Total antibody, conjugated antibody or antibody-conjugated drug, and free drug are key analytes required to establish exposure-response relationships for ADCs. Therefore, bioanalytical strategies for ADCs include ligand-binding assays (LBA) and LC-MS/MS methods. Here we describe detailed methodology to develop a solid-phase-based enzyme-linked immunosorbent assay (ELISA), which is the most widely used LBA to quantify large-molecule components of ADC in biological matrices such as plasma, serum, tumor, or tissue homogenates. The approach presented here is designed to quantify total antibody concentrations in ADC containing samples, and can be easily adapted to quantify conjugated antibody concentrations.
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http://dx.doi.org/10.1007/978-1-4939-9929-3_26 | DOI Listing |
NPJ Breast Cancer
January 2025
Cancer Science Institute of Singapore, National University of, Singapore, Singapore.
Point mutations in the ligand binding domain of retinoic acid receptor alpha (RARα) are linked to breast fibroepithelial tumor development, but their role in solid tumorigenesis is unclear. In this study, we assessed the functional effects of known RARα mutations on retinoic acid signaling using biochemical and cellular assays. All tested mutants exhibited reduced transcriptional activity compared to wild-type RARα and showed a dominant negative effect, a feature associated with developmental defects and tumor formation.
View Article and Find Full Text PDFJ Immunol Methods
December 2024
Institute for Animal Health, Henan Academy of Agricultural Sciences, Zhengzhou, China; College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoon-Ose, Yangzhou University, Yangzhou, China. Electronic address:
Background: Bovine IgG1 Fc receptor (boFcγRI) is a homologue to human FcγRI (CD64) that has three extracellular Ig-like domains and can bind bovine IgG1 with high affinity. Identification of the linear epitope for Fc-binding on boFcγRI provides new insights for the IgG-Fcγ interaction and FcγR-targeting drugs development.
Methods: The boFcγRI molecules were expressed on cell surface of the boFcγRI -transfected COS-7 cells.
J Pharmacol Toxicol Methods
December 2024
Department of Immunoassay and Immunochemistry, Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 101408, China; Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China. Electronic address:
Background: Antibody-drug conjugates (ADCs) are an emerging class of targeted therapeutics and are receiving growing attention in the pharmaceutical field. Here we aimed to validate two ligand binding assays for the quantitation of GQ1001, an ADC made of Trastuzumab site-specifically conjugated with DM1, in cynomolgus monkey serum, and then apply the validated assays to a nonclinical study.
Methods: The quantitative methods for conjugated GQ1001 and total GQ1001 were validated against regulatory guidance documents on bioanalytical method validation under a Good Laboratory Practice (GLP)-compliant environment.
Front Physiol
December 2024
National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Introduction: Adrenergic activation of protein kinase A (PKA) in cardiac muscle targets the sarcolemma, sarcoplasmic reticulum, and contractile apparatus to increase contractile force and heart rate. In the thin filaments of the contractile apparatus, cardiac troponin I (cTnI) Ser22 and Ser23 in the cardiac-specific N-terminal peptide (NcTnI: residues 1 to 32) are the targets for PKA phosphorylation. Phosphorylation causes a 2-3 fold decrease of affinity of cTn for Ca associated with a higher rate of Ca dissociation from cTnC leading to a faster relaxation rate of the cardiac muscle (lusitropy).
View Article and Find Full Text PDFJ Biomol Struct Dyn
December 2024
School of Biotechnology, KIIT Deemed To be University, Bhubaneswar, Odisha, India.
The FIKK protein family, encompassing 21 serine-threonine protein kinases, is a distinctive cluster exclusive to the Apicomplexa phylum. Predominantly located in which is a malarial parasite, with a solitary gene identified in a distinct apicomplexan species, this family derives its nomenclature from - phenylalanine, isoleucine, lysine, lysine (FIKK), a conserved amino acid motif. Integral to the parasite's life cycle and consequential to malaria pathogenesis, the absence of orthologous proteins in eukaryotic organisms designates it as a promising antimalarial drug target.
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