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Current Protocols Aug 2023Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method...
Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry, along with practical procedures for four different types of validation. The first, Basic Protocol 1 (the limited validation protocol), is recommended for research and non-regulated laboratories. Next, Basic Protocol 2) presents a reasonable, fit-for-purpose validation approach appropriate for biopharma and research settings. Basic Protocol 3 addresses the type of validation performed in clinical laboratories for moderate-risk tests developed in house. Finally, Basic Protocol 4 describes the process that should be applied whenever a method is being transferred from one facility to another. All four validation plans follow the fit-for-purpose validation approach, in which the validation parameters are selected based on the intended use of the assay. These validation protocols represent the minimal requirement and may not be applicable for every intended use such as high-risk clinical assays or data to be used as a primary endpoint in a clinical trial. The recommendations presented here are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (CLSI, 2021). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Limited validation Basic Protocol 2: Fit-for-purpose validation for biopharma and research settings Basic Protocol 3: Validation for moderate clinical risk laboratory developed tests Basic Protocol 4: Transfer validation.
Topics: Flow Cytometry; Research Design; Academies and Institutes; Biological Assay; Clinical Laboratory Services
PubMed: 37606503
DOI: 10.1002/cpz1.868 -
Frontiers in Immunology 2023Laminin 332 is a heterotrimeric structural protein of the basal membrane zone (BMZ) of the skin and adjacent mucosal tissues. The importance of laminin 332 for the... (Review)
Review
Laminin 332 is a heterotrimeric structural protein of the basal membrane zone (BMZ) of the skin and adjacent mucosal tissues. The importance of laminin 332 for the structural integrity of the BMZ is demonstrated by mutations in any of the three genes encoding for its three chains causing variants of junctional epidermolysis bullosa. Autoimmunity against laminin 332 is observed in mucous membrane pemphigoid (MMP) and in the rare patients with orf-induced pemphigoid. MMP is an autoimmune blistering disease with predominant mucosal manifestations and autoantibodies against the BMZ of the skin and orifice-close mucous membranes. The main autoantigens of MMP are type XVII collagen (BP180) and laminin 332 targeted in about 80% and 10-20% of patients, respectively. An increasing number of studies has highlighted the association of anti-laminin 332 MMP and malignancies that can be revealed in about a quarter of these patients. This data has led to the recommendation of current guidelines to assay for anti-laminin 332 reactivity in all MMP patients. The present review focuses on anti-laminin 332 MMP describing clinical features, its pathophysiology, and detection of serum anti-laminin 332 IgG. In addition, the available data about the occurrence of malignancies in anti-laminin 332 MMP, the underlying tumor entities, and its biology are detailed.
Topics: Humans; Autoimmunity; Pemphigoid, Bullous; Autoantibodies; Skin; Biological Assay
PubMed: 37638011
DOI: 10.3389/fimmu.2023.1250115 -
International Journal of Molecular... Feb 2024Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their... (Review)
Review
Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.
Topics: Protein Stability; Proteins; Amino Acids; Fluorometry; Biological Assay; Protein Denaturation
PubMed: 38339045
DOI: 10.3390/ijms25031764 -
Drug Discovery Today Mar 2024G-protein-coupled receptors (GPCRs) are the target of >30% of approved drugs. Despite their popularity, many of the >800 human GPCRs remain understudied. The... (Review)
Review
G-protein-coupled receptors (GPCRs) are the target of >30% of approved drugs. Despite their popularity, many of the >800 human GPCRs remain understudied. The Illuminating the Druggable Genome (IDG) project has generated many tools leading to important insights into the function and druggability of these so-called 'dark' receptors. These tools include assays, such as PRESTO-TANGO and TRUPATH, billions of small molecules made available via the ZINC virtual library, solved orphan GPCR structures, GPCR knock-in mice, and more. Together, these tools are illuminating the remaining 'dark' GPCRs.
Topics: Humans; Animals; Mice; Receptors, G-Protein-Coupled; Biological Assay; Ligands
PubMed: 38052317
DOI: 10.1016/j.drudis.2023.103848 -
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 -
Analytica Chimica Acta Oct 2023Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not... (Review)
Review
Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not surprising that many researchers have thought about MIPs as artificial receptors in immunoassay-like analytical applications, where the general machinery of the assay is maintained, but the molecular recognition is no longer assured by an antibody but by an artificial receptor. However, the number of papers devoted explicitly to applications of MIPs in the immunoassay field is quite limited if compared to the huge number of papers covering the multifaceted molecular imprinting technology. For this reason, this critical review wants to give a general view of MIP-based immunoassays, trying to highlight the critical points that have so far prevented a wider application of molecular imprinting technology in the immunoassay field and, possibly, try to suggest strategies to overcome them.
