Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques.

Does triple-negative phenotype accurately identify basal-like tumour? An immunohistochemical analysis based on 143 ‘triple-negative’ breast cancers

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

A sensitive radioreceptor assay (RRA) to determine the serum concentrations of fentanyl, pentazocine and morphine was developed on the basis of the drug's competition with a labeled tracer ((3)H-naloxone) for the membrane bound opioid receptor in rat brain homogenates. The binding data were computer-fitted to a standard curve by means of nonlinear least square regression. Sensitivity of the assay applied directly to serum samples without extraction was limited to approximately 3, 5 and 25 ng/ml for fentanyl, morphine and pentazocine, respectively, because of endogenous plasma constituents that interfere with the opioid receptor binding. With the use of petrol-ether extraction the sensitivity was improved to 0.3 ng/ml fentanyl and 3 ng/ml pentazocine (0.3 ml serum samples). No RRA-active metabolites were detectable after HPLC separation of serum from a patient treated with fentanyl. The plasma concentration time course of fentanyl in a patient, measured by RRA, was similar to that obtained by a radioimmunoassay (RIA). The RRA represents a general procedure for the detection of clinically used opioid analgesics and their active metabolites.

Determination of α1-adrenoceptor antagonists in plasma by radioreceptor assay

IntroductionInsulin and proinsulin autoantibodies (IAA/PAA) are usually the first markers to appear in patients with type 1 Diabetes Mellitus (T1DM) and their prevalence ranges from 10 to 60% in the child-adolescent population. The reference method for IAA/PAA detection is the Radioligand Binding Assay (RBA), a highly specific and sensitive technique, but expensive and polluting. The aim of this work was to develop a novel flow cytometric microsphere-based immunoassay (FloCMIA) for PAA detection, employing recombinant human proinsulin (PI), as an alternative method to RBA, less expensive and harmful to the environment.Materials and MethodsHuman PI was expressed as Thioredoxin fusion protein (TrxPI) in E. coli and a fraction was biotinylated. A double paratope model was used in which samples were incubated with TrxPI–biotin and microspheres adsorbed with TrxPI. The immune complexes were revealed using Streptavidin–Phycoerythrin. The geometric mean of the signals was analyzed, and the results were expressed as Standard Deviation scores (SDs). Sera from 100 normal human control and from 111 type 1 diabetic patients were evaluated by FloCMIA. To correlate the novel assay with RBA, 51 diabetic patients were selected, spanning a wide range of PAA reactivity by RBA.ResultsThe study of ROC curves allowed choosing a cut-off value of 3.0 SDs and the AUC was 0.705, indicating that FloCMIA has fair ability to distinguish between samples from each group. A prevalence of 50% for PAA was obtained in the population of diabetic patients studied. The specificity was 96% and the analytical sensitivity (percentage of patients RBA positive, also positive by FloCMIA) was 69%. There was a substantial agreement between methods (kappa statistic=0.700).ConclusionsA novel immunoassay based on flow cytometry that uses easy-to produce recombinant PI was developed. This assay constitutes an innovative and cost-effective alternative to RBA for the determination of PAA in patients’ sera. The method developed here, presents good performance and a wide dynamic range together with a small required sample volume. Furthermore, these results make it possible to develop multiplex immunoassays that allow the combined detection of autoantibodies present in T1DM and other related autoimmune diseases.

Ultrasound strain imaging utilizes radio-frequency (RF) ultrasound echo signals to estimate the relative elasticity of tissue under deformation. Due to the diagnostic value inherent in tissue elasticity, ultrasound strain imaging has found widespread clinical and preclinical applications. Accurate displacement estimation using pre and post-deformation RF signals is a crucial first step to derive high quality strain tensor images. Incorporating regularization into the displacement estimation framework is a commonly employed strategy to improve estimation accuracy and precision. In this work, we propose an adaptive variation of the iterative Bayesian regularization scheme utilizing RF similarity metric signal-to-noise ratio previously proposed by our group. The regularization scheme is incorporated into a 2D multi-level block matching (BM) algorithm for motion estimation. Adaptive nature of our algorithm is attributed to the dynamic variation of iteration number based on the normalized cross-correlation (NCC) function quality and a similarity measure between pre-deformation and motion compensated post-deformation RF signals. The proposed method is validated for either quasi-static and cardiac elastography or strain imaging applications using uniform and inclusion phantoms and canine cardiac deformation simulation models. Performance of adaptive Bayesian regularization was compared to conventional NCC and Bayesian regularization with fixed number of iterations. Results from uniform phantom simulation study show significant improvement in lateral displacement and strain estimation accuracy. For instance, at 1.5% lateral strain in a uniform phantom, Bayesian regularization with five iterations incurred a lateral strain error of 104.49%, which was significantly reduced using our adaptive approach to 27.51% (p < 0.001). Contrast-to-noise (CNRe) ratios obtained from inclusion phantom indicate improved lesion detectability for both axial and lateral strain images. For instance, at 1.5% lateral strain, Bayesian regularization with five iterations had lateral CNRe of −0.31 dB which was significantly increased using the adaptive approach to 7.42 dB (p < 0.001). Similar results are seen with cardiac deformation modelling with improvement in myocardial strain images. In vivo feasibility was also demonstrated using data from a healthy murine heart. Overall, the proposed method makes Bayesian regularization robust for clinical and preclinical applications.

