title: Human monoclonal antibody HA-1A binds to endotoxin via an epitope in the lipid A domain of lipopolysaccharide.
abstract: HA-1A, a human IgM mAb, has been shown to significantly reduce mortality in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the reported specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a solid phase; a rate nephelometry assay, which measured the ability of HA-1A to bind and aggregate lipid A in solution; and a dot-blot immunoassay, which measured the ability of HA-1A to interact with lipid A adsorbed to Immobilon-P. In all three assay systems, HA-1A bound in a dose-dependent manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to competitively inhibit HA-1A reactivity with lipid A in a dose-dependent manner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against HA-1A was also able to block the binding of HA-1A to lipid A in these assay formats. HA-1A reactivity with synthetic lipid A confirmed that HA-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assessed. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these results conclusively establish that HA-1A binds to the lipid A region of LPS by an interaction with the V region of the antibody.
authors.affiliation: Centocor, Inc., Malvern, PA 19355.
authors.forename: W C
authors.initials: WC
authors.lastname: Bogard
authors.forename: S A
authors.initials: SA
authors.lastname: Siegel
authors.forename: A O
authors.initials: AO
authors.lastname: Leone
authors.forename: E
authors.initials: E
authors.lastname: Damiano
authors.forename: D J
authors.initials: DJ
authors.lastname: Shealy
authors.forename: T M
authors.initials: TM
authors.lastname: Ely
authors.forename: B
authors.initials: B
authors.lastname: Frederick
authors.forename: M A
authors.initials: MA
authors.lastname: Mascelli
authors.forename: R C
authors.initials: RC
authors.lastname: Siegel
authors.forename: B
authors.initials: B
authors.lastname: Machielse
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive; D004731:Endotoxins / Q000276:immunology*; D000939:Epitopes; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D066298:In Vitro Techniques; D008050:Lipid A / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology*; D011112:Polymyxin B / Q000276:immunology; D012475:Salmonella / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D004731:Endotoxins; D000939:Epitopes; D007075:Immunoglobulin M; D008050:Lipid A; D008070:Lipopolysaccharides; D011112:Polymyxin B