Quantification of blood glial fibrillary acidic protein using a second-generation microfluidic assay. Validation and comparative analysis with two established assays.

OBJECTIVES: Increased levels of glial fibrillary acidic protein (GFAP) in blood have been identified as a valuable biomarker for some neurological disorders, such as Alzheimer's disease and multiple sclerosis. However, most blood GFAP quantifications so far were performed using the same bead-based assay, and to date a routine clinical application is lacking. METHODS: In this study, we validated a novel second-generation (2nd gen) Ella assay to quantify serum GFAP. Furthermore, we compared its performance with a bead-based single molecule array (Simoa) and a homemade GFAP assay in a clinical cohort of neurological diseases, including 210 patients. RESULTS: Validation experiments resulted in an intra-assay variation of 10 %, an inter-assay of 12 %, a limit of detection of 0.9 pg/mL, a lower limit of quantification of 2.8 pg/mL, and less than 20 % variation in serum samples exposed to up to five freeze-thaw cycles, 120 h at 4 °C and room temperature. Measurement of the clinical cohort using all assays revealed the same pattern of GFAP distribution in the different diagnostic groups. Moreover, we observed a strong correlation between the 2nd gen Ella and Simoa (r=0.91 (95 % CI: 0.88-0.93), p<0.0001) and the homemade immunoassay (r=0.77 (95 % CI: 0.70-0.82), p<0.0001). CONCLUSIONS: Our results demonstrate a high reliability, precision and reproducibility of the 2nd gen Ella assay. Although a higher assay sensitivity for Simoa was observed, the new microfluidic assay might have the potential to be used for GFAP analysis in daily clinical workups due to its robustness and ease of use.

Development of an ultrasensitive microfluidic assay for the analysis of Glial fibrillary acidic protein (GFAP) in blood.

Introduction: A rapid and reliable detection of glial fibrillary acidic protein (GFAP) in biological samples could assist in the diagnostic evaluation of neurodegenerative disorders. Sensitive assays applicable in the routine setting are needed to validate the existing GFAP tests. This study aimed to develop a highly sensitive and clinically applicable microfluidic immunoassay for the measurement of GFAP in blood. Methods: A microfluidic GFAP assay was developed and validated regarding its performance. Subsequently, serum and cerebrospinal fluid (CSF) of Alzheimer's disease (AD), Multiple Sclerosis (MS) and control patients were analyzed with the established assay, and levels were compared to the commercial GFAP Simoa discovery kit. Results: The developed GFAP assay showed a good performance with a recovery of 85% of spiked GFAP in serum and assay variations below 15%. The established assay was highly sensitive with a calculated lower limit of quantification and detection of 7.21 pg/mL and 2.37 pg/mL, respectively. GFAP levels were significantly increased in AD compared to control patients with advanced age (p = 0.002). However, GFAP levels revealed no significant increase in MS compared to control patients in the same age range (p = 0.140). Furthermore, serum GFAP levels evaluated with the novel microfluidic assay strongly correlated with Simoa concentrations (r = 0.88 (95% CI: 0.81-0.93), p < 0.0001). Conclusion: We successfully developed a sensitive and easy-to-use microfluidic assay to measure GFAP in blood. Furthermore, we could confirm previous findings of elevated GFAP levels in AD by applying the assay in a cohort of clinically characterized patients.

Regulatory effects of berberine on microRNome in Cancer and other conditions.

Berberine (BBR) is an isoquinoline alkaloid found in different plant families such as Berberidaceae, Ranunculaceae, and Papaveraceae. BBR is well-known for its anti-inflammatory, lipid-modifying, anticancer, anti-diabetic, antibacterial, antiparasitic and fungicide activities. Multiple pharmacological actions of BBR stem from different molecular targets of this phytochemical. MicroRNAs (miRs) are single-stranded, evolutionary conserved, small non-coding RNA molecules with a length of 19-23 nucleotides that are involved in RNA silencing and post-transcriptional regulation of gene expression through binding to the 3'-untranslated region (3'UTR) of target mRNA. MiRs emerged as important regulatory elements in almost all biological processes like cell proliferation, apoptosis, differentiation and organogenesis, and numerous human diseases such as cancer and diabetes. BBR was shown to regulate the expression of miRs in several diseases. Here, we reviewed the target miRs of BBR and the relevance of their modulation for the potential treatment of serious human diseases like multiple myeloma, hepatocellular carcinoma, colorectal cancer, gastric cancer, ovarian cancer and glioblastoma. The role of miR regulation in the putative anti-diabetic effects of BBR is discussed, as well.