The liver is an essential organ of the body that performs several vital functions, including the metabolism of biomolecules, foreign substances, and toxins, and the production of plasma proteins, such as coagulation factors. There are hundreds of genetic disorders affecting liver functions and, for many of them, the only curative option is orthotopic liver transplantation, which nevertheless entails many risks and long-term complications. Some peculiar features of the liver, such as its large blood flow supply and the tolerogenic immune environment, make it an attractive target for in vivo gene therapy approaches. In recent years, several genome-editing tools mainly based on the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system have been successfully exploited in the context of liver-directed preclinical or clinical therapeutic applications. These include gene knock-out, knock-in, activation, interference, or base and prime editing approaches. Despite many achievements, important challenges still need to be addressed to broaden clinical applications, such as the optimization of the delivery methods, the improvement of the editing efficiency, and the risk of on-target or off-target unwanted effects and chromosomal rearrangements. In this review, we highlight the latest progress in the development of in vivo liver-targeted genome editing approaches for the treatment of genetic disorders. We describe the technological advancements that are currently under investigation, the challenges to overcome for clinical applicability, and the future perspectives of this technology.

Achieving the required sensitivity can be a challenge in the development of ligand binding assays for pharmacokinetic (PK) determinations of biotherapeutics. To address this need, BioScale's Acoustic Membrane Microparticle (AMMP) technology was evaluated for the quantification of a PEGylated domain antibody (dAb) biotherapeutic. Previous uses of this technology had shown utility in biomarker and process development applications and this is the first application, to our knowledge, for PK determinations. In this evaluation, AMMP was capable of delivering a sensitivity of 0.750 ng/mL, which surpasses the sensitivity requirements for the majority of assays to support PK determinations. This evaluation demonstrates that this emerging technology has the ability to produce the required sensitivity, reproducibility, and selectivity needed to meet the industry's standards for PK analysis.

Acoustic-resolution photoacoustic micro-scopy (AR-PAM) is an emerging biomedical imaging modality that combines superior optical sensitivity and fine ultrasonic resolution in an optical quasi-diffusive regime (~1-3 mm in tissues). AR-PAM has been explored for anatomical, functional, and molecular information in biological tissues. Heretofore, AR-PAM systems have suffered from a limited field-of-view (FOV) and/or slow imaging speed, which have precluded them from routine preclinical and clinical applications. Here, we demonstrate an advanced AR-PAM system that overcomes both limitations of previous AR-PAM systems. The new AR-PAM system demonstrates a super wide-field scanning that utilized a 1-axis water-proofing microelectromechanical systems (MEMS) scanner integrated with two linear stepper motor stages. We achieved an extended FOV of 36 ×80 mm2 by mosaicking multiple volumetric images of 36 ×2.5 mm2 with a total acquisition time of 224 seconds. For one volumetric data (i.e., 36 ×2.5 mm2), the B-scan imaging speed over the short axis (i.e., 2.5 mm) was 83 Hz in humans. The 3D volumetric image was also provided by using MEMS mirror scanning along the X-axis and stepper-motor scanning along the Y-axis. The super-wide FOV mosaic image was realized by registering and merging all individual volumetric images. Finally, we obtained multi-plane whole-body in-vivo PA images of small animals, illustrating distinct multi-layered structures including microvascular networks and internal organs. Importantly, we also visualized microvascular networks in human fingers, palm, and forearm successfully. This advanced MEMS-AR-PAM system could potentially enable hitherto not possible wide preclinical and clinical applications.

To obtain information on the occurrence and location of molecular events as well as to track target-specific probes such as antibodies or peptides, drugs or even cells non-invasively over time, optical imaging (OI) technologies are increasingly applied. Although OI strongly contributes to the advances made in preclinical research, it is so far, with the exception of optical coherence tomography (OCT), only very sparingly applied in clinical settings. Nevertheless, as OI technologies evolve and improve continuously and represent relatively inexpensive and harmful methods, their implementation as clinical tools for the assessment of children disease is increasing. This review focuses on the current preclinical and clinical applications as well as on the future potential of OI in the clinical routine. Herein, we summarize the development of different fluorescence and bioluminescence imaging techniques for microscopic and macroscopic visualization of microstructures and biological processes. In addition, we discuss advantages and limitations of optical probes with distinct mechanisms of target-detection as well as of different bioluminescent reporter systems. Particular attention has been given to the use of near-infrared (NIR) fluorescent probes enabling observation of molecular events in deeper tissue.

