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.
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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