A simple, sensitive, and low-cost FACS assay for detecting antibodies against the native SARS-CoV-2 spike protein.

BACKGROUND: Hamburg is a city state of approximately 1.9 Mio inhabitants in Northern Germany. Currently, the COVID-19 epidemic that had largely subsided during last summer is resurging in Hamburg and in other parts of the world, underlining the need for additional tools to monitor SARS-CoV-2 antibody responses. AIM: We aimed to develop and validate a simple, low-cost assay for detecting antibodies against the native coronavirus 2 spike protein (CoV-2 S) that does not require recombinant protein or virus. METHOD: We transiently co-transfected HEK cells or CHO cells with expression vectors encoding CoV-2 S and nuclear GFP. Spike protein-specific antibodies in human serum samples bound to transfected cells were detected with fluorochrome conjugated secondary antibodies by flow cytometry orimmunofluorescence microscopy. We applied this assay to monitor antibody development in COVID-19 patients, household contacts, and hospital personnel during the ongoing epidemic in the city state of Hamburg. RESULTS: All recovered COVID-19 patients showed high levels of CoV-2 S-specific antibodies. With one exception, all household members that did not develop symptoms also did not develop detectable antibodies. Similarly, lab personnel that worked during the epidemic and followed social distancing guidelines remained antibody-negative. CONCLUSION: We conclude that high-titer CoV-2 S-specific antibodies are found in most recovered COVID-19 patients and in symptomatic contacts, but only rarely in asymptomatic contacts. The assay may help health care providers to monitor disease progression and antibody responses in vaccination trials, to identify health care personnel that likely are resistant to re-infection, and recovered individuals with high antibody titers that may be suitable asplasma and/or antibody donors.

Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone.

UNLABELLED: Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts. INTRODUCTION: Chylomicrons (CM) and their remnants (CR) represent the postprandial plasma carriers of dietary lipids. Dietary vitamin K1 is known to be transported in the circulation as part of CM/CR and is required by osteoblasts as an essential co-factor for the gamma-carboxylation of bone matrix proteins. The molecular mechanisms underlying the delivery of lipophilic substances to bone are not understood. In this study, the expression and function of CM/CR receptors was examined in human osteoblasts. MATERIALS AND METHODS: Four human osteoblast-like cell lines were analyzed: two osteosarcoma lines (MG63, SaOS-2) and two telomerase-immortalized human bone marrow stromal cell lines (hMSC-TERT [4] and [20]) after 1,25(OH)2 vitamin D3 induction of osteoblastic differentiation (hMSC-TERT-OB). Receptor expression was examined by Western blotting and immunohistochemistry of normal human bone sections. Endocytotic receptor function was analyzed by cellular uptake assays using fluorescent and radiolabeled human CR. Vitamin K1-enriched CR (CR-K1) were generated in vivo after oral vitamin administration and vitamin K1 uptake by osteoblasts was measured by HPLC. The effect of CR-K1 uptake on osteocalcin carboxylation was measured by ELISA. RESULTS: Osteoblasts exhibit high levels of protein expression of the CR receptors LRP1 and LDLR. VLDLR is expressed to a lower degree. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells. Uptake of fluorescent CR by osteoblasts resulted in the typical pattern of receptor-mediated endocytosis. CR uptake was stimulated by the exogenous addition of the lipoprotein receptor ligands apolipoprotein E and lipoprotein lipase. Uptake was reduced by the known LRP1 inhibitors RAP, lactoferrin, and suramin, but not by LDL, which exclusively binds to the LDLR. Vitamin K1 uptake by hMSC-TERT-OB after incubation with CR-K1 was also shown to be sensitive to LPL stimulation and the LRP1 specific inhibitor lactoferrin. CR-K1 uptake into osteoblasts stimulated the gamma-carboxylation of osteocalcin. CONCLUSION: Human osteoblasts express receptors of the LDLR family with a capacity for vitamin K1 uptake through CR endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone. The current data suggest that, among the expressed receptors, LRP1 plays a predominant role.