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Journal of Visualized Experiments : JoVE May 2020Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid...
Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 μM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.
Topics: Biological Assay; Colorimetry; Hep G2 Cells; Humans; Limit of Detection; Lipid Peroxidation; Lipoproteins, LDL; Malondialdehyde; Oxidation-Reduction; Oxidative Stress; Reference Standards; Reproducibility of Results; Thiobarbituric Acid Reactive Substances
PubMed: 32478759
DOI: 10.3791/61122 -
Bioanalysis Jun 2022Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the...
Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.
Topics: Biological Assay; Real-Time Polymerase Chain Reaction
PubMed: 35703321
DOI: 10.4155/bio-2022-0109 -
SLAS Discovery : Advancing Life... Jun 2019Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its...
Many factors must be considered during the optimization of an enzyme assay. These include the choice of buffer and its composition, the type of enzyme and its concentration, as well as the type of substrate and concentrations, the reaction conditions, and the appropriate assay technology. The process of an enzyme assay optimization, in our experience, can take more than 12 weeks using the traditional one-factor-at-a-time approach. In contrast, the design of experiments (DoE) approaches have the potential to speed up the assay optimization process and provide a more detailed evaluation of tested variables. However, not all researchers are aware of DoE approaches or believe that it is easy to employ a DoE approach for the optimization of an assay. In order to facilitate enzyme assay developers to use DoE methodologies, we present in detail the steps required to identify in less than 3 days (1) the factors that significantly affect the activity of an enzyme and (2) the optimal assay conditions using a fractional factorial approach and response surface methodology. This is exemplified with the optimization of assay conditions for the human rhinovirus-3C protease, and the methodology used could be employed as a basic guide for the speedy identification of the optimum assay conditions for any enzyme.
Topics: 3C Viral Proteases; Biological Assay; Cysteine Endopeptidases; Enzyme Assays; Humans; Research Design; Substrate Specificity; Viral Proteins
PubMed: 30802413
DOI: 10.1177/2472555219830084 -
Nucleic Acids Research Jan 2023PubChem (https://pubchem.ncbi.nlm.nih.gov) is a popular chemical information resource that serves a wide range of use cases. In the past two years, a number of changes...
PubChem (https://pubchem.ncbi.nlm.nih.gov) is a popular chemical information resource that serves a wide range of use cases. In the past two years, a number of changes were made to PubChem. Data from more than 120 data sources was added to PubChem. Some major highlights include: the integration of Google Patents data into PubChem, which greatly expanded the coverage of the PubChem Patent data collection; the creation of the Cell Line and Taxonomy data collections, which provide quick and easy access to chemical information for a given cell line and taxon, respectively; and the update of the bioassay data model. In addition, new functionalities were added to the PubChem programmatic access protocols, PUG-REST and PUG-View, including support for target-centric data download for a given protein, gene, pathway, cell line, and taxon and the addition of the 'standardize' option to PUG-REST, which returns the standardized form of an input chemical structure. A significant update was also made to PubChemRDF. The present paper provides an overview of these changes.
Topics: Databases, Chemical; Drug Discovery; Biological Assay; Proteins; Cheminformatics
PubMed: 36305812
DOI: 10.1093/nar/gkac956 -
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 -
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 -
International Journal of Molecular... May 2021Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most... (Review)
Review
Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules.
Topics: Animals; Antibodies, Bispecific; Antigens; Biological Assay; Humans; Immunotherapy
PubMed: 34069573
DOI: 10.3390/ijms22105350 -
Sensors (Basel, Switzerland) Dec 2020Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of... (Review)
Review
Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.
Topics: Biological Assay; Fluorescence Polarization; Fluorescent Dyes; Metals; Nucleic Acids
PubMed: 33322750
DOI: 10.3390/s20247132 -
Trends in Biotechnology Jan 2023Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in... (Review)
Review
Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity, remain. Herein, we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights into remaining challenges and future research trends.
Topics: Microfluidics; High-Throughput Screening Assays; Drug Evaluation, Preclinical; Biological Assay; Microfluidic Analytical Techniques
PubMed: 35863950
DOI: 10.1016/j.tibtech.2022.06.006 -
Annual Review of Analytical Chemistry... Jul 2021Droplet-based microfluidics has emerged as an important subfield within the microfluidic and general analytical communities. Indeed, several unique applications such as... (Review)
Review
Droplet-based microfluidics has emerged as an important subfield within the microfluidic and general analytical communities. Indeed, several unique applications such as digital assay readout and single-cell sequencing now have commercial systems based on droplet microfluidics. Yet there remains room for this research area to grow. To date, most analytical readouts are optical in nature, relatively few studies have integrated sample preparation, and passive means for droplet formation and manipulation have dominated the field. Analytical scientists continue to expand capabilities by developing droplet-compatible method adaptations, for example, by interfacing to mass spectrometers or automating droplet sampling for temporally resolved analysis. In this review, we highlight recently developed fluidic control techniques and unique integrations of analytical methodology with droplet microfluidics-focusing on automation and the connections to analog/digital domains-and we conclude by offering a perspective on current challenges and future applications.
Topics: Biological Assay; Mass Spectrometry; Microfluidic Analytical Techniques; Microfluidics
PubMed: 33979546
DOI: 10.1146/annurev-anchem-122120-042627