Rapid lateral flow tests for the detection of SARS-CoV-2 neutralizing antibodies.

Background: Rapid Lateral Flow Test (LFT) has been broadly utilized in detection or diagnosis of numerous disease-related antigens and antibodies. It is the most popular format of point-of-care test (POCT) and quickest and easiest way to detect a targeted molecule. In the combat against COVID-19 pandemic, hundreds of POCTs have been developed and are commercially available now. They are designed to detect either a SARS-CoV-2 viral antigen or IgG and IgM antibodies binding to it. Among the binding antibodies, a special type of functional antibodies that block the interaction between SARS-CoV-2 virus and its human receptor, neutralizing antibodies (NAbs), are of particular interest to public as well as in vaccination management. However as of today, POCTs for the detection of SARS-CoV-2 NAbs remain under late stage of development.Scope and method:In this review, we first summarize the importance of awareness and monitoring of SARS-CoV-2 NAbs in the combat against COVID-19 pandemic. Secondly, we compare the available methods for the detection of SARS-CoV-2 NAbs. Next, we describe challenges in the development of a rapid lateral flow test for the detection of SARS-CoV-2 NAbs. Finally, we outline its product formats and applications in research and in disease management. Conclusion:Vaccine effectiveness is unknown for an individual unless measured. NAb level is the most viable measurement for vaccine effectiveness or immunity. A broadly accessible NAb POCT is urgently needed.

Measurement of fluid secretion from intact airway submucosal glands.

Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

Synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis.

Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl(-) and HCO(3) (-), and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.

Acinar origin of CFTR-dependent airway submucosal gland fluid secretion.

Cystic fibrosis (CF) airway disease arises from defective innate defenses, especially defective mucus clearance of microorganisms. Airway submucosal glands secrete most airway mucus, and CF airway glands do not secrete in response to VIP or forskolin. CFTR, the protein that is defective in CF, is expressed in glands, but immunocytochemistry finds the highest expression of CFTR in either the ciliated ducts or in the acini, depending on the antibodies used. CFTR is absolutely required for forskolin-mediated gland secretion; we used this finding to localize the origin of forskolin-stimulated, CFTR-dependent gland fluid secretion. We tested the hypothesis that secretion to forskolin might originate from the gland duct rather than or in addition to the acini. We ligated gland ducts at various points, stimulated the glands with forskolin, and monitored the regions of the glands that swelled. The results supported an acinar rather than ductal origin of secretion. We tracked particles in the mucus using Nomarski time-lapse imaging; particles originated in the acini and traveled toward the duct orifice. Estimated bulk flow accelerated in the acini and mucus tubules, consistent with fluid secretion in those regions, but was constant in the unbranched duct, consistent with a lack of fluid secretion or absorption by the ductal epithelium. We conclude that CFTR-dependent gland fluid secretion originates in the serous acini. The failure to observe either secretion or absorption from the CFTR and epithelial Na(+) channel (ENaC)-rich ciliated ducts is unexplained, but may indicate that this epithelium alters the composition rather than the volume of gland mucus.

An inwardly rectifying potassium channel in apical membrane of Calu-3 cells.

Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells.

A "virtual gland" method for quantifying epithelial fluid secretion.

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-microl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37 degrees C. Basal Jv was 2.7 +/- 0.1 microl x cm(-2) x h(-1) (n = 42), and TEP was -9.2 +/- 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6) x Jv and TEP were stimulated 64 and 59%, respectively, by 5 microM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal Jv and TEP were inhibited to 82 and 63%, respectively, with 10 microM bumetanide (n = 5), 71 and 82% with 100 microM acetazolamide (n = 5), and 47 and 56% with 600 microM glibenclamide (n = 4). Basal Jv and TEP were 52 and 89% of control values, respectively, after HCO3- replacement with HEPES (n = 16). The net HCO3- concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (approximately 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of Jv in Calu-3 cells.

Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands.

We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.