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Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) represents the successful combination of retentate chromatography and mass spectrometry, and this technology is an integral part of Ciphergen's ProteinChip System, which was designed to answer biomedical questions by performing protein analyses on a single experimental platform. The quantification capability of the ProteinChip System is essential in all proteomic applications for which this technology is used. Here we describe methods and results for three short experiments mimicking realistic analytical challenges to provide practical examples of quantification.