Room Temperature Al18F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging

Positron emission tomography (PET) is a non-invasive molecular imaging technology that is constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i. e. Fab, svFab, nanobodies) is increasing and has led us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which were investigated as such for {Al¹⁸F}²⁺-core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed under various conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240 min in human serum (HS), EDTA 5 mM, PBS and 0.9 % NaCl solutions. The in vivo stability of [Al¹⁸F(2-AMPDA-HB)]⁻ was assessed in healthy nude mice (n=6). Radiochemical yields between 55 % and 81 % were obtained at pH 5 and room temperature. High stability in HS was measured for [Al¹⁸F(2-AMPDA-HB)]⁻, with 90 % of F-18 complexed after 120 min. High stability in vivo, rapid hepatobiliary and renal excretion, with low accumulation of free F-18 in bones were measured. Thus, this new Al¹⁸F-chelator may have a great impact on immuno-PET radiopharmacy, by facilitating the development of new fluorine-18-labeled heat-sensitive biomolecules.     

18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[¹⁸F]fluorobenzamido)ethyl]maleimide ([¹⁸F]FBEM). ScFv_anti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[¹⁸F]fluorobenzamido)ethyl]maleimide ([¹⁸F]FBEM). Radiolabelled scFv was injected into mice with FeCl₃-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer ¹⁸F-scFv_anti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFv_anti-LIBS against activated platelets using site-specific bio conjugation.     

4,4,16-Trifluoropalmitate: Design,Synthesis, Tritiation,Radiofluorination and Preclinical PET Imaging Studies on Myocardial Fatty Acid Oxidation

Fatty acid oxidation (FAO) produces most of the ATP used to sustain the cardiac contractile work, although glycolysis is a secondary source of ATP under normal physiological conditions. FAO impairment has been reported in the advanced stages of heart failure (HF) and is strongly linked to disease progression and severity. Thus, from a clinical perspective, FAO dysregulation provides prognostic value for HF progression, the assessment of which could be used to improve patient monitoring and the effectiveness of therapy. Positron emission tomography (PET) imaging represents a powerful tool for the assessment and quantification of metabolic pathways in vivo. Several FAO PET tracers have been reported in the literature, but none of them is in routine clinical use yet. Metabolically trapped tracers are particularly interesting because they undergo FAO to generate a radioactive metabolite that is subsequently trapped in the mitochondria, thus providing a quantitative means of measuring FAO in vivo. Herein, we describe the design, synthesis, tritium labelling and radiofluorination of 4,4,16-trifluoro-palmitate ( 1 ) as a novel potential metabolically trapped FAO tracer. Preliminary PET-CT studies on [¹⁸F] 1 in rats showed rapid blood clearance, good metabolic stability – confirmed by using [³H] 1 in vitro – and resistance towards defluorination. However, cardiac uptake in rats was modest (0.24±0.04 % ID/g), and kinetic analysis showed reversible uptake, thus indicating that [¹⁸F] 1 is not irreversibly trapped.     

Pick’s Tau Fibril Shows Multiple Distinct PET Probe Binding Sites: Insights from Computational Modelling

In recent years, it has been realized that the tau protein is a key player in multiple neurodegenerative diseases. Positron emission tomography (PET) radiotracers that bind to tau filaments in Alzheimer’s disease (AD) are in common use, but PET tracers binding to tau filaments of rarer, age-related dementias, such as Pick’s disease, have not been widely explored. To design disease-specific and tau-selective PET tracers, it is important to determine where and how PET tracers bind to tau filaments. In this paper, we present the first molecular modelling study on PET probe binding to the structured core of tau filaments from a patient with Pick’s disease (Tau^PiD). We have used docking, molecular dynamics simulations, binding-affinity and tunnel calculations to explore Tau^PiD binding sites, binding modes, and binding energies of PET probes (AV-1451, MK-6240, PBB3, PM-PBB3, THK-5351 and PiB) with Tau^PiD. The probes bind to Tau^PiD at multiple surface binding sites as well as in a cavity binding site. The probes show unique surface binding patterns, and, out of them all, PM-PBB3 proves to bind the strongest. The findings suggest that our computational workflow of structural and dynamic details of the tau filaments has potential for the rational design of Tau^PiD specific PET tracers.     

