Bioanalysis最新文献

Singlicate anti-drug antibody (ADA) analysis is gaining recognition as a promising approach to optimize immunogenicity assessment. It offers significant benefits in terms of cost-effectiveness and aligns with patient-centric principles. However, its successful implementation requires a thorough understanding of its impacts on assay performance. In this perspective, we review recent case studies comparing singlicate and duplicate formats, focusing on cut point factors and sample result agreement, and seek to discuss how singlicate influences assay cut points and explore considerations for low positive control criteria under singlicate conditions. Furthermore, we propose a strategy for implementing singlicate analysis in both new and existing ADA assays, hoping to contribute to ongoing discussions in this area.

Aim: The purpose of this study is to establish a light-initiated chemiluminescence assay (LiCA) for the quantitative analysis of Humulus scandens pollen-specific IgE (sIgE) antibodies.

Methods: The best chemibeads coupling method in detecting Humulus scandens pollen - sIgE was selected. The working concentrations of the antigen-antibody and the reaction buffer were optimized as components of the reaction system. Then the assay performance was evaluated and the results of LiCA were compared with ImmunoCAP methods.

Results: In the range of 0.23 kU_A/L to 100.51 kU_A/L, LiCA demonstrated good linearity. The coefficients of variation for repeatability and intermediate precision ranged from 5.96% to 8.58% and 7.53% to 11.25%, respectively. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were determined to be 0.044 kU_A/L, 0.086 kU_A/L, and 0.11 kU_A/L, respectively. Furthermore, LiCA exhibited a statistically significant correlation with ImmunoCAP (r = 0.990).

Conclusion: The established LiCA-based quantitative detection method for Humulus scandens pollen-slgE has good analytical performance and potential clinical application prospects.

The analytical and technological approaches employed in doping analysis are constantly reviewed and updated to allow for keeping pace with progresses in pharmaceutical and medicinal research and the therein inherent options of misuse as performance enhancing drugs or methods. Enormous changes, improvements, and developments have been achieved in recent years, particularly, but not exclusively, in the bioanalytical sector. Several of these new strategies are examined systematically in this review using examples from the World Anti-Doping Agency (WADA) list of banned substances and methods. The review includes, among others, the application of sophisticated new in-vitro models mimicking multi compartment models, investigation into new long-term metabolites for anabolic agents, the impact of a distinct gene mutation on the analysis of erythropoietin, studies on the development of new therapeutic protein-based drugs with myostatin inhibiting properties, methods applying the new molecular biological section used to uncover gene doping, and finally new approaches uncovering the prohibited use of autologous blood transfusion. All of these challenges and investigations support the ongoing progress in modern doping controls in the future and will help to fill the gap between the advance of cheating athletes and sport drug testing.

Background: Plasma clearance of iohexol is used to measure glomerular filtration rate, for which a UHPLC-MS/MS analytical method was previously developed. In real-world conditions, samples may be thawed on arrival and sampled in unvalidated matrices, prompting the need for an improved validation. We aim to improve the method for iohexol determination in plasma with enhanced stability testing, optimized calibration curves, and partial validation in additional matrices.

Methods: Stability testing was conducted up to 9 weeks at room temperature, 4°C, 37°C, and at 37°C with daily two-hour exposure to 55°C. Quintuplicate QC samples were analyzed on 3 days, comparing results from eight-point and two-point calibration curves. Matrix comparison was performed on quintuplicate QC samples in serum, heparin plasma, urine, EDTA whole blood, and heparin whole blood.

Results: The method improvements were all compliant with the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency.

Conclusion: Human EDTA plasma samples can be stored up to 9 weeks at room temperature, 4°C, 37°C, and 37°C with 55°C daily temperature spikes. The samples can be analyzed using a two-point calibration curve and are partial validated for serum-, heparin plasma-, urine-, EDTA whole blood-, and lithium whole blood iohexol samples.

