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Bioanalysis Apr 2022To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Mild acidic assay conditions and capture and...
To mitigate assay interference in the drug and target assays to support the development of monoclonal antibody REGN-Z. Mild acidic assay conditions and capture and detection antibodies with different affinities and t under different assay pHs were used to mitigate interference in the total drug and total target assays. A free target assay was also developed using a lower-affinity capture antibody with a much slower association and dissociation rate. The impact of sample incubation, dilution and storage on the accurate detection of the free target was also evaluated. The total drug, total and free target assays can accurately quantitate drug and target concentrations when tested with a subset of clinical study samples.
Topics: Antibodies, Monoclonal; Biological Assay; Indicators and Reagents
PubMed: 35297286
DOI: 10.4155/bio-2021-0276 -
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 -
Biochemistry. Biokhimiia Dec 2017The high affinity and specificity of biological receptors determine the demand for and the intensive development of analytical systems based on use of these receptors.... (Review)
Review
The high affinity and specificity of biological receptors determine the demand for and the intensive development of analytical systems based on use of these receptors. Therefore, theoretical concepts of the mechanisms of these systems, quantitative parameters of their reactions, and relationships between their characteristics and ligand-receptor interactions have become extremely important. Many mathematical models describing different bioassay formats have been proposed. However, there is almost no information on the comparative characteristics of these models, their assumptions, and predictive insights. In this review we suggested a set of criteria to classify various bioassays and reviewed classical and contemporary publications on these bioassays with special emphasis on immunochemical analysis systems as the most common and in-demand techniques. The possibilities of analytical and numerical modeling are discussed, as well as estimations of the minimum concentrations that may be detected in bioassays and recommendations for the choice of assay conditions.
Topics: Biological Assay; Models, Theoretical; Research; Research Design
PubMed: 29523069
DOI: 10.1134/S0006297917130119 -
The Yale Journal of Biology and Medicine Mar 2017Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an... (Review)
Review
Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K uptake. These modified cells do not grow in low-K medium, but functional expression of connexin hemichannels allows K uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.
Topics: Animals; Biological Assay; Connexins; Humans; Potassium; Protein Isoforms
PubMed: 28356896
DOI: No ID Found -
SLAS Discovery : Advancing Life... Jun 2019Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and...
Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and drug discovery applications. Although the setting up and running of TSA reactions is a relatively simple process, the subsequent analysis of TSA data, especially in high-throughput format, requires substantial amount of effort if conducted manually. We therefore developed the Thermal Shift Assay-Curve Rapid and Automatic Fitting Tool (TSA-CRAFT), a freely available software that enable automatic analysis of TSA data of any throughput. TSA-CRAFT directly reads real-time PCR instrument data files and displays the analyzed results in a web browser. This software features streamlined data processing and Boltzmann equation fitting, which is demonstrated in this study to provide more accurate data analysis than the commonly used first-derivative method. TSA-CRAFT is freely available as a cross-operating system-compatible standalone tool ( https://sourceforge.net/projects/tsa-craft/ ) and also as a freely accessible web server ( http://tbtlab.org/tsacraft.html ).
Topics: Biological Assay; Data Analysis; Drug Discovery; Ligands; Software; Web Browser
PubMed: 30744467
DOI: 10.1177/2472555218823547 -
The AAPS Journal May 2021In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been... (Review)
Review
In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.
Topics: Antibodies, Bispecific; Biological Assay; Cell- and Tissue-Based Therapy; Chromatography, Liquid; Drug Development; Flow Cytometry; Genetic Therapy; Guidelines as Topic; Mass Spectrometry; Oligonucleotides; Prodrugs; RNA; Recombinant Fusion Proteins
PubMed: 33942188
DOI: 10.1208/s12248-021-00594-w -
SLAS Discovery : Advancing Life... Dec 2021
Topics: Biological Assay; Drug Discovery; Humans
PubMed: 34813395
DOI: 10.1177/24725552211054044 -
Mutagenesis Apr 2022BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been...
BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.
Topics: Animals; Biological Assay; DNA Damage; Mammals; Mutagenicity Tests; Mutagens; Odorants
PubMed: 35302169
DOI: 10.1093/mutage/geac004 -
SLAS Discovery : Advancing Life... Dec 2021A diverse range of biochemical and cellular assays are used by medicinal chemists to guide compound optimization. The data collected from these assays influence... (Review)
Review
A diverse range of biochemical and cellular assays are used by medicinal chemists to guide compound optimization. The data collected from these assays influence decisions taken on structure-activity relationship (SAR) campaigns. Therefore, it is paramount that medicinal chemists have a solid understanding of the strengths and limitations of each assay being used to characterize synthesized analogs. For the successful execution of a medicinal chemistry campaign, it is our contention that an early partnership among assay biologists, informaticians, and medicinal chemists must exist. Their combined skill sets are necessary to not only design and develop robust assays but also implement an effective screening cascade in which multiple orthogonal and counter assays are selected to validate the activity and target(s) of the synthesized compounds. We review multiple cases of drug and chemical probe discovery from collaborative National Center for Advancing Translational Sciences/National Institutes of Health projects and published scientific literature in which the evaluation of compounds in secondary or orthogonal assays led to the discovery of unexpected activities, forcing a reconsideration of the original assay design that was used to discover the biological activity of the compound. Using these retrospective case studies, the goal of this Perspective is to hedge toward the development of physiologically relevant assays that are able to capture the true bioactivity of compounds being developed in a medicinal chemistry campaign.
Topics: Animals; Biological Assay; Chemistry, Pharmaceutical; Drug Discovery; Humans; Structure-Activity Relationship
PubMed: 34225522
DOI: 10.1177/24725552211026238 -
American Journal of Physiology. Renal... Nov 2018Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted... (Review)
Review
Investigators have for decades used mouse voiding patterns as end points for studying behavioral biology. It is only recently that mouse voiding patterns were adopted for study of lower urinary tract physiology. The spontaneous void spot assay (VSA), a popular micturition assessment tool, involves placing a mouse in an enclosure lined by filter paper and quantifying the resulting urine spot pattern. The VSA has advantages of being inexpensive and noninvasive, but some investigators challenge its ability to distinguish lower urinary tract function from behavioral voiding. A consensus group of investigators who regularly use the VSA was established by the National Institutes of Health in 2015 to address the strengths and weaknesses of the assay, determine whether it can be standardized across laboratories, and determine whether it can be used as a surrogate for evaluating urinary function. Here we leverage experience from the consensus group to review the history of the VSA and its uses, summarize experiments to optimize assay design for urinary physiology assessment, and make best practice recommendations for performing the assay and analyzing its results.
Topics: Animals; Biological Assay; Disease Models, Animal; Mice; Reproducibility of Results; Time Factors; Urinary Bladder; Urination; Urination Disorders; Urodynamics
PubMed: 30156116
DOI: 10.1152/ajprenal.00350.2018