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The inhibitory effect of galactose on phagocyte function was investigated in normal and hypergalactosemic chicks by monitoring the in vitro killing of Escherichia coli by leukocytes and the in vivo clearance of colloidal 125I-labeled bovine serum albumin ([125I]BSA) from the circulation. Elevated levels of galactose (30 mM) significantly impaired the bactericidal activities of leukocytes from both control and hypergalactosemic chicks. However, the latter cells were more susceptible to the galactose-dependent inhibition. Leukocytes from hypergalactosemic chicks displayed near-normal bactericidal activity when assayed in vitro under simulated normal conditions in the absence of galactose. Mean corrected phagocytic indexes, obtained from data on the clearance of colloidal [125I]BSA, were calculated to be 0.358 and 0.299 for control and hypergalactosemic chicks, respectively. Moreover, increased concentrations of galactose significantly impaired the bactericidal activity of circulating leukocytes but did not significantly affect the phagocytic activity of the reticuoendothelial system.

To determine whether the deleterious action of D-galactose upon phagocyte function could be related to inhibition of glucose uptake, the properties of glucose transport were investigated by following the incorporation of [G-3H]2-deoxyglucose into human and guinea pig polymorphonuclear leukocytes (PMN). Uptake of [G-3H]2-deoxyglucose by guinea pig PMN proceeded in vitro with a Km of 1.8 mM and Vmax of 0.67 nmol/min per 10(6) cells. This system was competitively inhibited by glucose and mannose but was not significantly affected by galactose, fructose, or 3-0-methylglucose. Maximal uptake of 2-deoxyglucose occurred at 41 degrees C, and phosphorylation was necessary for its intracellular concentration. Transport of 2-deoxyglucose, although not altered by uncouplers of oxidative phosphorylation, was sensitive to inhibitors of glycolysis. Preincubation of cells with 2 mM iodoacetate for 30 min significantly reduced the uptake of 2-deoxyglucose and the intracellular levels of adenosine-5'-triphosphate without decreasing cell viability. These results indicated that uptake of 2-deoxyglucose in guinea pig PMN occurred by facilitated diffusion with subsequent phosphorylation. Similar results were obtained with PMN isolated from human peripheral blood.

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.

Recent innovations in particle design have led to the development of highly sensitive and reproducible immunoassay methods for the Du Pont aca discrete clinical analyzer. Key advances include the synthesis and use of particles less than 1 micron in diameter with high refractive index cores surrounded by thin, chemically reactive shells. The cores are prepared by emulsion polymerization to a well-defined size that depends on the choice of monomer and the requirements for turbidimetric signal. Methods for measuring therapeutic drugs (e.g., theophylline) involve particles with polystyrene cores; other methods require cores with higher refractive indices such as polyvinylnaphthalene. The shells are critical for overall method performance because they bind covalently the immunochemicals of interest. Polyglycidyl methacrylate shells have been used effectively to attach antigens and haptens to the particle surface.

To account for enhanced susceptibility to infection among galactosemics, the acute effects of D-galactose on metabolic and functional activities of phagocytic cells in vitro were investigated. Human and guinea pig polymorphonuclear leukocytes (PMN) when incubated in medium containing 30 mM galactose displayed substantially less killing of Escherichia coli than when incubated in medium with 5 mM glucose. Impaired bactericidal activity was dependent upon galactose concentration but could be partially averted by supplementing the galactose-containing medium with 15 mM glucose. Phagocytic activities of guinea pig PMN and peritoneal macrophages were assayed by following ingestion of 32P-labeled E. coli and were also depressed by elevated galactose. Galactose was readily epimerized to glucose by resting PMN, and this conversion was stimulated by phagocytosis. Incubation of macrophages in the presence of galactose resulted in depletion of intracellular levels of adenosine 5' -triphosphate as well as other metabolities.

Although highly purified cytochrome P-450 of Pseudomonas putida (P-450 cam), and that from phenobarbital-induced rabbit liver microsomes (P-450 LM), differ markedly in their catalytic and physical properties, they show immunological cross reaction by competitive binding and inhibition of catalytic activity, and are of similar amino acid composition. Upon treatment with cyanogen bromide they yield small heme-containing peptides of highly similar amino acid composition.

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.