BACKGROUND The availability of a suitable matrix reference material is essential for standardization of the immunoassays used to measure serum proteins. The earlier serum protein reference material ERM-DA470 (previously called CRM470), certified in 1993, has led to a high degree of harmonization of the measurement results. A new serum protein material has now been prepared and its suitability in term of homogeneity and stability has been verified; after characterization, the material has been certified as ERM-DA470k/IFCC. METHODS We characterized the candidate reference material for 14 proteins by applying a protocol that is considered to be a reference measurement procedure, by use of optimized immunoassays. ERM-DA470 was used as a calibrant. RESULTS For 12 proteins [α2 macroglobulin (A2M), α1 acid glycoprotein (orosomucoid, AAG), α1 antitrypsin (α1-protease inhibitor, AAT), albumin (ALB), complement 3c (C3c), complement 4 (C4), haptoglobin (HPT), IgA, IgG, IgM, transferrin (TRF), and transthyretin (TTR)], the results allowed assignment of certified values in ERM-DA470k/IFCC. For CRP, we observed a bias between the lyophilized and liquid frozen materials, and for CER, the distribution of values was too broad. Therefore, these 2 proteins were not certified in the ERM-DA470k/IFCC. Different value transfer procedures were tested (open and closed procedures) and found to provide equivalent results. CONCLUSIONS A new serum protein reference material has been produced, and values have been successfully assigned for 12 proteins.

Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.

A cluster of 14 genes located on the large plasmid of enteropathogenic Escherichia coli (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant E. coli host. The fifth gene in the cluster, bfpU, encodes a protein that is predicted to be localized to the periplasmic space. To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar bfpU mutant EPEC strain by allelic exchange. The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP. These phenotypes were restored to the bfpU mutant by a plasmid containing bfpU. There was no difference between the wild-type and bfpU mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes. Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm. Thus, BfpU represents a novel protein required for type IV pilus assembly.

A new serum protein reference material has been produced with the properties required for a serum protein reference material for 14 proteins. An apparent loss of CRP of approximately 20% was observed upon freeze-drying of the material.

Glyceraldehyde-3-phosphate dehydrogenase was purified from Clostridium acetobutylicum by sequential ammonium sulfate precipitation, gel filtration and anion-exchange chromatography (to a specific activity of 27 U mg-l). The enzyme had a molecular mass of 40 kDa as determined by SDS-PAGE and a native molecular mass of 160 kDa as determined by nondenaturing PAGE, indicating that it has a homotetrameric composition. Its pH optimum was between 8 5 and 9.3. The corresponding gene (gap) was cloned and sequenced from C. acetobutylicum DSM 792 and found to cluster with other genes of enzymes from the glycolytic pathway (pgk, phosphoglycerate kinase; tpi, triosephosphate isomerase; pgm(i), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase). No sequences resembling rho-independent transcription terminators were found in the intergenic regions. A plasmid carrying the clostridial gap gene complemented an Escherichia coli gap mutant.