Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19.

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

Development of fully automated and ultrasensitive assays for urinary adiponectin and their application as novel biomarkers for diabetic kidney disease.

Glomerular filtration rate (GFR) and urinary albumin excretion rate (UAER) are used to diagnose and classify the severity of chronic kidney disease. Total adiponectin (T-AN) and high molecular weight adiponectin (H-AN) assays were developed using the fully automated immunoassay system, HI-1000 and their significance over conventional biomarkers were investigated. The T-AN and H-AN assays had high reproducibility, good linearity, and sufficient sensitivity to detect trace amounts of adiponectin in the urine. Urine samples after gel filtration were analyzed for the presence of different molecular isoforms. Low molecular weight (LMW) forms and monomers were the major components (93%) of adiponectin in the urine from a diabetic patient with normoalbuminuria. Urine from a microalbuminuria patient contained both high molecular weight (HMW) (11%) and middle molecular weight (MMW) (28%) adiponectin, although the LMW level was still high (52%). The amount of HMW (32%) and MMW (42%) were more abundant than that of LMW (24%) in a diabetic patient with macroalbuminuria. T-AN (r = - 0.43) and H-AN (r = - 0.38) levels showed higher correlation with estimated GFR (eGFR) than UAER (r = - 0.23). Urinary levels of both T-AN and H-AN negatively correlated with renal function in diabetic patients and they may serve as new biomarkers for diabetic kidney disease.

Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells.

Autophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells.

Quantitative monitoring of autophagic degradation.

We developed a quantitative method for analyzing the induction of autophagy using a CHO-K1 cell line stably expressing a green fluorescent protein (GFP) in mitochondrial matrix (mtGFP-CHO). When mtGFP-CHO cells were incubated with a medium depleted of amino acids and serum, the GFP fluorescence was decreased concomitant with degradation of the protein. Biochemical and morphological analyses strongly suggested the degradation of mtGFP was mediated by bulk and non-selective degradation of mitochondria by autophagy. Quantitative measurement of the mtGFP degradation was performed by measuring the GFP fluorescence and DNA content by a fluorometric method and calculating the relative GFP intensity of DNA content, which approximated mean GFP fluorescence per cell. Using this method, we showed for the first time that different inducers, such as amino acids and serum starvation or rapamycin treatment, promote autophagy with different kinetics. This method is easy, relatively quick, and may be easily adapted to high throughput screening for novel drugs that enhance or inhibit autophagy, and also for genes that regulate or modulate autophagy.