Comparison of in Silico,Electrochemical, in Vitro and in Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography

The imaging of σ₁ receptors in the brain by fluorinated radiotracers will be used for the validation of σ₁ receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1′‐benzyl‐3‐(ω‐fluoromethyl to ω‐fluorobutyl)‐3H‐spiro[2]benzofuran‐1,4′‐piperidine] ([¹⁸F] 1 – 4 ) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [¹⁸F] 1 – 4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC–MS) analysis allowed structural identification of non‐radioactive metabolites. Radio‐HPLC and radio‐TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [¹⁸F] 2 – 4 , but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [¹⁸F] 4 reached the brain, rendering [¹⁸F] 4 less appropriate as a PET tracer than [¹⁸F] 2 and [¹⁸F] 3 . Compounds [¹⁸F] 2 and [¹⁸F] 3 possess the most promising properties for imaging of σ₁ receptors in the brain. High σ₁ affinity (K_i=0.59 nm ), low lipophilicity (logD_7.4=2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [¹⁸F] 2 slightly over the fluoropropyl derivative [¹⁸F] 3 for human use.     

18F-Sodium Fluoride PET as a Diagnostic Modality for Metabolic,Autoimmune, and Osteogenic Bone Disorders: Cellular Mechanisms and Clinical Applications

In a healthy body, homeostatic actions of osteoclasts and osteoblasts maintain the integrity of the skeletal system. When cellular activities of osteoclasts and osteoblasts become abnormal, pathological bone conditions, such as osteoporosis, can occur. Traditional imaging modalities, such as radiographs, are insensitive to the early cellular changes that precede gross pathological findings, often leading to delayed disease diagnoses and suboptimal therapeutic strategies. ¹⁸F-sodium fluoride (¹⁸F-NaF)-positron emission tomography (PET) is an emerging imaging modality with the potential for early diagnosis and monitoring of bone diseases through the detection of subtle metabolic changes. Specifically, the dissociated ¹⁸F^- is incorporated into hydroxyapatite, and its uptake reflects osteoblastic activity and bone perfusion, allowing for the quantification of bone turnover. While ¹⁸F-NaF-PET has traditionally been used to detect metastatic bone disease, recent literature corroborates the use of ¹⁸F-NaF-PET in benign osseous conditions as well. In this review, we discuss the cellular mechanisms of ¹⁸F-NaF-PET and examine recent findings on its clinical application in diverse metabolic, autoimmune, and osteogenic bone disorders.     

Differential biohydrogenation and isomerization of [U-13C]Oleic and [1-13C]Oleic acids by mixed ruminal microbes

The additional mass associated with ¹³C in metabolic tracers may interfere with their metabolism. The comparative isomerization and biohydrogenation of oleic, [1-¹³C]oleic, and [U-¹³C]oleic acids by mixed ruminal microbes was used to evaluate this effect. The percent of stearic, cis-14 and- 15, and trans-9 to-16 18∶1 originating from oleic acid was decreased for [U-¹³C]oleic acid compared with [1-¹³C]oleic acid. Conversely, microbial utilization of [U-¹³C]oleic acid resulted in more of the ¹³C label in cis-9 18∶1 compared with [1-¹³C]oleic acid (53.7 vs. 40.1%). The isomerization and biohydrogenation of oleic acid by ruminal microbes is affected by the mass of the labeled tracer.     

Apoptosis Imaging in Oncology by Means of Positron Emission Tomography: A Review

To date, a wide variety of potential PET-apoptosis imaging radiopharmaceuticals targeting apoptosis-induced cell membrane asymmetry and acidification, as well as caspase 3 activation (substrates and inhibitors) have been developed with the purpose of rapidly assessing the response to treatment in cancer patients. Many of these probes were shown to specifically bind to their apoptotic target in vitro and their uptake to be enhanced in the in vivo-xenografted tumours in mice treated by means of chemotherapy, however, to a significantly variable degree. This may, in part, relate to the tumour model used given the fact that different tumour cell lines bear a different sensitivity to a similar chemotherapeutic agent, to differences in the chemotherapeutic concentration and exposure time, as well as to the different timing of imaging performed post-treatment. The best validated cell membrane acidification and caspase 3 targeting radioligands, respectively ¹⁸F-ML-10 from the Aposense family and the radiolabelled caspase 3 substrate ¹⁸F-CP18, have also been injected in healthy individuals and shown to bear favourable dosimetric and safety characteristics. However, in contrast to, for instance, the ^99mTc-HYNIC-Annexin V, neither of both tracers was taken up to a significant degree by the bone marrow in the healthy individuals under study. Removal of white and red blood cells from the bone marrow through apoptosis plays a major role in the maintenance of hematopoietic cell homeostasis. The major apoptotic population in normal bone marrow are immature erythroblasts. While an accurate estimate of the number of immature erythroblasts undergoing apoptosis is not feasible due to their unknown clearance rate, their number is likely substantial given the ineffective quote of the erythropoietic process described in healthy subjects. Thus, the clinical value of both ¹⁸F-ML-10 and ¹⁸F-CP18 for apoptosis imaging in cancer patients, as suggested by a small number of subsequent clinical phase I/II trials in patients suffering from primary or secondary brain malignancies using ¹⁸F-ML-10 and in an ongoing trial in patients suffering from cancer of the ovaries using ¹⁸F-CP18, remains to be proven and warrants further investigation.     

