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RSC Advances Mar 2024This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures...
A prototype of ultrasensitive time-resolved fluoroimmunoassay for the quantitation of lead in plasma using a fluorescence-enhanced europium chelate label for the detection system.
This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures were conducted in 96-microwell plates and involved the competitive binding format. The assay used a mouse monoclonal antibody, designated as 2C33, that specifically recognized the diethylenetriamine pentaacetic acid chelate of Pb (Pb-DTPA) but did not recognize Pb-free DTPA chelator. The antigen used for coating onto the inner surfaces of assay plate microwells was Pb-DTPA conjugated with bovine serum albumin protein (Pb-DTPA-BSA). The competitive binding reaction occurred between Pb-DTPA chelates, formed in the sample solutions by treating the samples with an excess DTPA, and the coated Pb-DTPA-BSA for a limited quantity of 2C33 antibody binding sites. The antigen-antibody complex formed in the plate wells was quantified by a europium-DTPA-labeled secondary antibody and a fluorescence enhancement solution. The conditions of the assay were refined, and its optimum procedures were established. The TRFIA was validated following the immunoassay validation guidelines, and all of the validation criteria were acceptable. The working range of the assay was 20-300 pg mL and its limit of quantitation was 20 pg mL. Metals that are commonly encountered in blood plasma did not interfere with Pb in the analysis by the proposed TRFIA. The assay was applied to the quantitation of Pb in plasma samples with satisfactory accuracy and precision. The results were compared favorably with those obtained by atomic emission spectroscopy. In conclusion, the present study represents the first TRFIA for the quantitation of Pb in plasma. The assay is superior to the existing atomic spectrometric methods and other immunoassays for Pb in terms of sensitivity, convenience, and analysis throughputs. The proposed TRFIA is anticipated to effectively contribute to assessing Pb concentrations and controlling the exposure of humans to its potential toxicity.
PubMed: 38495999
DOI: 10.1039/d3ra07673c -
Biotechnology and Bioengineering Jun 2024The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative....
The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developing and optimizing perfusion operations requires screening various parameters, which is expensive and time-consuming when using benchtop bioreactors. Scale-down models (SDMs) are the most feasible option for high-throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi-perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24-well MWP (V = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady-state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (V = 30 mL), comparing a squared six-well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs. SF) and scales (MWP vs. DWP + SF). The results show that the well-plate-based methods are suitable to perform HCD and quasi steady-state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.
Topics: CHO Cells; Cricetulus; Animals; High-Throughput Screening Assays; Bioreactors; Cell Culture Techniques; Cell Count
PubMed: 38393309
DOI: 10.1002/bit.28682 -
Biosensors & Bioelectronics Feb 2024Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1-2 days) and requires specialized...
Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1-2 days) and requires specialized laboratory and personnel. This study reported a deep learning enhanced digital microfluidic platform for multiplex detection of viable foodborne pathogens. The new method used a Time-Lapse images driven EfficientNet-Transformer Network (TLENTNet) to type and quantify the bacteria through spatiotemporal features of bacterial growth and digital enumeration of bacterial culture. First, the bacterial sample was prepared with LB medium and injected into a pre-vacuumed microfluidic chip with an array of 800 microwells to encapsulate at most one bacterium in each well. Then, a programmed sliding microscopic platform was used to scan all microwells every 15 min, capturing time-lapse images of bacterial growth within each microwell. Finally, the TLENTNet was used to facilitate bacterial typing and quantification. Under optimal conditions, this platform was able to detect four bacterial species (S.typhimurium, E. coli O157:H7, S. aureus and B. cereus) with an average accuracy of 97.72% and a detection limit of 63 CFU/mL in 7 h.
Topics: Food Microbiology; Microfluidics; Staphylococcus aureus; Deep Learning; Biosensing Techniques; Escherichia coli O157; Bacteria
PubMed: 38000308
DOI: 10.1016/j.bios.2023.115837 -
Biotechnology and Bioengineering Apr 2024Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from...
A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.
Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.
Topics: Humans; Induced Pluripotent Stem Cells; Oxygen; Cell Differentiation; Liver; Cell Culture Techniques; Organoids; Dimethylpolysiloxanes; Metalloendopeptidases; GPI-Linked Proteins
PubMed: 38184815
DOI: 10.1002/bit.28640 -
Biotechnology and Bioengineering Jun 2024The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high...
The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d. The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 10 cells mL were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations.
