This position paper presents a novel perspective on addressing the challenges of digital transformation in higher education through the development of a qualification-based learning model (QBLM) qualification management methodology. It argues that the rapid pace of technological advancement and the resulting need for continuous upskilling and reskilling necessitate a more dynamic and adaptive approach to human-resource management and development. The paper posits that by extending QBLM through the integration of artificial intelligence (AI) and machine learning (ML), a more effective system for analyzing competence requirements and designing personalized learning pathways can be created. The paper proposes a three-fold approach: (1) developing the FPHR ontology to support semantic annotation of HR qualifications in higher-education institutions (HEIs), (2) integrating this ontology into QBLM to ensure the machine-readability of qualifications, and (3) modeling a knowledge-based production process for HRs in skills-based learning. This paper outlines the current state of the art, presents conceptual models, and describes planned proof-of-concept implementations and evaluations. It contends that this approach will significantly enhance the effectiveness of human-resource development in the rapidly evolving digital knowledge society. By presenting this position, the paper aims to stimulate discussion and collaboration within the academic community on innovative approaches to qualification management in higher education. The work addresses critical issues arising from technological development and offers a forward-thinking solution to bridge the gap between current and future skill requirements in industry and academia.

Correction for ‘Smartphone-read phage lateral flow assay for point-of-care detection of infection’ by Maede Chabi, et al. , Analyst , 2023, 148 , 839–848, https://doi.org/10.1039/D2AN01499H.

Measurement of antibody and antibody fusion protein concentration is vital for process development and manufacturing. Continuous, in-line monitoring of antibody concentration could be useful in a variety of applications, such as controlling the loading of protein A columns to prevent breakthrough, monitoring bioreactor titer, and detecting leaks past ultrafiltration/diafiltration membranes. Molecule-specific monitoring techniques are advantageous for antibody detection in cell culture fluid in the presence of complex process impurities. Here we report a continuous in-line, real-time IgG monitoring platform using a fiber-optic biosensor with a replaceable sensor tip covalently functionalized with a fluor-labeled protein consisting of a pentamer of the Z domain (a more stable form of the B domain) of protein A. The sensor demonstrates concentration-dependent fluorescence enhancement in the presence of human IgG (0.01-0.75 g/L), with consistent signals during five runs each with 1 and 0.1 g/L IgG, and maintains its specificity in the presence of Chinese hamster ovary (CHO) cell culture fluid. A 5% breakthrough of a typical 10 g/L load would be detected in less than 20 s in a flowing stream emerging from a protein A column without prior sample preparation. This sensor platform may be suitable for monitoring IgG and fragment, crystallizable (Fc) fusion proteins in diverse upstream and downstream bioprocess applications.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow. We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers. In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

Correction for 'Smartphone-read phage lateral flow assay for point-of-care detection of infection' by Maede Chabi, et al., Analyst, 2023, 148, 839-848, https://doi.org/10.1039/D2AN01499H.

In this chapter, a competency-based approach, based on the qualifications-based learning model (QBLM), will be presented, which makes it possible to store the acquired competencies and qualifications (CQ) in a so-called CQ-profile (CQP) while playing an educational game. The game and the course are based on a didactical structural template (DST), making it possible to switch between the course and the game. Before the educational game can be played, it must be checked whether a learner has all the required CQs for the course or learning unit in which the educational game is included. Therefore, this chapter shows an approach to how the CQPs can be implemented, and the data from the CQP can be used to check course prerequisites. Finally, a prototypical implementation with its evaluation and the remaining challenges will be presented in this chapter.

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL-over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

Glow enzyme-linked immunosorbent assay (glow ELISA) uses inexpensive and shelf-stable glow stick reagents to chemically excite fluorescent reporters, obviating the need for excitation light sources, filters, and complex optics. It achieves excellent limits of detection while offering portability and equipment cost comparable to lateral flow immunoassays.

PDF file - 1272K, Comparison of MDM2 binding properties of RG7112 and nutlin-3a (S1); Overlay of the crystal structures of MDM2 bound to RG7112 and Nutlin-2 (S2); RG7112 is a non-genotoxic p53 activator (S3); Effect of RG7112 on the viability of HCT116 cancer cells and their p53-null clone HCT116R1 (S4); RG7112 blocks the proliferation of mouse NIH-3T3 cells (S5).

The COVID-19 pandemic has highlighted the urgent need for sensitive, affordable, and widely-accessible testing at the point-of-care. Here we report the development of a sensitive chemiluminescence-based smartphone-readable lateral flow assay for the detection of SARS-CoV-2 nucleoprotein using M13 phage conjugated with antibodies and HRP enzymes as LFA reporter particles. We screened 84 anti-nucleoprotein monoclonal antibody pairs in phage LFA and identified an antibody pair that gave an LoD of 25 pg/mL nucleoprotein in nasal swab extract using a FluorChem gel documentation system and 100 pg/mL when the test was imaged and analyzed by an in-house-developed smartphone reader. The smartphone-read LFA signals for positive clinical samples tested (N = 15, with known Ct) were statistically different (p < 0.001) from negative clinical samples tested (N = 11). The translation-ready phage LFA technology combined with smartphone chemiluminescence imaging can enable the timely development of ultrasensitive, affordable point-of-care testing platforms for SARS-CoV-2 and beyond.

