The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).
VWF is required for platelet adhesion to sites of vessel injury, a process vital for both hemostasis and thrombosis. Enhanced VWF secretion and oxidative stress are both hallmarks of inflammation. We recently showed that the neutrophil oxidant hypochlorous acid (HOCl) inhibits VWF proteolysis by ADAMTS13 by oxidizing VWF methionine 1606 (M1606) in the A2 domain. M1606 was readily oxidized in a substrate peptide, but required urea in multimeric plasma VWF. In the present study, we examined whether shear stress enhances VWF oxidation. With an HOCl-generating system containing myeloperoxidase (MPO) and H 2 O 2 , we found that shear stress accelerated M1606 oxidation, with 56% becoming oxidized within 1 hour. Seven other methionine residues in the VWF A1A2A3 region (containing the sites for platelet and collagen binding and ADAMTS13 cleavage) were variably oxidized, one completely. Oxidized methionines accumulated preferentially in the largest VWF multimers. HOCl-oxidized VWF was hyperfunctional, agglutinating platelets at ristocetin concentrations that induced minimal agglutination using unoxidized VWF and binding more of the nanobody AU/VWFa-11, which detects a gain-offunction conformation of the A1 domain. These findings suggest that neutrophil oxidants will both render newly secreted VWF uncleavable and alter the largest plasma VWF forms such that they become hyperfunctional and resistant to proteolysis by ADAMTS13. (Blood. 2011; 118(19):5283-5291) IntroductionVWF is an enormous plasma glycoprotein synthesized in endothelial cells and megakaryocytes, the primary function of which is to attach platelets to sites of blood vessel injury. 1 VWF also chaperones coagulation factor VIII in plasma, protecting it from degradation and delivering it to the platelet surface, where it participates in blood coagulation. 2 VWF comprises homopolymers of a 2050-amino acid polypeptide synthesized through N-terminal disulfide bonding of C-terminal disulfide-bonded dimers. 3 Each monomer has many domains, including 3 tandem A domains (A1A2A3) containing, respectively, the platelet glycoprotein (GP) Ib-binding site, the cleavage site for ADAMTS13, and a collagen-binding site. 3 The polymers can reach a mass of Ͼ 20 million Da 4 and are either constitutively secreted or stored in granules, the Weibel-Palade bodies of endothelial cells, or the ␣-granules of platelets, from which they are released upon stimulation. 5 Newly released VWF multimers (termed ultra-large VWF or ULVWF) are hyperadhesive for platelets compared with the VWF normally found in plasma, 6 but are rapidly converted to smaller, less reactive plasma forms by cleavage by the metalloprotease ADAMTS13. 1 In addition to its hemostatic roles, VWF is also involved in thrombosis. The most obvious example is when ADAMTS13 fails to process ULVWF, resulting in the sometimes fatal microvascular clotting syndrome, thrombotic thrombocytopenic purpura. 7 VWF has also been implicated in thrombosis associated with disorders such as HELLP syndrome (hemolysis, elevated liver enzymes, a...
The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form...
The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.
The rapid onset of the global COVID-19 pandemic has led to multiple challenges for accurately diagnosing the infection. One of the main bottlenecks for COVID-19 detection is reagent and material shortages for sample collection, preservation, and purification prior to testing. Currently, most authorized diagnostic tests require RNA extraction from patient samples and detection by reverse transcription polymerase chain reaction (RT-PCR). However, RNA purification is expensive, time consuming, and requires technical expertise to perform. Additionally, there have been reported shortages of the RNA purification kits needed for most tests. With these challenges in mind, we report on extraction-free amplification of SARS-CoV-2 RNA directly from patient samples. In addition, we have developed a multiplex RT-PCR using the CDC singleplex targets. This multiplex has a limit of detection of 2 copies/μL. We have demonstrated these improvements to the current diagnostic workflow, which reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
2112 The interaction between von Willebrand factor (VWF) and the platelet glycoprotein Ib-IX-V complex mediates the first step of platelet adhesion to the vessel wall at sites of injury in the hemostatic response to blood loss. This interaction is also involved in pathologic thrombosis, the most extreme case being thrombotic thrombocytopenic purpura, but the interaction has been proposed to have important pathogenic roles in disparate syndromes such as sepsis, HELLP syndrome, antiphospholipid syndrome, acute lung injury, sickle cell anemia, and cerebral malaria. These syndromes have in common an association with severe inflammation, one of the consequences of which is production of oxidants, in particular by neutrophils. We recently showed that one of the most potent neutrophil oxidants, hypochlorous acid (HOCl), which is produced by the myeloperoxidase-catalyzed reaction of H2O2 with chloride ion, markedly reduces ADAMTS13 proteolysis of VWF by oxidizing M1606 at the ADAMTS13 cleavage site within the A2 domain of VWF (Blood, 115(3) 706-12, 2010). In that study, M1606 present in a substrate A2 peptide was readily oxidized by HOCl, but only minimally oxidized in multimeric plasma VWF, except in the presence of the denaturing agent urea. As this requirement resembled the requirement of urea for ADAMTS13 proteolysis of plasma VWF, we wondered whether the application of shear stress would similarly enhance M1606 oxidation by HOCl. Using a system containing 25 nM MPO (a plasma concentration often seem in inflammatory conditions) and varying concentrations of H2O2, we found that application of 0.6 dynes/cm2 shear stress through a closed circuit of plastic tubing rendered M1606 much more sensitive to oxidation: 80% oxidized within 1 hr. This suggestion of shear-induced unfolding and enhanced oxidation was verified when we examined 7 other methionine residues in the A1A2A3 region of VWF, the region containing the binding sites for platelets and collagen and the ADAMTS13 cleavage site. The Met residues were variably sensitive to oxidation, but all became increasingly oxidized over time in the presence of shear stress. Although the shear stresses we used in this experiment are far below the shear stress considered necessary to unfold even very large VWF multimers, the VWF solution also experienced constant elongational flow generated by a peristaltic pump, necessitating flow acceleration through the region narrowed by the rollers. Elongational flow can impart up to 100-fold more tensile stress to suspended VWF than the constant shear stress (Biophys. J., 98 L35, 2010). Two other findings favor the interpretation that oxidation of the A1A2A3 region is facilitated by domain unfolding. First, we further separated the oxidized VWF by gel-filtration into large, intermediate, and small multimeric fractions and found that methionine oxidation was much more prevalent in the fraction with the largest multimers and rare in the fraction with the smallest multimers. Second, we found that ristocetin, a VWF modulator that simulates the effect of shear stress on VWF, also accelerated oxidation of M1606. In functional tests, we found that HOCl-oxidized plasma VWF agglutinated fixed platelets at concentrations of ristocetin that induced minimal agglutination using unoxidized VWF. These findings have several important clinical implications. First, inflammatory conditions will not only activate endothelial cells and induce release of VWF, especially the largest and most adhesive forms (ultralarge VWF), the oxidants produced from endothelial cells themselves and from the neutrophil respiratory burst will render the VWF resistant to proteolysis. Second, these same oxidants will also convert the largest preexisting plasma VWF multimers that were previously rendered quiescent by ADAMTS13, into hyperfunctional and uncleavable forms. All of these mechanisms converge to generate a highly prothrombotic state, perhaps initially evolved as a mechanism to trap and isolate microorganisms, but which also has the potential to cause tremendous harm to those affected by these inflammatory conditions. Disclosures: No relevant conflicts of interest to declare.
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