Search

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.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.

Influenza is a viral respiratory tract infection responsible for up to 5 million cases of severe infection and nearly 600,000 deaths worldwide each year. While treatments for influenza exist, diagnostics for the virus at the point of care are limited in their sensitivity and ability to differentiate between subtypes. We have developed an integrated two-dimensional paper network (2DPN) for the detection of the influenza virus by the surface glycoprotein, hemagglutinin. The hemagglutinin assay was developed using proteins computationally designed to bind with high affinity to the highly-conserved sialic acid binding site. The integrated 2DPN uses a novel geometry that allows automated introduction of an enzymatic amplification reagent directly to the detection zone. This assay was integrated into a prototype device and demonstrated successful detection of clinically relevant virus concentrations spiked into 70 μL of virus-free pediatric nasal swab samples. Using this novel geometry, we found improved assay performance on the device (compared to a manually-operated dipstick method), with a sensitivity of 4.45 × 10(2) TCID(50)/mL on device.

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.

SUMMARYThe actin cytoskeleton assembles into diverse load-bearing networks including stress fibers, muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl-1 & Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force-sensitivity. Zyxin rapidly localizes via its LIM domains to failing stress fibers in cells, known as strain sites, to initiate stress fiber repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We observe that many, but not all, LIM domains from functionally diverse proteins localize to spontaneous or induced stress fiber strain sites in mammalian cells. Additionally, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to stress fiber strain sites in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. Sequence analysis and mutagenesis of the LIM domain region of zyxin indicate a requirement of tandem LIM domains, which contribute additively, for sensing stress fiber strain sites. In vitro, purified LIM domains from mammalian zyxin and fission yeast Pxl1 bind to mechanically stressed F-actin networks but do not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin's nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.

Immunoassays are important for the detection of proteins to enable disease identification and monitor treatment, but many immunoassays suffer from sensitivity limitations. The development of digital assays has enabled highly sensitive biomarker detection and quantification, but the necessary devices typically require precisely controlled volumes to reduce biases in concentration estimates from compartment size variation. These constraints have led to systems that are often expensive, cumbersome, and challenging to operate, confining many digital assays to centralized laboratories. To overcome these limitations, we have developed a simplified digital immunoassay performed in polydisperse droplets that are prepared without any specialized equipment. This polydisperse digital droplet immunoassay (ddIA) uses proximity ligation to remove the need for wash steps and simplifies the system to a single reagent addition step. Using interleukin-8 (IL-8) as an example analyte, we demonstrated the concept with samples in buffer and diluted whole blood with limits of detection of 0.793 pM and 1.54 pM, respectively. The development of a one-pot, washless assay greatly improves usability compared to traditional immunoassays or digital-based systems that rely heavily on wash steps and can be run with common and readily available laboratory equipment such as a heater and simple fluorescent microscope. We also developed a stochastic model with physically meaningful parameters that can be utilized to optimize the assay and enable quantification without standard curves, after initial characterization of the parameters. Our polydisperse ddIA assay serves as an example of sensitive, lower-cost, and simpler immunoassays suitable for both laboratory and point-of-care applications.

BACKGROUND Depletion of haptoglobin (Hp) and hemopexin (Hx) with increase in free hemoglobin and heme are important etiologies of vaso-occlusive complications in sickle cell disease (SCD). This study is the first to show an association between clinical improvement in SCD and repletion of Hp and Hx by therapeutic plasma exchange (TPE) using plasma replacement. STUDY DESIGN AND METHODS Thirteen fresh-frozen plasma (FFP) units derived from consecutive whole blood donations were thawed at 37°C after 10 months of storage; Hp and Hx concentrations immediately postthaw and after 5 days of refrigerated storage were analyzed by enzyme-linked immunosorbent assay (ELISA). All SCD patients presenting to a single institution over a 2-year period with acute multiorgan failure syndrome resistant to red blood cell exchange (RCE) were treated with TPE with FFP replacement; concentrations of Hp, Hx, and heme were evaluated before and after TPE by ELISA. RESULTS Plasma concentrations of Hp and Hx decreased approximately 20% (p ≤ 0.002) after 5 days of refrigerated storage. Significant mean fold increases after TPE of 10 for Hp (p

Reaction of the complex [(TpPh,Me)FeII(NCMe)3]BF4, where TpPh,Me = hydrotris(3-phenyl,5-methyl-1-pyrazolyl)borate, with the iodonium heteroylide PhI═NTs (1.5 equiv) is proposed to result in the insertion of N-tosylnitrene into one C–H bond at the ortho ring position of a 3-pyrazole phenyl substituent; subsequent deprotonation of the nascent aniline and one-electron oxidation of iron forms TsNH2 (0.5 equiv) as a coproduct. The covalent ligand modification and oxidation results in an intense purple-brown anilinato–iron(III) LMCT chromophore. This intramolecular reaction is utilized as a consistent clock to determine relative rates of competitive intermolecular nitrene transfer to added substrates, specifically to para-substituted styrenes and thioanisoles. Prior addition of substrate to the reaction of PhI═NTs with the iron(II) complex attenuates the CT absorbance of the equilibrium solution. Fitting of the concentration-dependent absorption data gives the ratio of intra- versus intermolecular nitrene trans...

