigenic heterogeneity of H. influenzae lipid A. Thin-layer chromatography demonstrated that the 3D2 epitope is associated with a chloroform-soluble lipid moiety in the lipid A. Fluorescent antibody analysis of H. influenzae indicated that the epitope is on the cell surface. The monoclonal antibody was not bactericidal for strain 3524, and it did not inhibit the bactericidal action of normal human serum against the same strain. These studies demonstrate that the lipid As of H. influenzae are antigenically heterogeneous.