Currently, the standard method for identifying biological agents of potential threats to national security and public health, such as pathogens, virus, and toxins, mainly rely on microbiological cultivation. This method is time-consuming and it requires sophisticated equipment and well-trained personnel, which are often unavailable in remote areas or at point-of-need. Therefore, an alternative rapid, simple, and sensitive method for detecting bio-threat agents is in crucial need. We report a paper-based Vertical Flow Immunoassay (VFI) device that can overcome these limitations. The VFI device utilizes a nanoporous nitrocellulose membrane encapsulated in a stainless steel filter holder. As the sample is pushed through the membrane, which is pre-functionalized with capture antibody, a sandwich assay is formed and colorimetric signal is generated to reflect the presence of target antigens. Through theoretical analyses of antigen-antibody binding process inside a porous membrane, we identified two critical factors - membrane pore size and sample flow rate that can be optimized to improve the assay sensitivity. Then, the effects were demonstrated through experimental studies using Burkholderia pseudomallei (the causative agent of melioidosis) as a model pathogen. The B. pseudomallei VFI was based on an immunoassay targeting the B. pseudomallei surface capsular polysaccharide (CPS). The experimental results agreed well with the theory showing that increasing the flow speed (up to 1.06 mm/s) and reducing the membrane pore size (down to 0.1 µm) could improve the sensitivity by at least 5 times. The VFI's limit-of-detection for CPS spiked in buffer solution was determined to be 0.02 ng/mL. The developed VFI shows great potential as a point-of-care tool for detection of bio-threat agents in a variety of clinical and resource-restricted conditions.

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Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The “gold standard” for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, B. pseudomallei capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the in vivo fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.

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Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as “large VFI” and “mini VFI.” Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

Point mutations in the human fibroblast growth factor (FGF) receptor 3 gene (Fgfr3) produce a constitutively active receptor, which disrupts chondrocyte differentiation in the growth plate and results in skeletal dysplasias with severe shortening of the limbs. Alternative splicing of the Fgfr3 transcript gives rise to two isoforms, IIIc and IIIb, which vary in their specificity for FGF ligands. We examined the expression of these FGFR3 isoforms in the bovine fetal rib growth plate to determine whether levels of FGFR3 expression are zone-related. Transcripts for both Fgfr3 isoforms are expressed in rib growth plate, with maximum expression in the hypertrophic region and the least expression in the reserve zone. Fgfr3 IIIc is the predominant isoform in the growth plate. Western-blot analysis revealed the presence of full-length FGFR3 (135 kDa) for both isoforms in the reserve zone, a major 98 kDa fragment in all zones and smaller fragments primarily in the hypertrophic zone. Immunostaining localized FGFR3 to the pericellular region of reserve chondrocytes and to the extracellular matrix in the hypertrophic zone. These results suggest that the transmembrane form of FGFR3 increasingly undergoes proteolytic cleavage towards the hypertrophic zone to produce an extracellular-domain fragment of FGFR3, which is present in large amounts in the matrix of hypertrophic cells. These findings suggest a proteolytic regulatory mechanism for FGFR3, whereby Fgfr3 fragments could control availability of FGF for the intact receptor, and by which proteolysis could inactivate the receptor.

Background Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. Principal findings The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1–2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. Conclusions LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

The incidence of fatal drug overdoses in the United States is an alarming public health threat that has been exacerbated by the COVID-19 pandemic, resulting in over 100,000 deaths between April 2020 and April 2021. A significant portion of this is attributable to widespread access to fentanyl and other synthetic opioids, alone or in combination with heroin or psychostimulants such as cocaine or methamphetamine. Monoclonal antibodies (mAb) offer prophylactic and therapeutic interventions against opioid overdose by binding opioids in serum, reducing distribution of drug to the brain and other organs. Here, we investigated the efficacy of a lead anti-fentanyl mAb, clone HY6-F9, in reversal and prevention of fentanyl-induced toxicity compared to the opioid receptor antagonist naloxone (NLX) in rats. In post-exposure models, rats were challenged with fentanyl, followed by HY6-F9, NLX, or both. HY6-F9 reversed fentanyl-induced antinociception, respiratory depression, and bradycardia, and rats retained protection against additional challenges for at least 1 week. Although intravenous NLX reversed fentanyl-induced respiratory depression more rapidly than mAb alone, kinetics of reversal by intravenous mAb were similar to subcutaneous NLX. Co-administration of mAb and NLX provided greater protection than individual treatments against high doses of fentanyl. Prophylactic administration of mAb reduced the ED 50 of NLX approximately 2-fold against 2.25 mg/kg fentanyl. Finally, mAb sequestered fentanyl and its metabolite norfentanyl in serum, and reduced brain concentrations of fentanyl. These results support the translation of mAb as medical interventions alone or in combination with NLX to prevent and reverse fentanyl-related overdose. Significance statementFentanyl-related overdoses have increased dramatically in the US and worldwide. Currently, approved pharmacotherapies for treatment of opioid use disorder and reversal of overdose are not sufficient to curb the incidence of opioid-related deaths. Additionally, fentanyl and its potent analogs present a potential risk from use in deliberate poisoning or chemical attacks. This study demonstrates the use of mAb as a countermeasure to fentanyl-induced toxicity in pre-and postexposure scenarios, supporting their use in combination with the opioid antagonist NLX.