A simple, sensitive and rapid ELISA method for the quantification and characterization of antibodies to lipid A has been developed, which can also be applied to other hydrophobic antigens. For coating, antigens were applied to the wells of ELISA plates as solutions in a mixture of chloroform and ethanol 1:9 (v/v), and the solvent evaporated in a stream of warm air. Under these conditions a high coating efficiency was achieved, which made the assay highly sensitive. The use of the above organic solvent considerably reduced the nonspecific adsorption of immunoglobulins to the solid phase, making the usual blocking of unspecific binding sites with BSA or gelatine unnecessary. For screening of lipid A antibodies in the sera of immunized animals, coating with 0.2 microgram of the corresponding antigen per well was found to be suitable. For optimal measurement of antibodies in pre-immune sera, sera of healthy human donors, and of monoclonal antibodies, higher amounts of antigen (1-2 micrograms/well) had to be used. The coating method described here proved excellent also for other antigens directly soluble in organic solvents, such as Re-lipopolysaccharide (LPS) or gangliosides. In addition, the method was successfully applied to less hydrophobic antigens, such as LPS of the classes Ra to Rd and S forms, and lipoteichoic acid. These could be brought into solution in chloroform/ethanol by diluting their aqueous solutions with a large volume of the organic mixture.

Adolescent↗

PubMedDate not supplied

Chemical and biological properties of a phenol-water extract from Leptospira interrogans. Evidence for the absence of lipopolysaccharide.

Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.

Animals↗

PubMedDate not supplied

Differential cytokine production in stimulated blood cultures from intensive care patients with bacterial infections.

Mice infected with bacteria develop an interferon-gamma (IFN-gamma) dependent hypersensitivity to lipopolysaccharide (LPS) and other bacterial components. The broader aim of this study is to find out whether such hypersensitivity also occurs in patients suffering from bacterial infections. The capacity of stimulated peripheral blood cells from infected, intensive-care patients to produce cytokines (IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)) was compared to that of healthy donors. Culturing of the cells was carried out preferentially in whole blood diluted 1:3. Whole blood cultures (WBC) were stimulated with lipopolysaccharide (LPS), whole killed Salmonella typhimurium and Staphylococcus aureus and concanavalin A (ConA), and the cytokine production was determined. Two main findings emerged from this study: The IFN-gamma production by WBC of patients was, compared to healthy donors, markedly suppressed, regardless of stimulus used. Further, patients' WBC exhibited a suppressed TNF-alpha production after stimulation with LPS. Surprisingly, following stimulation with bacteria (S. typhimurium and S. aureus) an elevated TNF-alpha and IL-6 response was obtained. Thus, in severely infected patients the cytokine responses of peripheral blood cells to LPS may be suppressed, while the response to other bacterial components is enhanced.

Adolescent↗