Abstract
Gram-negative bacteria contain lipopolysaccharide, a toxic and highly biologically active component of the outer cell wall. Of the three regions of lipopolysaccharides, the hydrophobic portion, lipid A, is biologically active, toxic, and immunogenic. Here investigations into the nature of the immunodominant structure of lipid A were carried out. The antigen, lipid A of Salmonella comprises a diglucosamine backbone substituted with ester-bound C12, C14, C16, hydroxy-C14 and amide-bound hydroxy fatty acids. Initially purification by electrodialysis, polyamine removal and thin layer chromatographic 'heterogeneity was investigated in order to obtain a serologically homogeneous preparation. The specific immune globulin fraction was isolated from whole immune serum by an immune absorbent column of lipid A-bovine serum albumin. With this purified antigen and antibody, serological assays were adapted to this hydrophobic antigen. These included immune precipitation, passive haemolysis, enzyme-linked immunosorbent assay (ELISA), complement fixation and the inhibition of each respective assay. Liposomal incorporation increased antigenicity and aided solubility of the hydrophobic antigen which was otherwise highly susceptible to non-specific interaction with ions, proteins, etc. With these tools, degradation of the antigen was carried out by treatment with acid ( removal of KDO),alkali (removal of ester-bound fatty acids) and hydrazine (removal of ester-bound and eventually, amide-bound fatty acids). Using 30 min hydrazinolysate fractions containing amide-bound fatty acid, glucosamine and phosphate with high serological activity were obtained. Further treatment with immobilised alkaline phosphatase did not reduce this activity whereas it was lost by further hydrazine treatment, proportionally to the loss of hydroxy-C14. The diglucosamine backbone (20 h hydrazinolysate) was inactive, as was the acetylated diglucosamine. Preliminary results indicated that the amide-bound hydroxy-C14 substituted glucosamine was still as mitogenic as lipid A but displayed reduced toxicity. In summary, this study indicates a role of an amide- bound fatty acid (hydroxy-C14) substituted on the glucosamine backbone in the immunodominant structure of lipid A.