Topics: Bioreactors; CHO Cells; Cricetulus; Animals; Perfusion; Cell Culture Techniques; High-Throughput Screening Assays; Cell Count; Batch Cell Culture Techniques
PubMed: 38433473
DOI: 10.1002/bit.28685 -
Bacterial Aggregation in Cerebral Spinal Fluid: The Extent it Occurs and the Clinical Ramifications.Current Microbiology Jun 2024Bacteria can form aggregates in synovial fluid that are resistant to antibiotics, but the ability to form aggregates in cerebral spinal fluid (CSF) is poorly defined....
Bacteria can form aggregates in synovial fluid that are resistant to antibiotics, but the ability to form aggregates in cerebral spinal fluid (CSF) is poorly defined. Consequently, the aims of this study were to assess the propensity of four bacterial species to form aggregates in CSF under various conditions. To achieve these aims, bacteria were added to CSF in microwell plates and small flasks at static and different dynamic conditions with the aid of an incubating shaker. The aggregates that formed were assessed for antibiotic resistance and the ability of tissue plasminogen activator (TPA) to disrupt these aggregates and reduce the number of bacteria present when used with antibiotics. The results of this study show that under dynamic conditions all four bacteria species formed aggregates that were resistant to high concentrations of antibiotics. Yet with static conditions, no bacteria formed aggregates and when the CSF volume was increased, only Staphylococcus aureus formed aggregates. Interestingly, the aggregates that formed were easily dispersed by TPA and significant (p < 0.005) decreases in colony-forming units were seen when a combination of TPA and antibiotics were compared to antibiotics alone. These findings have clinical significance in that they show bacterial aggregation does not habitually occur in central nervous system infections, but rather occurs under specific conditions. Furthermore, the use of TPA combined with antibiotics may be advantageous in recalcitrant central nervous system infections and this provides a pathophysiological explanation for an unusual finding in the CLEAR III clinical trial.
Topics: Humans; Anti-Bacterial Agents; Cerebrospinal Fluid; Bacteria; Staphylococcus aureus; Tissue Plasminogen Activator; Drug Resistance, Bacterial; Microbial Sensitivity Tests
PubMed: 38831167
DOI: 10.1007/s00284-024-03727-4 -
Surgical Infections Feb 2024Chronic prosthetic joint infections (PJI) are associated with substantial morbidity because conventional antibiotic agents lack activity to bacteria in biofilms that...
Chronic prosthetic joint infections (PJI) are associated with substantial morbidity because conventional antibiotic agents lack activity to bacteria in biofilms that necessitates prosthetic removal to attempt definitive cure. However, these are complex infections that go beyond biofilms and bacteria can be present in various other different states such as synovial fluid aggregates. Consequently, the purpose of this study was to assess the propensity of historically preserved PJI clinical isolates to form synovial fluid aggregates and if aggregation occurred then what is proclivity to be tolerant to high doses of antibiotic agents. Historically preserved chronic PJI clinical isolates from 2021 were evaluated for their ability to form synovial fluid aggregates under static and dynamic conditions in 24-microwell plates. Tolerance to vancomycin, gentamicin, or amphotericin was conducted by adding high concentrations of these antibiotic agents to synovial fluid microbial aggregates. All clinical isolates formed synovial fluid aggregates under dynamic conditions, which with the use of scanning electron microscopy showed dense collections of bacteria with synovial fluid polymers. However, under static conditions only formed aggregates. Importantly, all the microbes in these aggregates were tolerant to high concentrations of antibiotic agents. This study demonstrates that synovial fluid aggregation occurred with all bacterial and fungal species assessed. Therefore, the findings here have important clinical ramifications given the extent that this phenomenon occurs across microbial species and the propensity for the microbes in these aggregates to be tolerant to antibiotic agents.
Topics: Humans; Synovial Fluid; Anti-Bacterial Agents; Biofilms; Arthritis, Infectious; Vancomycin; Bacteria; Prosthesis-Related Infections
PubMed: 38150525
DOI: 10.1089/sur.2023.242 -
Biotechnology Journal Jan 2024The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia...
The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease affecting the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli; however, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The expression of three vaccine antigens Oml1, Oml7, and ApxII in the four strongest bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates (MWP) led to improved production. Finally, the production yields reached unprecedented levels of 2.43 g L of Oml1, 2.59 g L of Oml7, and 1.21 g L of ApxII, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study establishes an efficient bicistronic T7 expression system that can be used to express recombinant proteins in E. coli and achieves the hyper-production of PCP vaccine proteins.
Topics: Swine; Animals; Bacterial Proteins; Escherichia coli; Pleuropneumonia, Contagious; Recombinant Proteins; Actinobacillus Infections; Vaccines, Subunit
PubMed: 38178735
DOI: 10.1002/biot.202300187