A “second-generation” scorpionate ligand was utilized to prepare the nickel(II) borohydride complex [(TpPh,Me)Ni(η3-BH4)], wherein the borohydride was coordinated through two bridging B–H bonds, leaving two terminal B–H bonds uncoordinated as determined by X-ray crystallography. The distorted square-pyramidal complex is paramagnetic (S = 1), with 18 valence electrons, and contrasts with a previously reported 20-electron pseudo-octahedral “first-generation” analogue, [(TpMe,Me)Ni(η4-BH4)] (P. J. Desrochers, et al. Inorg. Chem. 2003, 42, 7945–7950). These distinct borohydride coordination modes were distinguished by FTIR spectroscopy, and rationalized by DFT calculations on simplified models, [(Tp)Ni(ηn-BH4)] (n = 3, 4). The difference in borohydride hapticities is attributed to the steric effect of the scorpionate 3-pyrazole phenyl substituents disposed proximally to the metal, thus demonstrating the subtle versatility of Trofimenko's scorpionate ligands in controlling ligand field geometries. The precursor complex [(TpPh,Me)Ni(κ2-NO3)] was also prepared and characterized.

Pseudotetrahedral scorpionato halide complexes [(TpPh,Me)Ni–X] [TpPh,Me = hydrotris(3-phenyl-5-methyl-1-pyrazolyl)borate] were synthesized by metathesis of Tl(TpPh,Me) and NiX2 (X = Cl, Br, I). Pyrazole adducts [(TpPh,Me)Ni(HpzPh,Me)(X)] (X = Cl, I) were also obtained. [(TpPh,Me)Ni–X] (X = Br, I) are isomorphous with previously reported [(TpPh,Me)Ni–Cl], and a new solvated pseudo-polymorph [(TpPh,Me)Ni–Cl]·2CH2Cl2 was found in this work. Ni–X bond lengths correspond to ionic radii of the halide ions, but some variations in Ni–Cl and Ni–N bond lengths are respectively attributed to bending of the chloride from the ideal threefold HB···Ni axis and to the size of the 3-pyrazole substituents. Electronic spectra were rationalized by means of time-dependent (TD) DFT calculations on simplified C3v-symmetric [(Tp)Ni–X] models. Exogenous pyrazole binds to give the pentacoordinate adducts [(TpPh,Me)Ni(HpzPh,Me)(X)] (X = Cl, I). We obtained a crystal structure of the iodide adduct, as well as that of [(TpPh,Me)Ni(HpzPh,Me)(Cl)]·0.5MeCN, isomorphous with a previously reported aquo monosolvate. A hallmark of adduct formation is an intramolecular N–H···X hydrogen bond between the added pyrazole and the adjacent halide ligand, thus giving rise to a strong IR absorption band. The stretching frequency correlates to the N(H)···X separation for the range of known adducts.

Synthesis and characterization of several new pseudotetrahedral arylthiolate complexes [(Tp(Ph,Me))Ni-SAr] (Tp(Ph,Me) = hydrotris{3-phenyl-5-methyl-1-pyrazolyl}borate; Ar = Ph, 2,4,6-(i)Pr3C6H2, C6H4-4-Cl, C6H4-4-Me, C6H4-4-OMe) are reported, including X-ray crystal structures of the first two complexes. With prior results, two series of complexes are spanned, [(Tp(Ph,Me))Ni-S-2,4,6-RC6H2] (R'' = H, Me, (i)Pr) plus the xylyl analogue [(Tp(Ph,Me))Ni-S-2,6-Me2C6H3], as well as [(Tp(Ph,Me))Ni-S-C6H4-4-Y] (Y = Cl, H, Me, OMe), intended to elucidate steric and/or electronic effects on arylthiolate coordination. In contrast to [(Tp(Me,Me))Ni-SAr] analogues that adopt a sawhorse conformation, the ortho-disubstituted complexes show enhanced trigonal and Ni-S-Ar bending, reflecting the size of the 3-pyrazole substituents. Moreover, weakened scorpionate ligation is implied by spectroscopic data. Little spectroscopic effect is observed in the series of para-substituted complexes, suggesting the observed effects are primarily steric in origin. The relatively electron-rich and encumbered complex [(Tp(Ph,Me))Ni-S-2,4,6-(i)Pr3C6H2] behaves uniquely when dissolved in CH3CN, forming a square planar solvent adduct with a bidentate scorpionate ligand, [(κ(2)-Tp(Ph,Me))Ni(NCMe)(S-2,4,6-(i)Pr3C6H2)]. This adduct was isolated and characterized by X-ray crystallography. Single-point DFT and TD-DFT calculations on a simplified [(κ(2)-Tp)Ni(NCMe)(SPh)] model were used to clarify the electronic spectrum of the adduct, and to elucidate differences between Ni-SAr bonding and spectroscopy between pseudotetrahedral and square planar geometries.