Development of the First Potential Nonpeptidic Positron Emission Tomography Tracer for the Imaging of CCR2 Receptors

Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as ¹⁸F-labeled PET tracers. The derivatives were evaluated extensively for their off-target profile at 48 different targets. The most potent and selective candidate was applied in vivo in a biodistribution study, demonstrating a promising profile for further preclinical development. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.     

Using stable isotopes to trace diet‐induced shifts in pathways of lipid metabolism

Advances in the use of stable isotopes in tracer methodology have clearly improved our capacity to investigate in vivo lipid metabolism in humans. The introduction of the isotope ratio mass spectrometry (IRMS) provides higher precision of measurement of isotopic enrichment to track shifts in diet on the flux of tracers through metabolic pathways. Novel applications using stable isotopes including ²H, ¹³C, and ¹⁸O have allowed important discoveries to be made in areas of fat and cholesterol metabolism in ways that have assisted in understanding diet‐disease relationships.     

Prognostic and Theranostic Applications of Positron Emission Tomography for a Personalized Approach to Metastatic Castration-Resistant Prostate Cancer

Metastatic castration-resistant prostate cancer (mCRPC) represents a condition of progressive disease in spite of androgen deprivation therapy (ADT), with a broad spectrum of manifestations ranging from no symptoms to severe debilitation due to bone or visceral metastatization. The management of mCRPC has been profoundly modified by introducing novel therapeutic tools such as antiandrogen drugs (i.e., abiraterone acetate and enzalutamide), immunotherapy through sipuleucel-T, and targeted alpha therapy (TAT). This variety of approaches calls for unmet need of biomarkers suitable for patients’ pre-treatment selection and prognostic stratification. In this scenario, imaging with positron emission computed tomography (PET/CT) presents great and still unexplored potential to detect specific molecular and metabolic signatures, some of whom, such as the prostate specific membrane antigen (PSMA), can also be exploited as therapeutic targets, thus combining diagnosis and therapy in the so-called “theranostic” approach. In this review, we performed a web-based and desktop literature research to investigate the prognostic and theranostic potential of several PET imaging probes, such as ¹⁸F-FDG, ¹⁸F-choline and ⁶⁸Ga-PSMA-11, also covering the emerging tracers still in a pre-clinical phase (e.g., PARP-inhibitors’ analogs and the radioligands binding to gastrin releasing peptide receptors/GRPR), highlighting their potential for defining personalized care pathways in mCRPC.     

Head-to-Head Comparison of Fibroblast Activation Protein Inhibitors (FAPI) Radiotracers versus [18F]F-FDG in Oncology: A Systematic Review

Several recent studies comparing radiolabeled fibroblast activation protein inhibitors (FAPI) and fluorine-18 fluorodeoxyglucose ([¹⁸F]F-FDG) as positron emission tomography (PET) radiotracers in oncology have been published. The aim of this systematic review is to perform an updated evidence-based summary about the comparison of these PET radiotracers in oncology to better address further research in this setting. Studies or subsets of studies comparing radiolabeled FAPI and [¹⁸F]F-FDG as PET radiotracers in oncology were eligible for inclusion in this systematic review. A systematic literature search of PubMed/MEDLINE and Cochrane library databases was performed until August 2021. Literature data about the comparison of [¹⁸F]F-FDG and radiolabeled FAPI are rapidly increasing. Overall, taking into account radiotracer uptake and tumor-to-background uptake ratio, compared to [¹⁸F]F-FDG PET, an equal or higher detection of primary tumors and/or metastatic lesions was usually demonstrated by using radiolabeled FAPI PET. In particular, the cancer entities with better detection rate of tumor lesions by using radiolabeled FAPI PET, compared to [¹⁸F]F-FDG PET, were gastrointestinal tumors, liver tumors, breast cancer and nasopharyngeal carcinoma. Further comparison studies are needed to better evaluate the best field of application of radiolabeled FAPI PET.     