Topics: Humans; Antibodies; Biological Assay; Immunoassay; Molecular Imprinting; Molecularly Imprinted Polymers
PubMed: 37604627
DOI: 10.1016/j.aca.2023.341547 -
Carbohydrate Polymers Jul 2024More than 3000 proteins are now known to bind to glycosaminoglycans (GAGs). Yet, GAG-protein systems are rather poorly understood in terms of selectivity of recognition,... (Review)
Review
More than 3000 proteins are now known to bind to glycosaminoglycans (GAGs). Yet, GAG-protein systems are rather poorly understood in terms of selectivity of recognition, molecular mechanism of action, and translational promise. High-throughput screening (HTS) technologies are critically needed for studying GAG biology and developing GAG-based therapeutics. Microarrays, developed within the past two decades, have now improved to the point of being the preferred tool in the HTS of biomolecules. GAG microarrays, in which GAG sequences are immobilized on slides, while similar to other microarrays, have their own sets of challenges and considerations. GAG microarrays are rapidly becoming the first choice in studying GAG-protein systems. Here, we review different modalities and applications of GAG microarrays presented to date. We discuss advantages and disadvantages of this technology, explain covalent and non-covalent immobilization strategies using different chemically reactive groups, and present various assay formats for qualitative and quantitative interpretations, including selectivity screening, binding affinity studies, competitive binding studies etc. We also highlight recent advances in implementing this technology, cataloging of data, and project its future promise. Overall, the technology of GAG microarray exhibits enormous potential of evolving into more than a mere screening tool for studying GAG - protein systems.
Topics: Glycosaminoglycans; Binding, Competitive; Biological Assay; Microarray Analysis; Research
PubMed: 38616080
DOI: 10.1016/j.carbpol.2024.122106 -
Frontiers in Immunology 2023Tuberculosis (TB) remains a major underdiagnosed public health threat worldwide, being responsible for more than 10 million cases and one million deaths annually. TB... (Review)
Review
Tuberculosis (TB) remains a major underdiagnosed public health threat worldwide, being responsible for more than 10 million cases and one million deaths annually. TB diagnosis has become more rapid with the development and adoption of molecular tests, but remains challenging with traditional TB diagnosis, but there has not been a critical review of this area. Here, we systematically review these approaches to assess their diagnostic potential and issues with the development and clinical evaluation of proposed CRISPR-based TB assays. Based on these observations, we propose constructive suggestions to improve sample pretreatment, method development, clinical validation, and accessibility of these assays to streamline future assay development and validation studies.
Topics: Humans; Biological Assay; Public Health; Tuberculosis
PubMed: 37600797
DOI: 10.3389/fimmu.2023.1172035 -
Analytical Biochemistry Dec 2023Microarrays are powerful tools for high-throughput bioassays that can extract information from tens of thousands of micro-spots consisting of biomolecules. This... (Review)
Review
Microarrays are powerful tools for high-throughput bioassays that can extract information from tens of thousands of micro-spots consisting of biomolecules. This information is invaluable to many applications, such as drug discovery and disease diagnostics. Different applications of these microarrays need spots of different shapes, sizes, and chemistries to achieve their goals. Micro/nano-fabrication techniques are used to make microarrays with different feature structures and array densities for required assay procedures. Understanding these fabrication methods is essential to creating an effective microarray. The purpose of this article is to critically review fabrication methods used in recent microarray-based bioassay studies. We summarized commonly used microarray fabrication techniques and filled the gap in recent literature on relevant topics. We discussed recent examples of how microarrays were fabricated and used in a variety of bioassays. Specifically, we examined microarray printing, various microlithography techniques, and microfluidics-based microarray fabrication. We evaluated how their application shaped the fabrication methods and compared their performance based on different applications. In the end, we discussed current challenges and outlined potential future directions. This review addressed the gap in literature and provided important insights for choosing appropriate fabrication techniques towards different applications.
Topics: Microarray Analysis; Microfluidics; Biological Assay
PubMed: 37914004
DOI: 10.1016/j.ab.2023.115369 -
Journal of Medicinal Chemistry Nov 2023Tricyclic tetrahydroquinolines (THQs) have been repeatedly reported as hits across a diverse range of high-throughput screening (HTS) campaigns. The activities of these... (Review)
Review
Tricyclic tetrahydroquinolines (THQs) have been repeatedly reported as hits across a diverse range of high-throughput screening (HTS) campaigns. The activities of these compounds, however, are likely due to reactive byproducts that interfere with the assay. As a lesser studied class of pan-assay interference compounds, the mechanism by which fused THQs react with protein targets remains largely unknown. During HTS follow-up, we characterized the behavior and stability of several fused tricyclic THQs. We synthesized key analogues to pinpoint the cyclopentene ring double bond as a source of reactivity of fused THQs. We found that these compounds degrade in solution under standard laboratory conditions in days. Importantly, these observations make it likely that fused THQs, which are ubiquitously found within small molecule screening libraries, are unlikely the intact parent compounds. We urge deprioritization of tricylic THQ hits in HTS follow-up and caution against the investment of resources to follow-up on these problematic compounds.
Topics: High-Throughput Screening Assays; Small Molecule Libraries; Quinolines; Biological Assay
PubMed: 37874947
DOI: 10.1021/acs.jmedchem.3c01277