A Comparison of the Performance of SARS-CoV-2 Antibody Assays in Healthcare Workers with COVID-19.

Aims Since its emergence, significant interest surrounds the use of SARS-CoV-2 serological tests as an alternative or as an adjunct to molecular testing. However, given the speed of this pandemic, paralleled with the pressure to develop and provide serological tests in an expediated manner, not every assay has undergone the rigorous evaluation that is usually associated with medical diagnostic assays. We aimed to examine the performance of several commercially available SARS-CoV-2 IgG antibody assays among participants with confirmed COVID-19 disease and negative controls. Methods Serum taken between day 17 and day 40 post onset of symptoms from 41 healthcare workers with RT-PCR confirmed COVID-19 disease, and pre-pandemic serum from 20 negative controls, were tested for the presence of SARS-CoV-2 IgG using 7 different assays including point-of-care (POC) and laboratory-based assays. Results Assay performance varied. The lab-based Abbott diagnostics SARS-CoV-2 IgG assay proved to be the assay with the best positive and negative predictive value, and overall accuracy. The POC Nal von Minden GmbH and Biozek assays also performed well. Conclusion Our research demonstrates the variations in performance of several commercially available SARS-CoV-2 antibody assays. These findings identify the limitations of some serological tests for SARS-CoV-2. This information will help inform test selection and may have particular relevance to providers operating beyond accredited laboratories.

Meta-analysis of the cardiovascular benefits of intensive lipid lowering with statins.

OBJECTIVE: To evaluate the efficacy of intensive lipid lowering with higher-dose statins. METHODS: Meta-analysis of seven randomized controlled trials comprising 50,972 participants. RESULTS: Mean follow-up was 3.1 years with mean age 63 years. Final LDL-C levels in intensive lipid-lowering group were 1.42-2.07 mmol/l compared to 2.1-3.5 mmol/l in the less intensive or control group. The intensive arm had significantly lower risks for stroke OR 0.80 (95% CI 0.71-0.89); major coronary events OR 0.74 (95% CI 0.65-0.83); cardiovascular disease (CVD) or coronary heart disease (CHD) deaths OR 0.84 (95% CI 0.74-0.95). Significantly higher liver enzyme abnormalities occurred in intensive group* (OR 3.96; 95% CI 2.08-7.53), but it was not associated with drug discontinuations (OR 1.20; 95% CI 0.88-1.64). CONCLUSION: In those at high risk of cardiovascular events, intensive lipid lowering with statins to LDL-C level <2.1 mmol/l significantly reduces risk of stroke, major coronary events and CVD or CHD deaths compared to LDL-C level ≥ 2.1 mmol/l. [*Correction added on 11 January 2011 after first online publication on 27 October 2010. The phrase, "Significantly higher liver enzyme abnormalities occurred in less intensive group", was amended to "Significantly higher liver enzyme abnormalities occurred in intensive group".].

Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation.

Immunohistochemical localization of the INsP4 receptor GTPase-activating protein GAP1IP4BP in the rat brain.

The distribution of GAP1(IP4BP), a GTPase-activating protein showing high affinity and stereospecificity for inositol 1,3,4,5-tetrakisphosphate (InsP4), was investigated by Western blot and immunohistochemistry of rodent brain with polyclonal antibodies generated against the carboxy-terminus of the cloned protein. GAP1(IP4BP)-like immunoreactivity was found throughout the brain, most notably in the pyriform cortex, neocortex, hippocampus, striatum, and cerebellar cortex. However, the most striking immunolabeling was consistently localized to area CA1 of the hippocampus and the central, medial, and intercalated nuclei of the amygdala. Western blot analysis of the corresponding brain regions corroborated these immunohistochemical observations. The regionally specific expression of GAP1(IP4BP) provides the prerequisite neuroanatomical substrate toward elucidating the functional role of InsP4 and GAP1(IP4BP) in the central nervous system.

Antisense knock out of the inositol 1,3,4,5-tetrakisphosphate receptor GAP1(IP4BP) in the human erythroleukemia cell line leads to the appearance of intermediate conductance K(Ca) channels that hyperpolarize the membrane and enhance calcium influx.

To study the role of the inositol 1,3,4,5-trisphosphate-binding protein GAP1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisense DNA (V-HEL cells). GAP1(IP4BP) protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-beta-D- galactoside to relieve LacI repression. The loss of GAP1(IP4BP) was associated with both a membrane hyperpolarization and a substantially increased Ca2+ entry induced by thrombin or thapsigargin. The activation of intermediate conductance Ca2+-activated K+ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca2+ entry, and both were blocked by charybdotoxin. Stimulated V-HEL cells did not hyperpolarize and basal Ca2+ influx was unaffected by charybdotoxin. In V-HEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stimulated Ca2+ influx was increased. Expression of mRNA for the human Ca2+-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (IK(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels. Our results demonstrate that GAP1(IP4BP), likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca2+ entry subsequent to the release of internal Ca2+ stores.

Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein.

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 microM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin-agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527-530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.

Purification and characterization of the human type 1 Ins(1,4,5)P3 receptor from platelets and comparison with receptor subtypes in other normal and transformed blood cells.

We report the first purification of a native human form of the Ins(1,4,5)P3 (InsP3) receptor. This receptor, isolated from platelets, has an apparent molecular mass on SDS/PAGE of 252 kDa and is chromatographed by gel filtration as an oligomer of about 1 x 10(6) kDa. [3H]InsP3 bound to a single class of sites on the purified receptor protein with a Kd of 27 nM and a Bmax. of 2.2 nmol/mg of protein. The platelet InsP3 receptor, like the rodent cerebellar receptors, was identified immunochemically as a type 1 receptor, but unlike its brain counterparts bound poorly to concanavalin A and other lectins and was not significantly phosphorylated by protein kinase A. All cultured megakaryocytic leukaemia cell lines (e.g. Dami, CHRF-288 and Meg-01) and HEL cells were also immunopositive for type 1 receptor, which was substantially increased in some cases by DMSO or phorbol 12-myristate 13-acetate (PMA) which induce further megakaryocytic differentiation. Normal mixed lymphocyte and granulocyte fractions and an enriched T-cell fraction from human blood had measurable InsP3-binding activity, but no detectable type 1 protein. In contrast, Jurkat E6-1 (T-cell lymphoma) cells and the transformed B-cell line RPMI 8392 were immunopositive for type 1 receptor. HL-60 (human promyelocytic leukaemia) cells had no detectable type 1 receptor unless they were stimulated to differentiate along monocyte/macrophage lines by PMA. We conclude that: (1) of the major normal blood cells only platelets contain type 1 InsP3 receptors; (2) some neoplastic transformed blood cell lines also express type 1 receptors, in contrast to their normal counterparts; and (3) increased levels of type 1 InsP3 receptor are induced in some transformed cells under conditions that favour their further terminal differentiation.

Suppression of the delayed rectifier type of voltage gated K+ outward current in megakaryocytes from patients with myelogenous leukemias.

In normal human megakaryocytes, we identified a delayed rectifier type of voltage-gated outward K+ current (DRK). In two human megakaryoblastic tumor cell lines (DAMI, CHRF-288-11) and the human erythroleukemia cell line (HEL) the DRK current was not detected. To determine if the absence of the DRK current in the tumor cells is the result of the underlying malignant state, we examined megakaryocytes from myelogenous leukemia patients. In 24 of 29 megakaryocytes from the myelogenous leukemia patients, the DRK current was greatly suppressed, whereas in the remaining 5 megakaryocytes a normal large amplitude DRK current was present. We had the opportunity to reexamine megakaryocytes from a patient with acute promyelocytic leukemia (M3), after chemotherapy. Whereas the DRK current was suppressed before treatment, the current reappeared after chemotherapy. Exposure to the adenylate cyclase activator, forskolin, caused the appearance of a voltage-gated outward current in the megakaryocytes of patients with acute myelogenous leukemia. This finding suggests either that the channels underlying the DRK current are present but somehow suppressed in megakaryocytes from these patients or that forskolin induces a different voltage-gated outward current. We suggest that the megakaryocytes from the myelogenous leukemia patients with suppressed DRK current are abnormal, whereas the others may be normal megakaryocytes. The suppression of the DRK current may be a contributory factor to the dysregulation of thrombopoiesis (Zittoun et al: Semin Hop Paris 44:183, 1968 and Rabellino et al: Blood 63:615, 1984) in myelogenous leukemias.

Ca2+ release by inositol 1,4,5-trisphosphate is blocked by the K(+)-channel blockers apamin and tetrapentylammonium ion, and a monoclonal antibody to a 63 kDa membrane protein: reversal of blockade by K+ ionophores nigericin and valinomycin and purification of the 63 kDa antibody-binding protein.

