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Methods in Molecular Biology (Clifton,... 2021Microscale thermophoresis (MST) is a biophysical assay to quantify the interaction between molecules, such as proteins and small molecules. In recent years, the MST...
Microscale thermophoresis (MST) is a biophysical assay to quantify the interaction between molecules, such as proteins and small molecules. In recent years, the MST assay has been used to detect protein-protein and protein-drug interactions. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent molecules in response to a temperature gradient. In practice, the fluorescent molecule is mixed with different concentrations of the nonfluorescent ligand, and the mixture of molecules in solution is loaded to capillaries. A temperature gradient is applied to samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded. The effect of different concentrations of the nonfluorescent ligand on the movement of the fluorescent molecule is quantified to test for the interaction between molecules. If the fluorescent molecule interacts with the ligand, the molecular properties of the molecules, such as charge, size, and hydration shell, will influence the molecular motility. MST has the advantages of being quantitative and robust. In this chapter, we will use Endosidin2 and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1), as an example to show the procedure of using MST to test the interaction between a GFP-tagged protein and a small molecule.
Topics: Arabidopsis Proteins; Biological Assay; Data Analysis; Fluorescence; Ligands; Limonins; Proteins; Small Molecule Libraries
PubMed: 33270204
DOI: 10.1007/978-1-0716-0954-5_17 -
Basic & Clinical Pharmacology &... May 2021Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been... (Review)
Review
Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.
Topics: Biological Assay; Biomarkers; Diagnostic Test Approval; European Union; Pharmacogenomic Testing; Precision Medicine; Reagent Kits, Diagnostic; United States; United States Food and Drug Administration
PubMed: 33665955
DOI: 10.1111/bcpt.13578 -
Bioanalysis Jul 2023The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing...
2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays ( - Recommendations on Gene...
The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.
Topics: Prescription Drugs; Technology; Biological Assay; Biomarkers; Cell- and Tissue-Based Therapy
PubMed: 37526071
DOI: 10.4155/bio-2023-0135 -
Annual Review of Analytical Chemistry... Jul 2021Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge... (Review)
Review
Adverse effects of environmental toxicants to human health have traditionally been assayed using in vitro assays. Organ-on-chip (OOC) is a new platform that can bridge the gaps between in vitro assays (or 3D cell culture) and animal tests. Microenvironments, physical and biochemical stimuli, and adequate sensing and biosensing systems can be integrated into OOC devices to better recapitulate the in vivo tissue and organ behavior and metabolism. While OOCs have extensively been studied for drug toxicity screening, their implementation in environmental toxicology assays is minimal and has limitations. In this review, recent attempts of environmental toxicology assays using OOCs, including multiple-organs-on-chip, are summarized and compared with OOC-based drug toxicity screening. Requirements for further improvements are identified and potential solutions are suggested.
Topics: Animals; Biological Assay; Ecotoxicology; Humans; Lab-On-A-Chip Devices; Oligonucleotide Array Sequence Analysis
PubMed: 33974806
DOI: 10.1146/annurev-anchem-091620-091335 -
Methods in Molecular Biology (Clifton,... 2020Assaying microtubule nucleation is essential to understand the organization of microtubule networks in any cell type. In this chapter we describe methods for measuring...
Assaying microtubule nucleation is essential to understand the organization of microtubule networks in any cell type. In this chapter we describe methods for measuring nucleation activity at centrosomes and at mitotic chromatin in cell lines, to study interphase and mitotic microtubule organization, and for measuring non-centrosomal nucleation in cultured primary neurons, to study microtubule organization in the absence of a microtubule organizing center. While a number of different approaches and variations thereof have been reported in the literature, here we aim to keep the methodology as simple as possible and thus accessible to most research laboratories.
Topics: Active Transport, Cell Nucleus; Biological Assay; Biomarkers; Cell Line; Cell Nucleus; Fluorescent Antibody Technique; Humans; Microtubules; Mitosis; Neurons; Protein Binding; Protein Multimerization
PubMed: 31879904
DOI: 10.1007/978-1-0716-0219-5_11 -
The AAPS Journal May 2022Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic...
Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.
Topics: Biological Assay; Biomarkers; Chromatography, Liquid; Mass Spectrometry; Reference Standards
PubMed: 35534647
DOI: 10.1208/s12248-022-00707-z -
Cold Spring Harbor Protocols Jul 2023Topical application and bottle bioassays measure the responses of adult mosquitoes to specific doses of an insecticide (dose-response). Topical application bioassays are...
Topical application and bottle bioassays measure the responses of adult mosquitoes to specific doses of an insecticide (dose-response). Topical application bioassays are generally used to measure the dose-response of adult mosquitoes to insecticides in which the amount (dose) of insecticides the mosquitoes receive is known in the laboratory. Here, a 0.5-µL drop of the insecticide dissolved in a relatively nontoxic solvent, such as acetone, is applied to the thorax of insects, and the insects' susceptibility to the insecticide is determined in terms of either the median lethal dose (LD) or 90% of the mortality lethal dose (LD). Bottle bioassays measure the dose-responses in which the exact amount of insecticide in a bottle is known while the exact amount of insecticide that mosquitoes (field-collected or laboratory-susceptible) receive is not known. Bottle bioassays can be either single dose tests or multiple dose applications. The bottle bioassay described in this protocol is a modified form of the World Health Organization (WHO) and U.S. Centers for Disease Control (CDC) bottle bioassays. For the single bottle assay, a detailed protocol with the amount (dose/bottle) of each insecticide and threshold times is provided by the CDC; here we provide protocols for topical and bottle bioassay applications with multiple doses.
Topics: Animals; Insecticides; Culicidae; Insecticide Resistance; Biological Assay; Acetone
PubMed: 36882289
DOI: 10.1101/pdb.prot108041 -
Methods in Molecular Biology (Clifton,... 2023Ion-exchange chromatography (IEC) is a fractionation technique that allows for the separation of ionizable molecules on the basis of differences in their electrostatic... (Review)
Review
Ion-exchange chromatography (IEC) is a fractionation technique that allows for the separation of ionizable molecules on the basis of differences in their electrostatic properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, ability to perform simultaneous quantitation, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline basic laboratory protocols to partially purify a soluble serine peptidase from bovine whole brain tissue, covering crude tissue extract preparation through to partial purification of the target enzyme using a form of IEC, namely, anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.
Topics: Animals; Cattle; Chromatography, Ion Exchange; Chromatography, Liquid; Automation; Biological Assay; Brain
PubMed: 37646998
DOI: 10.1007/978-1-0716-3362-5_9 -
Natural Product Reports Jul 2020Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product... (Review)
Review
Covering: up to 2020The National Cancer Institute of the United States (NCI) has initiated a Cancer Moonshot program entitled the NCI Program for Natural Product Discovery. As part of this effort, the NCI is producing a library of 1 000 000 partially purified natural product fractions which are being plated into 384-well plates and provided to the research community free of charge. As the first 326 000 of these fractions have now been made available, this review seeks to describe the general methods used to collect organisms, extract those organisms, and create a prefractionated library. Importantly, this review also details both cell-based and cell-free bioassay methods and the adaptations necessary to those methods to productively screen natural product libraries. Finally, this review briefly describes post-screen dereplication and compound purification and scale up procedures which can efficiently identify active compounds and produce sufficient quantities of natural products for further pre-clinical development.
Topics: Biological Assay; Biological Products; Drug Discovery; High-Throughput Screening Assays; Humans; Small Molecule Libraries
PubMed: 32186299
DOI: 10.1039/c9np00068b -
Environmental Toxicology and... Nov 2021
Topics: Animals; Biological Assay; Environmental Monitoring; Fishes; Risk Assessment; Water Pollutants, Chemical
PubMed: 34648980
DOI: 10.1016/j.etap.2021.103754