Performance Evaluation of Open Channel Buhlmann Fecal Calprotectin Turbo Assay on Abbott Alinity C Analyzer.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal (GI) tract. Fecal calprotectin (fCAL) is a noninvasive laboratory test used in the diagnosis and monitoring of IBDs such as Crohn's disease and ulcerative colitis. The fCAL send-out test that our facility has been offering so far uses an ELISA-based method. In the current study, we sought to validate the performance of a Buhlmann fCAL turbo assay in an automated Abbott Alinity C analyzer (AFCAL) in our core laboratory. Five-day imprecision studies showed good performance for both within-run (5.3%) and between-day (2.5%) measurements. The reportable range was verified as 30-20,000 µg/g. Deming regression and Bland-Altman analysis indicated a strong correlation of r = 0.99 with a low, acceptable bias of 1.8% for AFCAL relative to the predicate Buhlmann fCAL ELISA results. AFCAL's clinical performance was determined retrospectively in 62 patients with ICD codes for IBD. Overall, the implementation of AFCAL in our routine clinical testing has improved our turnaround time, reduced the cost per test, and significantly increased our clinician satisfaction.

Periplaneta americana L. a potential source of traditional medicine: chemometric analysis, in vitro and in silico study.

'Mayurbhanj is the ethnic dominant tribal population district in Odisha, India. The triabl's of Mayurbhanj depends on traditional medicines since time immemorial for health-related issues. Due to the imperative ethnic claim of traditional healers, the financial stringency of the patient community and the necessity to produce a better therapeutic effect has led to investigate ethno zoological sources and to find out the biochemical moiety responsible for the healing process. Considering the ethnic communities' acceptability of the zoological source as traditional medicine, the current evidence-based research study is conducted to investigate the biochemical moiety present in Periplaneta americana, responsible for therapeutic activity. The whole powdered Periplaneta americana was extracted using maceration techniques with n-hexane and methanol as solvent. The obtained extracts were subjected to GC-MS analysis to identify the biochemical moiety. To check the potential biological activity, an in-vitro antimicrobial test was carried out in both turbidimetry and a viable count method against E. coli. Moreover, the obtained biochemical molecules were exposed to in silico studies for their binding modes and their affinity using Discovery studio software. The major compounds were found to be hexadecanoic acid, methyl ester, n-hexadecanoic acid, oleic acid, octadecanoic acid along with other minor constituents. The maximum inhibitory activity of n-hexane and methanol extract against S. aureus at a concentration of 400 µg/mL was found to be 89 and 87%, respectively. The binding models of almost all identified compounds confer very good binding affinities with some key and strong non-covalent interactions with various amino acid residues of receptor active site pocket, which predict the compounds to be potent inhibitors of various infectious bacteria. These findings suggested that the hexane extract of P. americana could be exploited as a potential natural source. Communicated by Ramaswamy H. Sarma.

Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC.

Solubility assessment and on-line exposure confirmation in a patch-clamp assay for hERG (human ether-a-go-go-related gene) potassium channel inhibition.

INTRODUCTION: The hERG (human ether-a-go-go-related gene) potassium channel (KV11.1) is an important anti-target in drug discovery as its inhibition by small molecules has considerable promiscuity and is linked to an increased risk of the potentially fatal ventricular arrhythmia torsade de pointes. Therefore, great efforts are taken in the pharmaceutical industry to early on screen out compounds that block the channel. Early screening activities most often include compounds with sub-optimal physicochemical properties such as limited solubility. Therefore, careful monitoring of achieved compound concentration importantly supports the validity of experimental data. METHODS: A novel principle of exposure confirmation in a constant flow patch-clamp assay for hERG interaction is presented. Quantification is based on-real time UV absorption spectroscopy of the perfusion solution using long light path fiber optic flow cells. Calibration is performed using solutions which are confirmed by turbidometry to be free of precipitates. RESULTS: Turbidometry is shown to be sensitive enough to ensure valid calibration of the UV spectroscopic measurement. For a typical drug-like small molecule (verapamil) it is shown that even 30 nM can be accurately quantified using a 100 cm fiber optic flow cell. DISCUSSION: The combination of turbidometry and long light path fiber optic UV spectroscopy offers accurate, almost real-time exposure determination in a wide range of concentrations with little effort, affordable instrumentation, and no delay for data reporting. For research compounds with challenging physicochemical properties this method provides valuable data to support the validity of the measurements.

Applications of Biophysics in High-Throughput Screening Hit Validation.

For approximately a decade, biophysical methods have been used to validate positive hits selected from high-throughput screening (HTS) campaigns with the goal to verify binding interactions using label-free assays. By applying label-free readouts, screen artifacts created by compound interference and fluorescence are discovered, enabling further characterization of the hits for their target specificity and selectivity. The use of several biophysical methods to extract this type of high-content information is required to prevent the promotion of false positives to the next level of hit validation and to select the best candidates for further chemical optimization. The typical technologies applied in this arena include dynamic light scattering, turbidometry, resonance waveguide, surface plasmon resonance, differential scanning fluorimetry, mass spectrometry, and others. Each technology can provide different types of information to enable the characterization of the binding interaction. Thus, these technologies can be incorporated in a hit-validation strategy not only according to the profile of chemical matter that is desired by the medicinal chemists, but also in a manner that is in agreement with the target protein's amenability to the screening format. Here, we present the results of screening strategies using biophysics with the objective to evaluate the approaches, discuss the advantages and challenges, and summarize the benefits in reference to lead discovery. In summary, the biophysics screens presented here demonstrated various hit rates from a list of ~2000 preselected, IC50-validated hits from HTS (an IC50 is the inhibitor concentration at which 50% inhibition of activity is observed). There are several lessons learned from these biophysical screens, which will be discussed in this article.