Ins(1,4,5)P3-induced Ca2+ release from platelet membrane vesicles was blocked by apamin, a selective inhibitor of low-conductance Ca(2+)-activated K+ channels, and by tetrapentylammonium ion, and was weakly inhibited by tetraethylammonium ion. Other K(+)-channel blockers, i.e. charybdotoxin, 4-aminopyridine and glybenclamide were ineffective. A monoclonal antibody (mAb 213-21) obtained by immunizing mice with the InsP3-sensitive membrane fraction from platelets also blocked Ca2+ release by InsP3 from membrane vesicles obtained from platelets, cerebellum, aortic smooth muscle, HEL cells and sea-urchin eggs. ATP-dependent Ca2+ uptake and binding of [3H]InsP3 to platelet membranes was unaffected by either K(+)-channel blockers or mAb 213-21. Blockade of Ca2+ release by apamin, tetrapentylammonium and mAb 213-21 was not affected by the Na+/H+ carrier monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), but could be completely reversed by the K+/H+ ionophore nigericin and partially reversed by the K+ carrier valinomycin. The antibody-binding protein (ABP) solubilized from platelets, cerebellum, and smooth muscle chromatographed identically on gel filtration, anion-exchange and heparin-TSK h.p.l.c. ABP was purified to apparent homogeneity from platelets and aortic smooth muscle as a 63 kDa protein by immunoaffinity chromatography on mAb 213-21-agarose. These results suggest that optimal Ca2+ release by InsP3 from platelet membrane vesicles may require the tandem function of a K+ channel. A counterflow of K+ ions could prevent the build-up of a membrane potential (inside negative) that would tend to oppose Ca2+ release. The 63 kDa protein may function to regulate K+ permeability that is coupled to the Ca2+ efflux via the InsP3 receptor.

The inositol 1,4,5-trisphosphate receptor binding sites of platelet membranes. pH-dependency, inhibition by polymeric sulphates, and the possible presence of arginine at the binding site.

The present study was initiated to characterize the inositol 1,4,5-trisphosphate (InsP3)-binding site in human platelets that is involved in Ca2+ release. InsP3 binding to platelet membranes was measured in two ways; (1) by displacement of labelled InsP3 with unlabelled InsP3, as in previous studies, and (2) directly, using only radioactive InsP3 as ligand, over the concentration range 0.25-100 nM. At physiological pH (7.1) the binding data were best fitted by a model for a single saturable binding site, with KD = 11.8 nM and Bmax. = 1.4 pmol/mg of protein. At alkaline pH values (8.3 and 9.4) binding was best fitted by a two-site model, the second site being of higher affinity (KD = 0.75-1.2 nM) but lower concentration (Bmax. = 0.195-0.6 pmol/mg of protein). All binding of InsP3 was blocked by polymeric sulphates (heparin, dextran sulphate, polyvinyl sulphate) regardless of pH. The specific arginine-modifying reagent p-hydroxyphenylglyoxal irreversibly blocked InsP3 binding, suggesting the presence of arginine at the recognition site for InsP3 binding. NN'-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (ECCD), which are carboxy-group-specific reagents, blocked Ca2+ release, but not InsP3 binding, indicating the existence of another site that regulates Ca2+ release apart from the active centre for InsP3.

Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.

Stimulus-response coupling in a cell-free platelet membrane system. GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3.

The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP-ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.

Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets.

Human platelet membrane vesicles that accumulated Ca2+ in the presence of ATP were isolated on an isoosmotic KCl-Percoll gradient. ATP-dependent Ca2+ uptake was stimulated by oxalate and phosphate to steady-state levels of greater than 100 nmol/mg protein, and the accumulated Ca2+ could be largely released by ionophore A23187. Inositol 1,4,5-trisphosphate, in a dose-dependent manner (0.5-5.0 microM), caused the rapid release (less than 5 s) of 40-70% of the total A23187-releasable store of accumulated Ca2+. The membrane vesicles that release accumulated Ca2+ in response to inositol 1,4,5-trisphosphate were enriched in enzymes characteristically found in smooth endoplasmic reticulum. These results support the hypothesis that inositol 1,4,5-trisphosphate, produced by the hydrolysis of phosphatidylinositol 1,4-bisphosphate in response to stimulation of cell surface receptors, is a second messenger mediating the release of Ca2+ from intracellular storage sites.

Effects of insulin on CO2 fixation in adipose tissue. Evidence for regulation of pyruvate transport.

Insulin was found to double the rate of incorporation of H14CO3- into protein by segments of rat epididymal adipose tissue provided the incubation medium contained a suitable energy substrate such as fructose. Overall protein synthesis was increased by insulin to a lesser extent, one-third as measured by tritiated water indicating that insulin also increased CO2 fixation into amino acids. The latter could be demonstrated only when the tissue amino acid pools were expanded by the addition of aspartate to the incubation medium. The pattern of labeling observed in the amino acids indicated that CO2 fixation occurred primarily at the pyruvate carboxylase step. Addition of pyruvate to the incubation medium also increased CO2 fixation and this effect was not additive with that of insulin, suggesting that insulin acted by increasing the availability of pyruvate to the carboxylase. No change in carboxylase activity could be measured. Mitochondria isolated from tissue exposed to insulin retained a higher capacity to fix CO2 into acid-soluble products provided they were not freeze-thawed or sonicated. Uptake of pyruvate by mitochondria incubated 1 min at 2 degrees C or 5 s at 15 degrees C was doubled by prior insulin treatment of the tissue. It is concluded that insulin increases the flux through pyruvate carboxylase in adipose tissue in part by increasing the transport of pyruvate through the inner mitochondrial membrane.