PDF file - 12K, Antitumor activity of RG7112 against a panel of solid tumor cells.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immu-noassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Early diagnosis of infectious diseases improves outcomes by enabling earlier delivery of effective treatment, and helps prevent further transmission by undiagnosed persons. We demonstrated a proof-of-concept assay combining isothermal amplification and lateral flow assay (LFA) for early diagnosis of cutaneous leishmaniasis, a vector-borne infectious disease that affects ca. 700,000 to 1.2 million people annually. Conventional molecular diagnostic techniques based on polymerase chain reaction (PCR) require complex apparatus for temperature cycling. Recombinase polymerase amplification (RPA) is an isothermal DNA amplification method that has shown promise for use in low-resource settings. Combined with lateral flow assay as the readout, RPA-LFA can be used as a point-of-care diagnostic tool with high sensitivity and specificity, but reagent costs can be problematic. In this work, we developed a highly-sensitive smartphone-based RPA-LFA for the detection of Leishmania panamensis DNA using blue-emitting [(Sr0.625Ba0.375)1.96Eu0.01Dy0.03]MgSi2O7 (SBMSO) persistent luminescent nanophosphors as LFA reporters. The greater detectability of nanophosphors allows the use of a reduced volume of RPA reagents, potentially reducing the cost of RPA-LFA. The limit of detection (LOD) of RPA with gold nanoparticle-based LFA readout is estimated at 1 parasite per reaction, but LOD can be 100-fold better, 0.01 parasites per reaction, for LFA based on SBMSO. This approach may be useful for sensitive and cost-effective point-of-care diagnosis and contribute to improved clinical and economic outcomes, especially in resource-limited settings.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of −33.0 kcal/mol and −32.7 kcal/mol, and −T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Biotechnology and BioengineeringVolume 120, Issue 2 p. 319-322 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 120, Number 2, February 2023 First published: 10 January 2023 https://doi.org/10.1002/bit.28135AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume120, Issue2February 2023Pages 319-322 RelatedInformation

Objective:Anti-Müllerian Hormone (AMH) is a quantitative marker for ovarian reserve and is used to predict response during ovarian stimulation. Streamlining testing to the clinic or even to the physician's office would reduce inconvenience, turnaround time, patient stress and potentially also the total cost of testing, allowing for more frequent monitoring. In this paper, AMH is used as a model biomarker to describe the rational development and optimization of sensitive, quantitative, clinic-based rapid diagnostic tests. Design and Methods:We developed a one-step lateral-flow europium (III) chelate-based fluorescent immunoassay (LFIA) for the detection of AMH on a portable fluorescent reader, optimizing the capture/detection antibodies, running buffer, and reporter conjugates. Results:A panel of commercial calibrators was used to develop a standard curve to determine the analytical sensitivity (LOD = 0.41 ng/ml) and the analytical range (0.41-15.6 ng/ml) of the LFIA. Commercial controls were then tested to perform an initial evaluation of the prototype performance and showed a high degree of precision (Control I CV 2.18%; Control II CV 3.61%) and accuracy (Control I recovery 126%; Control II recovery 103%). Conclusions: This initial evaluation suggests that, in future clinical testing, the AMH LFIA will likely have the capability of distinguishing women with low ovarian reserve (<1 ng/ml AMH) from women with normal (1-4 ng/ml AMH) ovarian reserve. Furthermore, the LFIA demonstrated a wide linear range, indicating the assay's applicability to the detection of other health conditions such as PCOS, which requires AMH measurement at higher concentrations (>6 ng/ml).

Persons with obesity requires support, including updated and regular communication in order to make informed lifestyle choices. The STOP project, a H2020 RISE project funded by European Commission (GA No 823978) aims to establish a data and knowledge ecosystem as a basis for the STOP Portal to enable healthcare professionals in decision support, and persons with obesity. The aim of this current study was to test the feasibility of the STOP Portal for ease of use, and to validate its functions. This paper focuses on the communication-related findings. This is a feasibility study for the STOP Portal. The participants were adults who are overweight and obese. The STOP Portal collects the following data: knowledge resources, and physiological, activity and food data. Participants accessed the STOP Portal for one week and recorded their activity and food intakes. Data were sought from the platform to gain a more complete understanding of participation and engagement. Data were analysed using SPSS and content analysis. From the preliminary results, 31 participants registered for the STOP Portal and 24 used it for up to one week. From these 24 participants, they found the Portal was acceptable and easy to use. Factors influencing motivation and engagement to participate were: 1. social support; 2. technical issues; and 3. time. The Portal was able to collate information from other sources such as wearable wrist bands or apps in monitoring the activities along with inputted data to provide informative, educational and motivational messages for the participants. The STOP Portal has potential and is deemed acceptable and fit for purpose to collect activity and food data from a range of sources to communicate messages to people with obesity to inform their lifestyle choice decision making. Further research is ongoing to establish the effectiveness of the STOP Portal.

PDF file - 136K, Includes HTRF, cell growth/viability, apoptosis assays and biacore, crystal structure and animal studies. Also includes supplemental references.

IntroductionThe gold standard for diagnosis of active lupus nephritis (ALN), a kidney biopsy, is invasive with attendant morbidity and cannot be serially repeated. Urinary ALCAM (uALCAM) has shown high diagnostic accuracy for renal pathology activity in ALN patients. MethodsLateral flow assays (LFA) for assaying uALCAM were engineered using persistent luminescent nanoparticles, read by a smartphone. The stability and reproducibility of the assembled LFA strips and freeze-dried conjugated nanoparticles were verified, as was analyte specificity. ResultsThe LFA tests for both un-normalized uALCAM (AUC=0.93) and urine normalizer (HVEM)-normalized uALCAM (AUC=0.91) exhibited excellent accuracies in distinguishing ALN from healthy controls. The accuracies for distinguishing ALN from all other lupus patients were 0.86 and 0.74, respectively. ConclusionPeriodic monitoring of uALCAM using this easy-to-use LFA test by the patient at home could potentially accelerate early detection of renal involvement or disease flares in lupus patients, and hence reduce morbidity and mortality.

In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid emerging from a Protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 2 minutes at a flow rate of 0.5 mL/min.