Discussion

Scientific Reports - System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

Scientific Reports - Amyloidogenic motifs revealed by n-gram analysis

Typing and classification of Escherichia coli (E. coli) according to cell wall components, like polysaccharides, is routinely done by serotyping. Given the presence of 188 known O-antigens, this process is complex. The authors present a proof-of-concept planar microbead array for multiplexed O-serotyping. Ten clinically relevant E. coli serotypes associated with high risk for diarrhea in humans were examined (O26, O55, O78, O118, O124, O127, O128, O142, O145 and O157). Antisera were assigned to specific microbead populations, which can be differentiated by size and fluorescence color. Automatted image processing and data analysis were conducted by a microscopic interpretation platform. Homogenous antiserum coating of the microbeads was demonstrated by an intra-population CV that ranges from 3.3 to 6.3% and by an inter-population CV of 9.5%. Typical detections limits are in the range from 0.31 to 0.71 refMFI. Significantly elevated fluorescence signals revealed that E. coli of a certain serogroup bound specifically to microbeads with the matching antiserum (p < 0.001). In our perception, the method represents a viable diagnostic tool for automated multiplex serotyping of E. coli. It enables simultaneous and high-throughput screening for different O-antigens by a simple staining and binding protocol.

Europe PMC is an archive of life sciences journal literature.

Europe PMC is an archive of life sciences journal literature.

Europe PMC is an archive of life sciences journal literature.

If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis thereof. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and thereafter the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides.