COVID-19 is an ongoing, global pandemic caused by the novel, highly infectious SARS-CoV-2 virus. Efforts to mitigate the effects of SARS-CoV-2, such as mass vaccination and development of monoclonal therapeutics, require precise measurements of correlative, functional neutralizing antibodies that block virus infection. The development of rapid, safe, and easy-to-use neutralization assays is essential for faster diagnosis and treatment. Here, we developed a vesicular stomatitis virus (VSV)-based neutralization assay with two readout methods, imaging and flow cytometry, that were capable of quantifying varying degrees of neutralization in patient serum samples. We tested two different spike-pseudoviruses and conducted a time-course assay at multiple multiplicities of infection (MOIs) to optimize the assay workflow. The results of this assay correlate with the results of previously developed serology and surrogate neutralization assays. The two pseudovirus readout methods produced similar values of 50% neutralization titer values. Harvest-free in situ readouts for live-cell imaging and high-throughput analysis results for flow cytometry can provide unique capabilities for fast evaluation of neutralization, which is critical for the mitigation of future pandemics.

Mobilized colistin resistance (mcr) genes confer resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. In this case report, we describe a novel lipid-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) diagnostic used to rapidly identify an mcr-1-positive Escherichia coli directly from a patient with a urinary tract infection without the need for ex vivo growth.

COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.

Extracellular vesicles (EVs) are membrane-surrounded vesicles that carry heterogeneous cellular components, including proteins, nucleic acids, lipids, and metabolites. EVs’ intravesicular and surface contents possess many biomarkers of physiological and pathological importance. Because of the heterogeneous cargo, EVs can mediate local and distal cell–cell communication. However, the way in which the genome signature regulates EV cargo has not been well studied. This study aimed to understand how genetics impact EV cargo loading. EVs were isolated from vector copy number cells with a fluorescent reporter (GFP) with varying inserted transgene copies and from NIST SRM 2373 cells (MDA-MB-231, MDA-MB-453, SK-BR-3, and BT-474), which contain varying copies of the HER2 gene. Spectradyne nCS1 was utilized to count EVs and measure size distribution. Imaging Flow Cytometry was used to analyze the surface protein content of single EVs and for total EV counts. The RNA content of the EVs was measured using ddPCR. Our results from stable reporter cell lines and breast cancer cell lines suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of a reporter gene (GFP) or a naturally occurring gene (HER2) from breast cancer cells correlated with increasing EV counts positive for the protein cargo compared to total EV counts until a copy threshold was reached. This study has broad implications for understanding EV biology in the context of cancer biology, diagnostics, EV biology/manufacturing, and therapeutic delivery.

An emulsion of silicone oil droplets in aqueous buffer produces a distinctive series of peaks or resonances in the side scatter histogram in a flow cytometer. As many as 12 peaks are observed in the violet‐side scatter channel at 405 nm, with half that number observed in the blue side scatter channel at 488 nm. Using the index of refraction of the oil and buffer, the wavelength of light, and the collection angle and gain of the instrument, we assign the peaks to specific diameters at which Mie resonances occur. With the close match for the index of refraction of silicone oil (n = 1.417 at 405 nm) to biological materials, these resonances could form the basis of a finely spaced size calibration ladder in the range 0.5–6 μm for estimating the size of biological particles in a flow cytometer. Resonances were also observed using mineral oil (n = 1.483 at 405 nm) suggesting that investigating and modeling resonances for emulsion systems may be useful for understanding these systems.