Biological accelerator mass spectrometry (AMS) provides ultrasensitive carbon-14 isotopic analysis enabling a deeper understanding of human health concerns by enabling quantification of pharmacokinetics and other molecular endpoints directly in humans. It enables environmentally and human relevant studies of metabolic pathways through the use of very low concentrations of labeled metabolic substrates in cells and organisms. Here, we discuss why AMS is an important tool for the biosciences, the development and evolution of biological AMS at Livermore and discuss technical refinements that will improve the efficiency of operation for the measurement of ultra-trace levels of ¹⁴C, which, long term, will enable greater ease of use and sample throughput.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has transformed molecular biology through its precise gene-editing capabilities. Beyond its initial applications in genetic modification, CRISPR has emerged as a powerful tool in diagnostics and biosensing. This review explores its transition from genome editing to innovative detection methods, including nucleic acid identification, single nucleotide polymorphism (SNP) analysis, and protein sensing. Advanced technologies such as SHERLOCK and DETECTR demonstrate CRISPR's potential for point-of-care diagnostics, enabling rapid and highly sensitive detection. The integration of chemical modifications, CRISPR-Chip technology, and enzymatic systems like Cas12a and Cas13a enhances signal amplification and detection efficiency. These advancements promise decentralized, real-time diagnostic solutions with significant implications for global healthcare. Furthermore, the fusion of CRISPR with artificial intelligence and digital health platforms is paving the way for more accessible, cost-effective, and scalable diagnostic approaches, ultimately revolutionizing precision medicine.

Undruggable targets account for roughly 85% of human disease-related targets and represent a category of therapeutic targets that are difficult to tackle with traditional methods, but their considerable clinical importance. These targets are generally defined by planar functional interfaces and the absence of efficient ligand-binding pockets, making them unattainable for conventional pharmaceutical strategies. The advent of oligonucleotide-based proteolysis-targeting chimeras (PROTACs) has instilled renewed optimism in addressing these challenges. These PROTACs facilitate the targeted degradation of undruggable entities, including transcription factors (TFs) and RNA-binding proteins (RBPs), via proteasome-dependent mechanisms, thereby presenting novel therapeutic approaches for diseases linked to these targets. This review offers an in-depth examination of recent progress in the integration of PROTAC technology with oligonucleotides to target traditionally undruggable proteins, emphasizing the design principles and mechanisms of action of these innovative PROTACs.

Immunogenicity knowledge and the analytical capabilities to characterize molecules have evolved within the last decade. This creates opportunities in Biosimilar development by applying new strategies to demonstrate similarity between a proposed Biosimilar to its Reference.Within Immunogenicity Risk Assessment for Biosimilars, in silico and in vitro immunogenicity assessment tools are being evaluated for their utility in Biosimilar development. An ISI including an Immunogenicity Risk Assessment is recommended for Biosimilars within the dossier of licensing applications to facilitate the review by Health Authorities and to explain the immunogenicity in conjunction with analytical and clinical data. Operational aspects should also be considered to refine immunogenicity testing of Biosimilars, e.g. S/N ratio and singlicate sample analysis.

Background: Most oligonucleotide bioanalytical assays currently only quantify the pharmacologically-active antisense strand, though there have been recent efforts to simultaneously quantify the sense strand using hybridization ELISA or solid phase extraction LC-MS. Hybrid LC-MS, which offers both high sensitivity and specificity unlike the currently used platforms, has not been applied to quantify both siRNA strands simultaneously.

Materials & methods: A hybrid LC-MS assay utilizing LNA capture probes was developed and applied to quantify both strands of a 21-mer lipid-conjugated siRNA (SIR-3) using tandem mass spectrometry (MS/MS). A similar approach using high-resolution mass spectrometry (HRMS) was also evaluated.

Results: The final LC-MS/MS method was capable of quantifying both strands of SIR-3 at concentrations between 0.600 and 1000 ng/mL in cynomolgus monkey tissue homogenates with acceptable accuracy and precision. The LC-HRMS assay demonstrated similar sensitivity and assay performance as the LC-MS/MS assay.

Conclusions: Overall, this manuscript presents orthogonal methods to existing siRNA bioanalytical workflows that with high sensitivity and specificity can provide greater information about the concentration and biotransformation of an siRNA analyte.