Mesure des nombres de transport des ions Cs+ et Br− en milieu eau—dioxanne a l'aide de radiotraceurs

The transference numbers of the Br^? and Cs⁺ ions have been measured in aqueous mixtures containing 60 and 82.82 wt% dioxane by means of a capillary tubes device using radioactive tracers. The reliability of this has been improved by the use of a highly stabilised constant current power supply. A theoretical data processing based on the Bjerrum association concept and Fuoss-Chen conductance equation expanded in series by Justice and written in its ionic form, has been shown to give a good prediction of ionic transport properties in an electric field, even in strongly associated media.     

DI-B experiment: planning, design and performance of an in situ diffusion experiment in the Opalinus Clay formation

The DI-B experiment is a long-term, natural-scale, in situ diffusion experiment, which is being performed in the Opalinus Clay formation at the Mont Terri Underground Rock Laboratory (URL), in Switzerland, employing nonradioactive tracers. One of the key aspects to be addressed for nuclear waste repository safety assessment purposes is the understanding of the transport mechanisms of the radionuclides contained in the radioactive waste. Consolidated clay formations display very low water hydraulic conductivities, so it is expected that the predominant transport process will be diffusion.The experimental set-up has been designed to withstand the site conditions and for monitoring and recording several physicochemical parameters (pH, conductivity, oxidation–reduction potential), as well as the pressures in the circuit and for the long-distance monitoring of the data acquisition system.The tracer selection has been made based on previous investigations carried out at CIEMAT, including a literature survey, laboratory sorption experiments and hydrogeochemical modeling for determining tracer stability under the physicochemical conditions to be expected in the site. The final selection includes ⁶Li, ⁸⁷Rb, D (as D₂O) and I (as I⁻). Hydrogeochemical modeling confirmed the stability of all the tracers selected. Batch sorption experiments showed that no sorption in the rock occurred in the case of ⁶Li, D and I (conservative tracers), whereas ⁸⁷Rb was 100% sorbed. However, ⁸⁷Rb was chosen because of its analogy with Cs, a relevant radionuclide commonly present in the nuclear spent fuel.Diffusion experiments have been carried out at laboratory scale with Opalinus Clay samples to provide diffusion parameters for modeling purposes. Effective diffusion coefficients, perpendicular and parallel to the bedding planes of the rock, respectively, were (1.68± 0.42)×10⁻¹¹ and (4.02± 0.30)×10⁻¹¹ m²/s for tritium, and (2.70± 0.27)×10⁻¹² and (1.38± 0.49)×10⁻¹¹ m²/s for iodide. Additional through-diffusion experiments (parallel to the bedding) were performed with the nonsorbing tracer ³⁶Cl⁻, in order to check the results obtained for iodide. The effective diffusion coefficient measured for chloride ions was (1.18± 0.27)×10⁻¹¹ m²/s, which is practically equal to the value obtained for iodide.Preliminary diffusion calculations have been carried out using two transport codes: GIMRT and CORE^2D, with conservative and nonconservative tracers, using effective diffusion coefficients (D_e) obtained experimentally in the laboratory (through-diffusion experiments) or selected from the literature. The diffusion profiles obtained from the calculations showed slight variations, which were consistent with the different modeling approaches employed. The predictive modeling results have been used to determine the initial tracer concentration that should be added to the circuit to assure well-defined profiles at the end of the experiment.This long-term in situ diffusion experiment will also provide useful data for the interpretation of previous diffusion experiments performed at the Mont Terri URL.     

Harnessing the Maltodextrin Transport Mechanism for Targeted Bacterial Imaging: Structural Requirements for Improved in vivo Stability in Tracer Design

Diagnosis and localization of bacterial infections remains a significant clinical challenge. Harnessing bacteria‐specific metabolic pathways, such as the maltodextrin transport mechanism, may allow specific localization and imaging of small or hidden colonies. This requires that the intrabacterial tracer accumulation provided by the transporter is matched by high serum stability of the tracer molecule. Herein, radiolabeled maltodextrins of varying chain lengths and with free nonreducing/reducing ends are reported and their behavior against starch‐degrading enzymes in the blood, which compromise their serum stability, is evaluated. Successful single‐photon emission computed tomography (SPECT) imaging is shown in a footpad infection model in vivo by using the newly developed model tracer, [^99mTc] MB1143 , and the signal is compared with that of ¹⁸F‐fluorodeoxyglucose positron emission tomography ([¹⁸F]FDG‐PET) as a nonbacterial specific marker for inflammation. Although the [^99mTc] MB1143 imaging signal is highly specific, it is low, most probably due to insufficient serum stability of the tracer. A series of stability tests with different ¹⁸F‐labeled maltodextrins finally yielded clear structural guidelines regarding substitution patterns and chain lengths of maltodextrin‐based tracers for nuclear imaging of bacterial infections.     