Magnetic resonance fingerprinting (MRF) is an approach to quantitative magnetic resonance imaging that allows for efficient simultaneous measurements of multiple tissue properties, which are then used to create accurate and reproducible quantitative maps of these properties. As the technique has gained popularity, the extent of preclinical and clinical applications has vastly increased. The goal of this review is to provide an overview of currently investigated preclinical and clinical applications of MRF, as well as future directions. Topics covered include MRF in neuroimaging, neurovascular, prostate, liver, kidney, breast, abdominal quantitative imaging, cardiac, and musculoskeletal applications.

Bioanalysis Rising Star Award 2018: announcing our finalists!

Targeted protein degradation (TPD) platforms, including PROTACs and molecular glues, have revolutionized drug discovery as transformative therapeutic strategies. Recent advancements in mass spectrometry-based proteomics have dramatically improved assay speed and sensitivity, providing actionable insights into degradation profiles and drug efficacy on timescales compatible with discovery chemistry. This study evaluates the practical utility of advanced proteomic assays in supporting TPD research, focusing on their role in streamlining drug discovery pipelines. State-of-the-art proteomic workflows were employed to quantify the proteome-wide effects of PROTACs and molecular glues across diverse cellular models. Assay sensitivity, reproducibility, and throughput were rigorously assessed to determine their real-world applicability. Our findings reveal that these enhanced technologies generate robust, high-resolution datasets, capturing the dynamic interplay between target degradation, protein interactions, and cellular responses. This enabled the rapid identification of optimal degrader candidates and informed structure-activity relationship (SAR) studies. Mass spectrometry-based proteomics has emerged as an essential tool for advancing TPD strategies. By combining heightened sensitivity with operational feasibility, these assays offer critical insights that accelerate the development of PROTACs and molecular glues, bridging the gap between foundational research and clinical application. Citation Format: Michael J. Ford, Ravi Amunugama, David L. Allen, Richard C. Jones. Mass spectrometry-based proteomics for accelerating PROTAC and molecular glue drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7027.

ABSTRACTAnti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug. We describe the generation and characterization of such reagents for the development of ligand binding assays for drug quantification. We also show how these Type 3 antibodies can be used to develop very specific and sensitive assays that avoid the bridging format.Abbreviations: BAP: bacterial alkaline phosphatase; CDR: complementarity-determining regions in VH or VL; Fab: antigen-binding fragment of an antibody; HRP: horseradish peroxidase; HuCAL®: Human Combinatorial Antibody Libraries; IgG: immunoglobulin G; LBA: ligand binding assay; LOQ: limit of quantitation; NHS: normal human serum; PK: pharmacokinetics; VH: variable region of the heavy chain of an antibody; VL: variable region of the light chain of an antibody.

The Journal of Bone and Joint Surgery. British volumeVol. 64-B, No. 5 ArticlesFree AccessVitamin D metabolism and its clinical applicationJA KanisJA KanisSearch for more papers by this authorPublished Online:1 Dec 1982https://doi.org/10.1302/0301-620X.64B5.6754741AboutSectionsPDF/EPUB ToolsAdd to FavouritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByDetection of Vitamin D Metabolites in Breast Milk: Perspectives and challenges for measurement by Liquid Chromatography Tandem-Mass SpectrometryClinical Biochemistry, Vol. 175Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatographyTalanta, Vol. 190Recent trends in the analysis of vitamin D and its metabolites in milk – A reviewFood Chemistry, Vol. 171Osteochondrosis or Epiphysitis and Other Miscellaneous AffectionsCharacterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to 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in epileptic patients on carbamazepine29 January 2009 | Acta Neurologica Scandinavica, Vol. 71, No. 5The effects of different vitamin D-states on intestinal absorption of vitamin D 3 and its metabolites in ratsActa Physiologica Scandinavica, Vol. 123, No. 4Discrepancy between serum 1,25-dihydroxycholecalciferol measured by radioimmunoassay and cytosol radioreceptor assay8 July 2009 | Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 45, No. 8A SENSITIVE AND SELECTIVE RADIOIMMUNOASSAY FOR SERUM 24, 25-DIHYDROXYCHOLECALCIFEROL IN MANClinical Endocrinology, Vol. 21, No. 1LiteraturMeasurement of 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 in clinical settings28 August 2009 | Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 44, No. 7A selective and simplified radioimmunoassay of 25-hydroxyvitamin D 35 August 2009 | Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 44, No. 2 Vol. 64-B, No. 5 Metrics Downloaded 617 times History Published online 1 December 1982 Published in print 1 December 1982 InformationCopyright © 1982, The British Editorial Society of Bone and Joint Surgery: All rights reservedPDF download