Development of a certified reference material for anti-ß2-glycoprotein I IgG - commutability studies.

Objectives: In this paper, we describe the steps followed for the development of a certified reference material for immunoglobulin G antibodies against ß2-glycoprotein I (also known as apolipoprotein H). These steps include processing of the material, commutability, the impact of dilution, the appropriate reconstitution conditions, homogeneity and stability during transport and storage. Methods: We analysed 69 clinical samples from patients suffering from antiphospholipid syndrome with several commercial enzyme-linked immunosorbent assays (ELISA) purchased from in vitro diagnostic manufacturers. Results: Analysis of the results indicated that the candidate reference material can be safely freeze-dried, and that the user should carefully follow the reconstitution instructions as small changes in e.g. temperature may have unwanted effects. The statistical analysis of the commutability studies indicated that the analytical response of the reference material upon dilution is similar to that of clinical samples, and that correlation between results may differ from assay to assay. Finally yet importantly, the presented and developed candidate reference material is commutable for most assays tested, homogeneous and stable. Conclusions: Immunoglobulin G antibodies against ß2-glycoprotein I are associated with a higher risk of thrombosis and pregnancy complications. Their measurement is essential for the diagnosis and monitoring of antiphospholipid syndrome. These antibodies are detected by specific immunoassays, routinely used in clinical diagnostics, but various of these methods show enormous variability, in part due to the lack of a reference material.

Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content.

Certified Reference Materials and their need for the diagnosis of autoimmune diseases.

Autoantibody measurement is the chosen tool, in addition to clinical observations, for the diagnosis of autoimmune diseases. Hence, it is essential for these measurements to be reliable and in the longer run to be standardised. Due to the intrinsic variability of analytes and reagents, and the heterogeneity of the available techniques, standardisation cannot be taken for granted, and results may vary between laboratories. As a consequence, diagnoses can be missed or wrong and unnecessary costs may burden individuals and healthcare systems. Standardisation of autoantibody testing is a demanding and multi-parameter task, but could be part of the solution. So as to achieve standardisation, the development and availability of suitable certified reference materials for calibration and/or quality control is crucial.

Development of a Certified Reference Material for myeloperoxidase-anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA).

A serum Certified Reference Material (CRM) for supporting reliable autoimmune diagnostics was recently released by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission. It was produced in collaboration with a Working Group on the Harmonisation of Autoimmune Tests of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC WG-HAT). This material is aimed at facilitating the standardisation of measurements of anti-myeloperoxidase immunoglobulin G antibodies. The CRM could be used as a common calibrant by clinicians and manufacturers thereby significantly improving the comparability of results from commercial immunoassays used for IgG anti-MPO measurements. This paper provides information on the new CRM and its intended use.

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood encephalopathy.

A synthetically modified hydrophobin showing enhanced fluorous affinity.

Hydrophobins are natural surfactant proteins endowed with exceptional surface activity and film-forming capabilities and their use as effective "fluorine-free fluorosurfactants" has been recently reported. In order to increase their fluorophilicity further, here we report the preparation of a unique fluorous-modified hydrophobin, named F-HFBI. F-HFBI was found to be more effective than its wild-type parent protein HFBI at reducing interface tension of water at both air/water and oil/water interfaces, being particularly effective at the fluorous/water interface. F-HFBI was also found to largely retain the exceptionally good capability of forming strong and elastic films, typical of the hydrophobin family. Further studies by interface shear rheology and isothermal compression, alongside Quartz Crystal Microbalance and Atomic Force Microscopy, demonstrated the tendency of F-HFBI to form thicker films compared to the wild-type protein. These results suggest that F-HFBI may function as an effective compatibilizer for biphasic systems comprising a fluorous phase.

Imaging mass spectrometry: a new tool for kidney disease investigations.

Matrix-assisted laser desorption ionization (MALDI)-profiling and imaging mass spectrometry are promising technologies for measuring hundreds of different molecules directly on tissues. For instance, small molecules, drugs and their metabolites, endogenous lipids, carbohydrates and complex peptides/proteins can be measured at the same time without significant disruption of sample integrity. In this review, the potential of MALDI-profiling/imaging technologies in disease proteomics, drug action and studies of cellular processes in the context of kidney tissue is described. Spatial and sequence information obtained in tissue MALDI-profiling/imaging studies can be correlated with other mass spectrometry-based techniques, auxiliary imaging technologies and routine (immuno) histochemical staining.

Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links.

Cross-linking of ß-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.

Digestibility and allergenicity assessment of enzymatically crosslinked beta-casein.

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of beta-casein (CN). beta-CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to beta-CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS-PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the beta-CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of beta-CN.

Crosslinking food proteins for improved functionality.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.