Stable Carbon Isotope Composition of c9,t11-Conjugated Linoleic Acid in Cow’s Milk as Related to Dietary Fatty Acids

This study explores the potential use of stable carbon isotope ratios (δ¹³C) of single fatty acids (FA) as tracers for the transformation of FA from diet to milk, with focus on the metabolic origin of c9,t11-18:2. For this purpose, dairy cows were fed diets based exclusively on C₃ and C₄ plants. The FA in milk and feed were fractionated by silver-ion thin-layer chromatography and analyzed for their δ¹³C values. Mean δ¹³C values of FA from C₃ milk were lower compared to those from C₄ milk (−30.1‰ vs. −24.9‰, respectively). In both groups the most negative δ¹³C values of all FA analyzed were measured for c9,t11-18:2 (C₃ milk = −37.0 ± 2.7‰; C₄ milk −31.4 ± 1.4‰). Compared to the dietary precursors 18:2n-6 and 18:3n-3, no significant ¹³C-depletion was measured in t11-18:1. This suggests that the δ¹³C-change in c9,t11-18:2 did not originate from the microbial biohydrogenation in the rumen, but most probably from endogenous desaturation of t11-18:1. It appears that the natural δ¹³C differences in some dietary FA are at least partly preserved in milk FA. Therefore, carbon isotope analyses of individual FA could be useful for studying metabolic transformation processes in ruminants.     

A Novel Way To Radiolabel Human Butyrylcholinesterase for Positron Emission Tomography through Irreversible Transfer of the Radiolabeled Moiety

The enzyme butyrylcholinesterase (BChE) is known to be involved in the detoxification of xenobiotics in blood plasma and is associated with the progress of neurodegenerative disorders, diabetes type 2, obesity, and diseases of the cardiovascular system. In the present study, we developed carbamate‐based inhibitors serving as positron emission tomography (PET) radiotracers with ¹⁸F and ¹¹C as radioisotopes to visualize BChE distribution. These inhibitors are radiolabeled at the carbamate site and transfer this moiety onto BChE, which thus results in covalent and permanent radiolabeling of the enzyme. There are no comparable radiotracers for cholinesterases described to date. By ex vivo autoradiography experiments on mice brain slices and kinetic investigations, selective and covalent transfer of the radiolabeled carbamate moiety onto BChE was proven. These tracers might provide high resolution of BChE distribution in vivo to enable investigations into the pathophysiological mechanisms of diseases associated with alterations in BChE occurrence.     

Paramagnetic Quantum Dots as Multimodal Probes for Potential Applications in Nervous System Imaging

An axonal tracer that can be detected by both magnetic resonance imaging (MRI) and fluorescence is of great interest for studying nerve regeneration, particularly for spinal cord injury repairs. In this study, we develop a new type of multifunctional nanoparticle that combines three different functionalities of paramagnetism, fluorescence, and axonal tracing into one nanomaterial. We demonstrate that the new synthesized quantum dot nanoparticles have good biocompatibilities and can be readily taken up by cells. In addition, the quantum dots show excellent longitudinal and transverse relaxivities (i.e. r ₁ ?=?11.22?±?0.10 mM^?1 s^?1 and r ₂ ?=?24.50?±?0.51 mM^?1 s^?1) at 1.5 T, MRI contrast properties better than those of Magnevist®, a commercially available MRI contrast agent. The UV–vis absorbance spectra of all the pQDs-BDA samples indicate that these tracers are stable at different temperatures. Taken together, this new nanomaterial demonstrates good performances for both optical and MR imaging modalities, suggesting its promising potential applications in non-invasive imaging, particularly as a novel multimodal axonal tracer for nervous system imaging.     

The Use of Distinctive Monoclonal Antibodies in FMD VLP- and P1-Based Blocking ELISA for the Seromonitoring of Vaccinated Swine

The serum neutralization (SN) test has been regarded as the “gold standard” for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R² = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16–32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R² = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.     

Fluidization studies in large gas—solid systems. Part III. The effect of bed depth and fluidizing velocity on solids circulation patterns

Information on the pattern of solids circulation in various depth beds of silica sand fluidized in a square vessel, area 1.5 m², has been obtained by adding both coloured tracers and plastic spheres to the system a known time prior to defluidization. The stagnant bed was sectioned and the tracer location recorded.