Eliminating heterophilic antibody interference for ferritin detection using Olympus F(ab')2 based reagent.

Interferences with the Olympus immunoturbidimetric assay for ferritin have been reported because the antibodies used in the immunoassay are derived from rabbits. Rabbits are familiar pets known to be a risk factor for developing heterophilic (or interfering) antibodies. This report shows how the current Olympus Ferritin assay has been improved to eliminate the interference from heterophilic antibodies.

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

The expression of selected non-ribosomal peptide synthetases in Aspergillus fumigatus is controlled by the availability of free iron.

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SidE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD ( approximately 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy.