A comparative study on the effectiveness of various methods for attachment of two proteins (l-asparagine and horseradish peroxidase) to the surface of liposomes. patches. Severe infections with gram-negative organisms are still an important cause of death. One of the outer membrane components of such bacteria, endotoxin (LPS) takes on a pivotal part in the pathogenesis of GNB. There is substantial evidence that LPS initiates a cascade of events leading to the onset of the sepsis syndrome (13). Since lipid A is the harmful moiety of LPS, many efforts to prepare protecting providers against LPS have focused on the preparation of ligands to the lipid A moiety. It has been postulated that antibodies directed against the conserved core or lipid A region of LPS may cross-react with LPS produced by phylogenetically varied gram-negative bacteria involved in GNB (5). Centoxin (also called HA-1A) is definitely a human being monoclonal antibody raised against the rough LPS of J5 (Rc chemotype) and selected on binding to lipid A. Both animal studies (16, 18) and phase III human medical trials offered discrepant results as to the protecting effectiveness of Centoxin against GNB (14, 19). Since the publication of these reports, questions about the epitope specificity of this MAb have arisen. While it has been explained by some authors like a lipid A-specific MAb (8, 10), others defined the antibody as polyreactive since cross-reaction was observed with i antigen present on wire erythrocytes, a ligand on human being B lymphocytes, and several anionic polymers such as ssDNA, chondroitin sulfate, and cardiolipin (6, 7). In the present study we demonstrate Hexachlorophene that there might be a unifying basic principle to explain the cross-reactivity with several apparently different antigens. The epitope specificities of a number of anti-lipid A MAbs developed in our laboratory that showed a binding profile comparable to that of HA-1A are explained. By ELISA, SDS-PAGE, and dot Hexachlorophene spot techniques it has been made plausible that these MAbs identify hydrophobic molecular patches present in lipid A, denatured proteins and in aliphatic chains. Hexachlorophene Abbreviations used. BSA, bovine serum albumin; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate; ELISA, enzyme-linked immunosorbent assay; GNB, gram-negative bacteremia; LPS, lipopolysaccharide; MAb, monoclonal antibody; OD, optical denseness; OMPs, outer membrane proteins; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA; Ig, immunoglobulin. The antibodies used in this study were murine MAbs of the IgM class (clones 28, 37, 38, 40, and 43) or of the IgG class (clone 3). Centoxin (HA-1A) is definitely a human being MAb of the IgM class and was acquired upon immunization with J5 (18). MAbs 28 and 40 were raised against alkaline- and acid-treated R595 cells (Re chemotype), respectively (2). MAbs 37 and 38 were raised against heat-killed R4 cells (Rd chemotype). The epitope specificity of anti-lipid A MAb 43 has been explained before (3, 11, 12); this MAb recognizes the hydrophillic portion of lipid A (8a). MAb 3 was raised against heat-killed bacteria (3). ELISA was carried out as explained previously (1), and the antibody titers, defined BMP4 as the lowest MAb concentrations at which significant binding occurred, i.e., with an OD at 492 Hexachlorophene nm above control ideals by more than 0.200 without a first antibody, were determined. At 10 ng/ml MAbs 28, 37, and 40 still reacted (data not demonstrated) with heat-killed bacterial cells of O111 and additional varieties and with isolated lipid A; in contrast, even at 2,000 ng/ml no reaction with clean LPS was observed. Centoxin reacted in a similar pattern, i.e., a good reaction with bacterial cells at 100 ng/ml and no reaction with clean LPS at 32,000 ng/ml. Anti-lipid A MAb 43 reacted with lipid A but not with LPS or heat-killed bacteria. It has been proposed that HA-1A binds to bacteria from the lipid A epitope. The binding of HA-1A to clean bacteria is enhanced by antibiotic treatment, which would unmask the lipid A epitope (17). To investigate the epitopes of MAbs 28, 37,.